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https://openalex.org/W4283030294	https://hal-insu.archives-ouvertes.fr/insu-03859253/file/tc-16-4251-2022.pdf	English	null	Effects of topographic and meteorological parameters on the surface area loss of ice aprons in the Mont Blanc massif (European Alps)	null	2,022	cc-by	16,470	To cite this version: Suvrat Kaushik, Ludovic Ravanel, Florence Magnin, Yajing Yan, Emmanuel Trouve, et al.. Effects of topographic and meteorological parameters on the surface area loss of ice aprons in the Mont Blanc massif (European Alps). The Cryosphere, 2022, 16, pp.4251-4271. 10.5194/tc-16-4251-2022. insu-03859253 Effects of topographic and meteorological parameters on the surface area loss of ice aprons in the Mont Blanc massif (European Alps) Suvrat Kaushik, Ludovic Ravanel, Florence Magnin, Yajing Yan, Emmanuel Trouve, Diego Cusicanqui Distributed under a Creative Commons Attribution 4.0 International License Correspondence: Suvrat Kaushik (suvrat.kaushik@univ-smb.fr) Correspondence: Suvrat Kaushik (suvrat.kaushik@univ-smb.fr) Received: 13 May 2022 – Discussion started: 16 June 2022 Revised: 15 September 2022 – Accepted: 26 September 2022 – Published: 12 October 2022 Received: 13 May 2022 – Discussion started: 16 June 2022 Revised: 15 September 2022 – Accepted: 26 September 2022 – Published: 12 October 2022 Abstract. Ice aprons (IAs) are part of the critical compo- nents of the Alpine cryosphere. As a result of the changing climate over the past few decades, deglaciation has resulted in a surface decrease of IAs, which has not yet been docu- mented, except for a few speciﬁc examples. In this study, we quantify the effects of climate change on IAs since the mid- 20th century in the Mont Blanc massif (western European Alps). We then evaluate the role of meteorological param- eters and the local topography in the behaviour of IAs. We precisely mapped the surface areas of 200 IAs using high- resolution aerial and satellite photographs from 1952, 2001, 2012 and 2019. From the latter inventory, the surface area of the present individual IAs ranges from 0.001 to 0.04 km2. IAs have lost their surface area over the past 70 years, with an alarming increase since the early 2000s. The total area, from 7.93 km2 in 1952, was reduced to 5.91 km2 in 2001 (−25.5 %) before collapsing to 4.21 km2 in 2019 (−47 % since 1952). We performed a regression analysis using tem- perature and precipitation proxies to better understand the ef- fects of meteorological parameters on IA surface area varia- tions. We found a strong correlation between both proxies and the relative area loss of IAs, indicating the signiﬁcant in- ﬂuence of the changing climate on the evolution of IAs. We also evaluated the role of the local topographic factors in the IA area loss. At a regional scale, factors like direct solar ra- diation and elevation inﬂuence the behaviour of IAs, while others like curvature, slope and size of the IAs seem to be rather important on a local scale. 1 Introduction The predicted shift in climate dynamics over the next decades will undoubtedly have severe consequences on the high mountain environments, primarily on glacier extent (Raﬁq and Mishra, 2016; Kraaijenbrink et al., 2017; IPCC, 2021), permafrost (Magnin et al., 2017) and ice and snow cover (Rastner et al., 2019; Guillet and Ravanel, 2020). The effects of climate change on glaciers constitute a remarkably well- discussed topic in the scientiﬁc community (Yalcin, 2019). Meteorological parameters (mainly temperature and pre- cipitation) are the main driving forces responsible for these changes (Scherler et al., 2011; Bolch et al., 2012; Davies et al., 2012). Shifting temperature and precipitation trends lead to the advance or retreat of glaciers both in volume and sur- face area (Liu et al., 2013; Yang et al., 2019). On a regional and global scale, many authors have studied the impacts of climate warming on glacier retreats and, consequently, on the hydrology of the mountain environments (e.g. Baraer et al., 2012; Sorg et al., 2014; Frans et al., 2016; Coppola et al., 2018). However, as observed by Furbish and Andrews (1984), Oerlemans et al. (1998), Hoelzle et al. (2003), and Salerno et al. (2017), glaciers present in the same climate regime can re- spond to climate change in different ways. The local climate variations can partly explain these variable responses. How- ever, many of these variations result from different morpho- metric (size, shape, length) and topographic (altitude, slope, aspect, curvature, terrain ruggedness) characteristics. HAL Id: insu-03859253 https://insu.hal.science/insu-03859253v1 Submitted on 18 Nov 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License The Cryosphere, 16, 4251–4271, 2022 https://doi.org/10.5194/tc-16-4251-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs Also, they are typically present in extremely challenging topographies on isolated steep slopes. Cogley et al. (2011) speciﬁed that IAs are “lying above the head of a glacial bergschrund which separates the ﬂowing glacier ice from the stagnant ice, or a rock headwall”. shrinkage since the 1990s. They linked the loss of IA area with meteorological parameters, mainly air temperature and precipitation. It was thus the ﬁrst documented evidence that IAs have been losing ice volume due to the changing climate. However, since this study was local and based on only a few IAs, the authors could not consider other factors, such as the local topography critical for small glacier bodies (Hock, 2003; Laha et al., 2017). Thus, to overcome these limitations, we propose a large- scale analysis to ascertain the relationship of the area loss of IAs with the meteorological parameters, mainly air tempera- ture and precipitation, using a more comprehensive database (ca. 200 IAs) covering the whole MBM. The large inventory of IAs has been surveyed thanks to high-resolution aerial and satellite images from 1952, 2001, 2012 and 2019. Fur- ther, based on our inventory, we also evaluate the impacts of the topographic/geometric controls on the area changes of IAs. For this, we consider the size of IA, elevation/alti- tude, slope, curvature, topographic ruggedness index (TRI), direct solar radiation and permafrost conditions (classiﬁed together as topographic factors) based on past studies on similar themes (e.g. Oerlemans et al., 1998; Warren, 2008; DeBeer and Sharp, 2009; Jiskoot et al., 2009; Davies et al., 2012; Salerno et al., 2017). Because of their presence on steep slopes, IAs are essen- tial natural elements for the practice of mountaineering, espe- cially in famous destinations like MBM (Barker, 1982). IAs are passing points for many classic mountaineering routes (Mourey et al., 2019). Hence, the loss of IAs is a severe threat to the iconic practice of mountaineering, inscribed in 2019 by UNESCO on the Representative List of the Intangi- ble Cultural Heritage of Humanity. IAs on steep rock walls also carry the critical role of covering steep rock slopes and preventing them from direct exposure to direct solar radia- tion, thus partly preventing the warming of the underlying permafrost. In addition, a recent study by Guillet et al. S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs Several studies have been devoted to understanding the linkage between topographic factors and the response of glacier/ice bodies (e.g. Davies et al., 2012; De Angelis, 2014; Salerno et al., 2017). Figure 1. The Mont Blanc massif (western European Alps). A total of 200 IAs (red stars) were digitized accurately on high- resolution images. The glacier outlines (in blue) come from Gardent et al. (2014). The green line shows the border between France, Italy and Switzerland. The World Glacier Monitoring Service (WGMS) moni- tors glacier changes in all the major mountain regions of the world. However, most mapping and monitoring studies on a global scale focus on massive glaciers since they are gener- ally assessable and easier to monitor compared to other ice features (Liu et al., 2013). Studies are rare for small glaciers or ice bodies, which generally show a more pronounced response to climate change (Oerlemans and Reichert, 2000; Triglav- ˇCekada and Gabrovec, 2013; Fischer et al., 2015). This has led to a criti- cal gap in our understanding of their behaviour and mass bal- ance estimates. As part of this trend, ice aprons (IAs), some- times also referred to as “rock faces partially covered with ice” (Gruber and Haeberli, 2007; Hasler et al., 2011), have also received poor attention from the scientiﬁc community. Figure 1. The Mont Blanc massif (western European Alps). A total of 200 IAs (red stars) were digitized accurately on high- resolution images. The glacier outlines (in blue) come from Gardent et al. (2014). The green line shows the border between France, Italy and Switzerland. These small ice accumulations on steep rock slopes are commonly found in all signiﬁcant glacierized basins world- wide. However, a concrete and well-summarized deﬁnition for IAs is still missing from the literature. Previously, many authors like Benn and Evans (2010), Singh et al. (2011), and Cogley et al. (2011) tried to deﬁne IAs, but the most pre- cise deﬁnition for IAs up to now can be found in Guillet and Ravanel (2020) for the Mont Blanc massif (MBM; Eu- ropean Alps). These authors deﬁned IAs as “very small (typ- ically smaller than 0.1 km2 in extent) ice bodies of irregular outline, lying on slopes > 40◦, regardless of whether they are thick enough to deform under their weight”. The small spatial extent of the IAs makes them very difﬁcult to map and monitor. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 4252 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs hik et al.: Effects of topographic and meteorological parameters on the surface area loss of IA 2 Study area and the impacts of climate change in the region area was 24 % of the total area from the end of the LIA to 2008 (Gardent et al., 2014). The reported loss of ice thick- ness is also noteworthy. For example, the loss of ice thickness at the front of the Mer de Glace glacier (1650 m a.s.l.) from 1986 to 2021 is 145 m; the Argentière glacier (1900 m a.s.l.) has lost 80 m in thickness from 1994 to 2013 (Bauder et al., 2007). At 3550 m a.s.l., the surface of the Géant glacier also lowered by 20 m between 1992 and 2012 (Ravanel et al., 2013). The glacier retreat and shrinkage concur with the equilibrium line altitude (ELA) that rose by 170 m between 1984 and 2010 in the western Alps (Rabatel et al., 2013). As a result of the loss of ice volume, the density of open crevasses has considerably increased, along with an increase in bare ice areas. In some instances, ice volume loss leads to instability of steep slopes, and serac falls from the front of warm and cold glaciers are more frequent (Fischer et al., 2006). This latter process can be typical during the warmest periods of the year (Deline et al., 2012). Warming trends also intensify moraine erosion, resulting in an increase in rock- fall and landslide events (Deline et al., 2015; Ravanel et al., 2018). Degradation/warming is another critical concern for permafrost (e.g. Haeberli and Gruber, 2009). The Mont Blanc massif (Fig. 1) is located in the north- western (external) Alps between France, Switzerland and Italy. It covers ca. 550 km2 and displays some of the highest peaks in the European Alps; a dozen peaks have elevations greater than 4000 m a.s.l. The MBM thus shows a signiﬁcant variation in the elevation range throughout the massif; the lowest point of the massif is at 1050 m a.s.l. (Chamonix), and the highest, the top of Mont Blanc, is at 4808 m a.s.l. Because of its high elevation, the MBM is also the most glacierized massif in the French Alps (Gardent et al., 2014). There are about 100 glaciers often bordered by steep rock walls, including 12 glaciers larger than 5 km2. The steep and irregular terrain facilitates the development of many unique ice bodies like cold-based hanging glaciers or IAs. Figure 2 shows two examples of the locations of the IAs on the steep N faces from the study region. 2 Study area and the impacts of climate change in the region y g As a result of an asymmetry of the massif, six of the largest glaciers are located on its NW French side, where slopes are gentler than the Italian side and glaciers are well fed by the westerly winds while melting is reduced by the protec- tion of the shaded north faces. The SE Italian side is char- acterized by smaller glaciers and generally steeper slopes bounded by very high sub-vertical rock walls. This asym- metry is also evidenced by the difference in the meteoro- logical conditions observed on the two sides of the mas- sif. For example, the mean annual air temperature (MAAT) recorded in Chamonix (at 1044 m a.s.l.) is +7.2 ◦C, while that in Courmayeur (1223 m a.s.l.) is +10.4 ◦C (Deline et al., 2012). Comparing the annual MAAT values from 1934 to to- day shows that MAAT increased by > 2.1 ◦C in Chamonix (Météo-France data). Moreover, the increase in MAAT from 1970 to 2009 was almost 4 times faster than from 1934 to 1970 (Mourey et al., 2019). The MAAT also increased by 1.4 ◦C not only at lower elevations but also at elevations ex- ceeding 4000 m a.s.l. between 1990 and 2014 (Gilbert and Vincent, 2013). The MBM has experienced nine summers characterized by heatwaves (where maximum temperatures for at least three consecutive days exceed a heatwave tem- perature threshold deﬁned for the region) since 1990 (1994, 2003, 2006, 2009, 2015, 2017, 2018, 2019 and 2020), with the one as recent as 2018 being the second (after 2003) hottest. The average annual precipitation recorded for Cha- monix is 1288 mm, and that for Courmayeur is 854 mm (Vin- cent, 2002). The precipitation rates in the MBM have re- mained relatively constant since the end of the LIA, but there is a noticeable decrease in the number of snowfall days rel- ative to the total precipitation days below 2700 m a.s.l. (Ser- quet et al., 2011). 3 Data description This section describes all the datasets obtained from diverse sources used in this study (Table 1). S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs (2021) showed that the ice present at the base of the Triangle du Tacul IA could be older than 3 ka, making IAs a potentially important glacial heritage. Guillet and Ravanel (2020) showed that IAs in the MBM have lost mass since the Little Ice Age (LIA). Based on six different IAs, their study also showed an acceleration in the https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 4253 3.1 Digital elevation model Ice aprons and their locations in the MBM: (a) IAs on the N face of Grandes Jorasses (4208 m a.s.l.), photograph courtesy Ludovic Ravanel, and (b) IAs on the headwall of the Argentière glacier separated by a bergschrund (3280 m a.s.l.), IGN orthophotos 2015. Table 1. Datasets used for the study. Table 1. Datasets used for the study. Data type Source Resolution (m/time) Acquisition time/period Optical Orthoimages IGN 0.2 July 2015 Pleiades 1A PAN 0.5 25 Aug 2019, 19 Aug 2012 Sentinel 2 10 12 Sep 2019 SPOT 6 2.2 14 Sep 2019 Pleiades 1A XS 2 19 Aug 2012 Orthoimages IGN 0.5 Jul 2001 Orthoimages IGN 0.5 1952 Meteorological Col du Grand Saint Bernard weather station (2469 m a.s.l.) daily 1952–2019 Aiguille du Midi weather station (3840 m a.s.l.) daily 2007–2018 SAFRAN reanalysis daily 1958–2019 Resolution (m/time) Acquisition time/period of the ice bodies, we could only work with high-resolution optical images covering the entire MBM. We were thus lim- ited by only one set of excellent quality images for 1952 and 2001, as very high-resolution images for this study period were unavailable from any other source. Hence our mapping exercise relied only on the orthoimages for these two time pe- riods. For 2012 and 2019, we had data from multiple sources (Pleiades, SPOT and orthoimages) to deal with the problems associated with the lack of coverage, cloud cover, illumina- tion, shadow and seasonal snow cover that made visual in- terpretation difﬁcult. We used a combination of Pleiades and SPOT 6 XS images for mapping the IA boundaries, with val- idation of results conducted with the help of the orthoim- ages. To avoid overestimating the extent of IAs, we utilized images acquired at the end of the summer period (late Au- gust or early September). Considering that our optical images came from many sources, it was necessary to accurately co- register all images. We used the automatic image-to-image co-registration tool in ENVI 5.6. The process included lo- cating and matching several feature points called tie points resolution, robust 4 m DEM was obtained at the end of the processing steps. More detailed information about the pro- cessing parameters for DEM generation and co-registration can be found in Kaushik et al. (2021). We used this DEM to compute topographic parameters like slope, aspect, curva- ture, elevation, TRI, mean annual rock surface temperature (MARST) and direct solar radiation. 3.1 Digital elevation model Since one of the main aims of our study was to perform a joint analysis of the behaviour of small ice bodies and the local topography, it was paramount to have a robust high- resolution and accurate digital elevation model (DEM) for the study region. To avoid the uncertainties that most global DEMs are plagued with and to overcome the problem of dif- ferent DEM origins on the French and Italian sides of the MBM, we built our own DEM. As part of the CNES Kalideos Alps project, stereoscopic sub-metre resolution optical im- ages from the Pleiades constellation were acquired. Using the pair of stereo panchromatic images (25 August 2019), a 4 m resolution DEM was computed using the Ames Stereo Pipeline (ASP), an open-source processing chain developed by Shean et al. (2016). The parameters used for the pro- cessing were kept the same as those of Marti et al. (2016). The second part of the processing involved accurately co- registering the newly built DEM with an existing refer- ence DEM of high precision and accuracy. For this pur- pose, we used the automatic DEM co-registration method- ology given by Nuth and Kääb (2011). To co-register the source 4 m Pleiades DEM (Fig. 3a) we used a 2 m lidar DEM for the area around the Argentière glacier (8 × 2.5 km spa- tial extent) (Fig. 3b) built by the Institut des Géosciences de l’Environnement (IGE). A precisely co-registered, high- Global warming has led to a general retreat trend of the MBM glaciers since the end of the LIA despite small re- advances culminating in 1890, the 1920s and the 1980s (Bauder et al., 2007). The recorded loss of glacier surface https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4254 Figure 2. Ice aprons and their locations in the MBM: (a) IAs on the N face of Grandes Jorasses (4208 m a.s.l.), photograph courtesy Ludovic Ravanel, and (b) IAs on the headwall of the Argentière glacier separated by a bergschrund (3280 m a.s.l.), IGN orthophotos 2015. Figure 2. 3.2 Optical aerial and satellite images This study relies on high-resolution aerial and satellite im- ages (Table 1). Working with data from different sources al- lowed us to tap into the wealth of data for comparison. Span- ning over seven decades and covering the entire MBM, or- thoimages for 1952, 2001 (0.5 m resolution) and 2015 (0.2 m resolution) were downloaded from Géoportail IGN (French Institut national de l’information géographique et forestière), while the panchromatic and XS images from SPOT 6 and Pleiades at 2.2 and 0.5 m respectively were downloaded from the Kalideos Alps website. Considering the small dimensions https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 4255 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs Figure 3. (a) The source Pleiades DEM used for further analysis; (b) the reference lidar DEM of the Argentière glacier used for co- registration. Figure 3. (a) The source Pleiades DEM used for further analysis; (b) the reference lidar DEM of the Argentière glacier used for co- registration. Figure 3. (a) The source Pleiades DEM used for further analysis; (b) the reference lidar DEM of the A registration. Pleiades DEM used for further analysis; (b) the reference lidar DEM of the Argentière glacier used for c GSB represents a similar climatological regime to the MBM, and the weather records were available for an extended pe- riod starting from the 1860s. Such long-term weather records were unavailable from any weather station in the MBM. Since all IAs are located at elevations above the elevation of the GSB weather station, it was necessary to transform the weather records to an elevation closer to the average ele- vation range of the IAs. For this reason, it was necessary to transform the data from the GSB station using the weather records from the AdM weather station (data available since 2007). Guillet and Ravanel (2020) found a strong correlation between the monthly averaged AdM and GSB temperature records and were able to transform the GSB temperatures using a linear model: in a reference image and a warped image selected for co- registration. Here, we used the Pleiades panchromatic image of 2019 as a reference, and all the warped images were ac- cordingly co-registered. 3.3 Meteorological data Proxies to deﬁne accumulation and ablation phases were built to explore the correlated variations in the surface area of IAs with the changing climate. A similar study for six IAs was performed by Guillet and Ravanel (2020); we aim to test the validity of their results with a more extensive database (ca. 200 IAs) in the entire MBM. Since the IAs are spread across different elevation ranges, we tested the results using the SAFRAN reanalysis product (Vernay et al., 2019) that produces gridded temperature, precipitation, wind speed, and other datasets of meteorological variables at an hourly time step. These data were available as NetCDF ﬁles from 1958 for all the French massifs; at elevation belts every 300 m; at 0, 20, and 40◦slopes; and for all eight aspects (N, NE, E, SE, S, SW, W, NW). Our study is based on two different me- teorological datasets described in the subsections below to compare the differences. TAdM = αTGSB i + β + ri, (1) where α = 0.87 (slope), β = −7.7 ◦C (intercept) and r TAdM = αTGSB i + β + ri, (1) where α = 0.87 (slope), β = −7.7 ◦C (intercept) and r (residuals) has zero mean. TAdM = αTGSB i + β + ri, (1) (1) where α = 0.87 (slope), β = −7.7 ◦C (intercept) and r (residuals) has zero mean. No transformation for the precipitation values was per- formed as this relation is tough to establish and not always linear (Smith, 2008). Hence, the original GSB precipita- tion values were used for the analysis. Using these weather records, Guillet and Ravanel (2020) found a robust correla- tion between ablation and accumulation proxies and the sur- face area change of six IAs. We used the same datasets to test for similar potential relationships for ca. 200 IAs, and the results are shown in Sect. 5.3. 3.2 Optical aerial and satellite images Both coarse and ﬁne co-registration procedures were performed, and the co-registration process was stopped when the RMSE values achieved were less than half the pixel resolution of the warped image based on the recommendations of Han and Oh (2018). A more detailed description of the co-registration process was discussed in Kaushik et al. (2021). 3.3.1 Meteorological datasets used by Guillet and Ravanel (2020) Since the study from Guillet and Ravanel (2020) involved a small number of IAs, the disparity arising from elevation differences of IAs (in turn, the temperature and precipitation coming from weather stations at a ﬁxed elevation) could have been minimized or not well represented. We decided to use the SAFRAN reanalysis product and checked for similar po- tential relationships of climate variables with the surface area change of IAs. The ﬁrst problem we encountered was that The ﬁrst part of our analysis follows a similar methodol- ogy followed by Guillet and Ravanel (2020) in their analysis. Like their study, we used homogenized weather records from the Col du Grand Saint Bernard (GSB), located close to the MBM at 2469 m a.s.l. and provided by MeteoSwiss and from the Aiguille du Midi (AdM) cable car station (3810 m a.s.l.). https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4256 4256 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of Figure 4. SAFRAN reanalysis product temperature time series from 1952–2019 for different elevations in the MBM. The ﬁgure show variation of the mean annual temperatures for the entire study period. Figure 4. SAFRAN reanalysis product temperature time series from 1952–2019 for different elevations in the MBM. The ﬁgure shows the variation of the mean annual temperatures for the entire study period. Figure 4. SAFRAN reanalysis product temperature time series from 1952–2019 for different elevations in the MBM. The ﬁgure shows the variation of the mean annual temperatures for the entire study period. Figure 4. SAFRAN reanalysis product temperature time series from 1952–2019 for different elevations in the MBM. The ﬁgure shows the variation of the mean annual temperatures for the entire study period. Figure 5. Correlation between the monthly averaged temperature measurements at the Col du Grand Saint Bernard (GSB) and the SAFRAN reanalysis data at 2400 m a.s.l. As previously stated, a similar relationship for precipita- tion was tough to establish. Hence, for the analysis, we used the SAFRAN precipitation data from 1958 and extrapolated the precipitation values from the GSB weather station to all elevation bands of SAFRAN data before 1958 (6 years up to 1952). 3.3.1 Meteorological datasets used by Guillet and Ravanel (2020) However, taking a cue from the previous study of Guillet and Ravanel (2020), we expect this impact to be in- signiﬁcant when considering the results over seven decades. 4.1 Mapping the surface area of IAs from high-resolution satellite images Figure 5. Correlation between the monthly averaged temperature measurements at the Col du Grand Saint Bernard (GSB) and the SAFRAN reanalysis data at 2400 m a.s.l. Figure 5. Correlation between the monthly averaged temperature measurements at the Col du Grand Saint Bernard (GSB) and the SAFRAN reanalysis data at 2400 m a.s.l. IA boundaries were manually delineated/digitized by the ﬁrst author of this paper to maintain data consistency in a ge- ographic information system (GIS) environment for 1952, 2001, 2012 and 2019. The problem of seasonal snow, which can lead to an overestimation of surface areas, was avoided using images at the end of the ablation period. The differen- tiation of IAs from other snow/ice bodies relies on the slope angle (we only consider ice bodies on slopes > 40◦to be IAs) and whether they are thick enough to deform under their own weight and show movement, like in the case of hanging glaciers. The slope mask to remove areas with slopes < 40◦ was built in ArcGIS 10.6 using the Pleiades DEM. Figure 6 shows the variations in the surface areas of IAs over the study period. It also highlights the importance of high-resolution images because of the small dimensions of our studied ice bodies. However, these data are not always available in the best quality for the past periods as we could only very ac- curately map 200 IAs (out of the total 423 IAs reported in Kaushik et al., 2021) for all the periods. These 200 IAs were selected carefully after a detailed visual inspection and con- sidering issues related to shadow and illumination. Since a point-based correlation analysis (with meteorological and to- the SAFRAN data starts from 1958, while our ﬁrst images date from 1952. Therefore, for comparison, it was essential to interpolate the missing data for the 6 years before 1958 (Fig. 4). Like the previous methodology, we looked for a lin- ear relationship between the SAFRAN temperature data (at 2400 m a.s.l. elevation belt) and the GSB temperature data. We again found a strong correlation between the two datasets (Fig. 5) which helped us transform the data using the SAFRAN data starts from 1958, while our ﬁrst images date from 1952. Therefore, for comparison, it was essential to interpolate the missing data for the 6 years before 1958 (Fig. 4). 4.1 Mapping the surface area of IAs from high-resolution satellite images Like the previous methodology, we looked for a lin- ear relationship between the SAFRAN temperature data (at 2400 m a.s.l. elevation belt) and the GSB temperature data. We again found a strong correlation between the two datasets (Fig. 5) which helped us transform the data using TSAFRAN2400 = αTGSB i + β + ri, (2) (2) where α = 1.01 (slope), β = −1.35 ◦C (intercept) and r (residuals) has zero mean. For the SAFRAN data estimated (2400 m a.s.l.) from 1952, we extrapolated the data for all elevation bands. We used a standard gradient of −0.53 ◦C (100 m)−1 increase in elevation based on the observations of Magnin et al. (2015) for the MBM. https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4257 Figure 6. IA extent delineated on high-resolution images: (a) orthophotos 1952, (b) orthophotos 2001, (c) Pleiades panchromatic 2012 and (d) Pleiades panchromatic 2019. The different coloured polygons represent the surface area for each date. The orthophotos are courtesy of the IGN, while the Pleiades images were acquired as part of the CNES Kalideos Alps project. hotos 1952, (b) orthophotos 2001, (c) Pleiades panchromatic 2012 and present the surface area for each date. The orthophotos are courtesy of S Kalideos Alps project. Figure 6. IA extent delineated on high-resolution images: (a) orthophotos 1952, (b) orthophotos 2001, (c) Pleiades panchromatic 2012 and (d) Pleiades panchromatic 2019. The different coloured polygons represent the surface area for each date. The orthophotos are courtesy of the IGN, while the Pleiades images were acquired as part of the CNES Kalideos Alps project. the size of IAs. The factors we considered for our analysis are elevation, slope, aspect, curvature, TRI, direct solar radi- ation (all estimated in ArcGIS 10.6), MARST and size of the IAs. The topographic parameters are generated using the 4 m Pleiades DEM described in Sect. 3.1. pographic parameters) requires very high accuracy and pre- cision of mapping, any signiﬁcant uncertainty would have resulted in a major bias in our correlation estimates. To avoid this, we used only 200 of the best mapped IAs for the correla- tion analysis. 4.2.1 Direct solar radiation Direct solar radiation (DSR) measures the potential total in- solation across a landscape or at a speciﬁc location. On a lo- cal scale, components such as topographic shading, slope and aspect control the radiation distribution (Olson and Rupper, 2019). For estimating the DSR, the viewshed algorithm was run based on a uniform sky and a ﬁxed atmospheric trans- missivity value of 1. Mahmoud Sabo et al. (2016) showed the application of these algorithms in areas of rough topography. The total DSR (DSRtot) for a given location is calculated as the sum of the DSR (Dirθ,α) from all the sun sectors (cal- culated for every sun position at 30 min intervals throughout 4.1 Mapping the surface area of IAs from high-resolution satellite images However, for the estimation of the total area of IAs in 1952, 2001, 2012 and 2019, as described in Sect. 5.2, we use the complete database of 423 IAs with the assump- tion that overall, for the entire database, the uncertainty in the mapping (± the surface area) cancels out eventually and becomes insigniﬁcant. 4.2 Generation of topo-climatic parameters The relative area loss of IAs for three time periods, i.e. 1952 to 2001, 2001 to 2012, and 2012 to 2019, is analysed with all topographic factors. The area loss is expressed as a relative percentage of the area lost between the ﬁrst observation and the next. Authors like Salerno et al. (2017) have also used absolute values, but for our study, this would not give a fair estimation for the analysis as it generates a bias based on 4.2.4 Mean annual rock surface temperature (2012) (rock model 2) for the entire European Alps using a set of 53 MARST measurement points. The MAAT of the 1961–1990 period was used to calculate MARST, represent- ing a steady state. 4.2.4 Mean annual rock surface temperature MARST estimates the average annual temperature of the rock surface governed mainly by the potential incoming so- lar radiation (PISR) and the mean annual air temperature (MAAT). The method for estimating MARST is described by Boeckli et al. (2012) and Magnin et al. (2019). The esti- mation is based on a multiple linear regression model with the form DSRθ,α = SConst · (βm(θ)) · SunDurθ,α · SunGapθ,α · cos(AngInθ,α), (4) (4) where SConst is the solar constant with a value of 1367 W m−2; β is the transmissivity of the atmosphere (aver- aged over all wavelengths) for the shortest path (in the direc- tion of the zenith); m(θ) is the relative optical path length, measured as a proportion relative to the zenith path length; SunDurθ,α is the time duration represented by the sky sector; SunGapθ,α is the gap fraction for the sun map sector; and AngInθ,α is the angle of incidence between the centroid of the sky sector and the axis normal to the surface. Y = α + Xk i=1θiXi + ε, (5) (5) where Y is the value for MARST, α is the intercept term, θiXi represents the model’s k variables (PISR and MAAT) and their respective coefﬁcients, and ε is the residual error term distributed equally with the mean equal to 0 and the variance σ 2 > 0. For predicting the values of MARST in steep slopes, we use the equation The ﬁnal map of DSR is the sum of values calculated at an hourly time step for every pixel, as per the resolution of the DEM used. The values of solar radiation are given in watts per square metre (W m−2). Higher values for solar radiation indicate higher insolation, while lower values suggest low in- solation. We prefer DSR over the aspect for our analysis to avoid bias due to local shading on sun-exposed faces, con- sidering the slope angle associated with the aspect. MARST(pred) = α + PISR · b + MAAT · c, (6) (6) MARST(pred) = α + PISR · b + MAAT · c, where α is the MARSTpred value when PISR and MAAT are equal to 0, and b and c are the respective coefﬁcients of PISR and MAAT at measured rock surface temperature (RST) positions. These coefﬁcients were calibrated by Boeckli et al. 4.2.5 Topographic ruggedness index The topographic ruggedness index (TRI) measures the ruggedness of the landscape. TRI was calculated based on the methodology proposed by Sappington et al. (2007). It is calculated as a three-dimensional dispersion of vectors (x, y, z components) normal to the grid cells considering the slope and aspect of the cell. The magnitude of the resultant vector in a standardized form (vector strength divided by the num- ber of cells in the neighbourhood) measures the ruggedness of the landscape. Higher values of TRI thus suggest a more rugged and sporadic terrain, which could block the down- ward movement of the snow and subsequently lead to the for- mation of a weak layer, destabilizing the snowpack and lead- ing to small avalanches resulting in mass wasting (Schweizer, 2003). Since IA surfaces are smooth, the TRI values calcu- lated at the surface of the IA are always low. Hence, we con- sider the TRI values by taking a buffer of 20 m around the 4.2.2 Elevation Elevation strongly inﬂuences the meteorological conditions within the same region, signiﬁcantly altering the precipita- tion, temperature and wind regime even at a local scale. Gen- erally, higher elevations receive more precipitation and expe- rience lower temperatures and higher wind speeds. Hence, regions at higher elevations, especially above the ELA, should favour more accumulation than ablation. However, wind-driven snow at higher elevations does not readily accu- mulate on steep slopes. Some IAs may take advantage of the leeward conditions at lower elevations and sustain for more extended periods. Similar results for large glaciers have pre- viously been reported by Bhambri et al. (2011) or Pandey and Venkataraman (2013). The values for MARST are calculated in ◦C and, for our study region, range from −12 to 10 ◦C. MARST is also an important criterion to check for the very likely presence of permafrost below the IAs, which likely al- lows the formation and existence of IAs. S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IA the day and month for a year): the day and month for a year): slopes, accumulation in the temperature range of −5–0 ◦C can accumulate on steep slopes. Slope likewise plays a key role when calculating other terrain parameters and indices. DSRtot = X DSRθ,α. (3) DSRtot = X DSRθ,α. (3) The direct solar radiation (Dirθ,α) with a centroid at zenith angle (θ) and azimuth angle (α) is calculated using the fol- lowing equation: https://doi.org/10.5194/tc-16-4251-2022 https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 4258 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs for each observation period (i.e. 1952–2001, 2001–2012 and 2012–2019). This value of CPDD is then used as a proxy for ablation (Braithwaite and Olesen, 1989; Vincent and Vallon, 1997). for each observation period (i.e. 1952–2001, 2001–2012 and 2012–2019). This value of CPDD is then used as a proxy for ablation (Braithwaite and Olesen, 1989; Vincent and Vallon, 1997). IA boundary delineated for the ﬁrst observation (1952). The mean TRI value from this buffer is considered for our analy- sis. 4.2.6 Curvature The calculation of the proxy for accumulation is more tricky because we only consider the yearly sum of precipita- tion occurring at a temperature between −5 and 0 ◦C, as only snowfall within this temperature range is believed to accu- mulate/adhere to steep slopes (Kuroiwa et al., 1967; Guillet and Ravanel, 2020; Eidevåg et al., 2022). The temperature- dependent indicator function can be written in the following form: Curvature, estimated as a second derivative of the surface, deﬁnes the shape of the slope. Curvature is considered an essential factor because it can deﬁne snow accumulation or ablation rates for a surface. Generally, two types of curva- ture proﬁles are known, plan and proﬁle. For our analysis, we only used the proﬁle curvature as it deﬁnes the shape of the slope in the steepest direction. From a theoretical point of view, erosion processes prevail in convex (negative val- ues) proﬁle curvature locations, while deposition is predom- inant in concave (positive values) proﬁle curvature locations. The curvature values deﬁne how strongly the slope is convex (lower negative values) or concave (higher positive values). That is why curvatures can be considered essential in the ac- cumulation and ablation rates of a glacier or ice body. Like TRI, the IAs tend to show ﬂat curvature proﬁles if we con- sider their surface. Hence, we estimate the curvature values around the same buffer as the TRI and use this for further analysis. χi(T,(t)) = 1 if −5 ◦C ≤T (t) ≤0 ◦C 0 otherwise . (8) (8) 4.4 Surface area model Using the proxy for ablation and accumulation, Guillet and Ravanel (2020) proposed a surface area model to estimate the differences in the surface areas of IAs between different time steps due to the time-integrated changes in meteorolog- ical parameters. The main goal is to look for a potential lin- ear relationship between climate variables and the changes in surface areas of IAs, using a multivariate regression model. The equation for the model can be written as 4.3 Proxies for ablation and accumulation Sm(t) = S(t0) − tZ to (α1CPDD(t) −χi (T (t))α2A(t))dt + β + ε(t), (9) Sm(t) = S(t0) − tZ to (α1CPDD(t) To eventually correlate changes in surface area of IAs with the changing climate, we use the temperature and precipi- tation data from the transformed GSB weather records and SAFRAN reanalysis product (see Sect. 3.3) to build proxies for accumulation and ablation. The proxy for ablation was built by estimating the annual sum of positive degree days (PDD) computed from the normal probability distribution centred around the mean monthly temperature. Estimation of the PDD is based on the empirical relation, which states that the melting rate is proportional to the surface air temperature excess above 0 ◦C. Several methods for estimating PDD have been proposed by Braithwaite and Olesen (1989), Braith- waite (1995), and Hock (2003). However, the method pro- posed by Calov and Greve (2005) also accounts for stochas- tic variations in temperature during the computation of PDD. The formula for the estimation of the PDD using this method is given by −χi (T (t))α2A(t))dt + β + ε(t), (9) (9) −χi (T (t))α2A(t))dt + β + ε(t), where Sm(t) corresponds to the modelled surface area at time t; similarly, CPDD(t) and A(t) represent the proxies for ab- lation and accumulation; S(t = 0) is the ﬁrst available mea- surement; α1 and α2 are the coefﬁcients of linear regression, β is the intercept, and ϵ the residual. χ(T,t) accounts for precipitation occurring in the [−5, 0 ◦C] temperature range and is given by the temperature-dependent indicator function given in Eq. (8). The area of IAs at each time step was calcu- lated using the surface area model (with the temperature and precipitation proxies), and we hereafter refer to this area as the modelled area. The measured area is the surface area we delineated using high-resolution optical images. 4.2.3 Mean slope Slope angle strongly inﬂuences ice velocities of glaciers, the mass ﬂux and the hydrology of the mountain environments. Its inﬂuence on avalanche transport of snow over the glacier surface has been discussed previously (e.g. Hoelzle et al., 2003; DeBeer and Sharp, 2009). Numerous studies have also reported that slope is the single most crucial terrain parame- ter that controls glacier responses to climate change (Furbish and Andrews, 1984; Oerlemans et al., 1998; Jiskoot et al., 2009; Scherler et al., 2011). In mountainous regions, the ter- rain slope strongly inﬂuences snow accumulation. On steep https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 4259 5.1 Accuracy of the DEM Figure 11 shows the trend of PDD increase over the years in the MBM. All elevations, except 4800 m a.s.l., show an increasing trend of PDD values from 1952 to 2019. Specif- ically, for the year 1952, since we have only 1 year for the longer-term analysis, it is interesting to look in detail at the climatic conditions prevailing in the region around this period. Looking at the GSB records, the average tempera- ture in the region during the past 10 years before 1952 was −0.987 ◦C, with average summer temperatures (July, Au- gust and September) being 6.783 ◦C. Year 1947 was par- ticularly hot, with the annual average temperature recorded at −0.275 ◦C and average summer temperatures at 8.266 ◦C. The next 30 years after 1952 were more favourable, with av- erage annual temperatures at −1.523 ◦C and average summer temperatures at 5.256 ◦C (GSB data provided in the Supple- ment as “supplementary material 1”). Since 1952 was com- ing at the back of considerably warmer years, a signiﬁcant reduction in the surface area of IAs can be expected during this period. Looking at the weather records, the conditions after 1952 for the next 30 years were more favourable. Figure 7a shows the stable surfaces (after eliminating glacier boundaries, trees and forests) we used for our co-registration process, and Fig. 7b displays the difference in elevation between the reference DEM and the source DEM before co-registration. Figure 7c presents the results after the co- registration process considering all the surfaces (stable and non-stable), and Fig. 7d shows the difference considering only the stable areas after masking out non-stable areas using the glacier boundaries provided by the Randolph Glacier In- ventory (RGI v6.0) (Consortium, 2017). The source DEM was translated using the corresponding shift values x = −5.03 m, y = 6.00 m and z = 3.22 m. The distribution of errors can be visualized by a histogram of the sampled errors, where the number of errors (fre- quency) within certain predeﬁned intervals is plotted (Höhle and Höhle, 2009). Figure 8 shows the histogram of the errors 1h (elevation difference between the reference and source DEM) in metres for the stable areas. The accuracy estimates before and after the co-registration are shown by the nor- malized median absolute deviation (nmad) and the median value calculated together. S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4260 from various sources, (3) errors produced while generating the high-resolution DEM from stereo images, and (4) con- ceptual errors linked with deﬁning the boundaries of IAs in all images. Quantifying the errors inherent in processing all datasets used is challenging, and this is out of the scope of this paper. A detailed accuracy assessment of the DEM gen- eration and co-registration process is provided in Sects. 5.1 and 3.1, respectively. Quantifying errors resulting from the manual delineation of IA boundary is also challenging, but we have previous guidelines from Paul et al. (2017) for the quality and consistency assessment of manual delineations. were 5.16 and −5.06, respectively. After the co-registration process, the value dropped to 1.98 for the nmad and −0.14 for the median value. This suggests a good correlation be- tween the high-resolution lidar DEM used as a reference and the Pleiades DEM we built. 5.2 Total area loss of ice aprons in the Mont Blanc massif over seven decades The total area of IAs mapped in 1952 was 7.932 km2. It dropped to 5.915 km2 in 2001. The surface area dropped to 4.919 km2 in 2012 and then to 4.21 km2 in 2019 (Fig. 9). This implies that from 1952 to 2019, IAs lost ∼47 % of their original area. It corresponds to an average surface area loss of 0.78 km2 per decade. However, the percentage area loss from 1952 to 2001 was ∼25 % compared to ∼29 % rela- tive area loss from 2001 to 2019. This is an alarming rate: IAs have lost more relative area during the 18 recent years (with an average area loss of 1.15 % per year) compared to the 50 years before 2001 (0.5 % per year average area loss). One way to assess the area uncertainty is to perform mul- tiple digitizations of the same surface and calculate the mean area deviation (MAD), taking the ﬁrst digitization as a ref- erence (Meier et al., 2018). Considering this, the ﬁrst au- thor performed three digitizations for 50 IAs on images from 1952, 2001, 2012 and 2019, considering different challenges associated with aerial and satellite images like shadow and illumination. MAD provides a percentage estimate of how the ﬁnal area calculated varies across multiple digitizations for each polygon. MAD values are affected by the size of the polygon manually digitized. Previously, authors like Paul et al. (2013), Fischer et al. (2014) and Pfeffer et al. (2014) have reported an increase in the uncertainty of manual digitiza- tions with a decrease in the size of the polygons. With this in mind, we also digitized IAs of different sizes ranging from 0.001 to 0.01 km2. Figure 10 shows the MAD values for 50 IAs in 1952, 2001, 2012 and 2019. We did not observe an increase in MAD values with decreasing size of the IAs, mainly because the number of samples we used is comparatively less than that in the previous studies. Overall, the mean MAD observed for all years was ±6.4 %. The MAD for the IAs digitized on the orthophotos from 1952 was ±6.68 %, while for 2001, it was ±7.2 %. The MAD for 2012 and 2019 was ±6.32 % and ±5.50 %, respectively. 4.5 Uncertainty estimations PDD = A Z 0 dt T 2 ac √2π exp −Tac 2σ 2 + Tac 2 erfc −Tac √2σ . (7) Since this study uses data from different sources and peri- ods, uncertainties of different origins might have been intro- duced to delineate the IA boundaries. A good estimation of these uncertainties is thus crucial to have a fair estimation of the signiﬁcance of the results (Racoviteanu et al., 2008; Shukla and Qadir, 2016; Garg et al., 2017). Some sources of uncertainty in this study could arise from (1) errors in- herent to the aerial images and satellite-derived datasets, (2) errors resulting from inaccurate co-registration of data Tac is the annual temperature cycle (monthly mean tempera- tures estimated in ◦C for the entire year); σ is the standard de- viation of the temperature from the annual cycle, A = 1 year; and erfc is the conventional error function built into all pro- gramming languages. Tac is the annual temperature cycle (monthly mean tempera- tures estimated in ◦C for the entire year); σ is the standard de- viation of the temperature from the annual cycle, A = 1 year; and erfc is the conventional error function built into all pro- gramming languages. After computing the PDD, we calculate the cumulative PDD (CPDD) by taking the sum of all the annual PDD values https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 5.1 Accuracy of the DEM As can be seen, the nmad and me- dian values before the co-registration process for stable areas Similarly, Fig. 12 shows the variations in the accumulation rates (average annual accumulation per period) for all eleva- tion bands. The results show only that part of the snowfall https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 4261 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area Figure 7. Stepwise Pleiades DEM accuracy assessment: (a) the surfaces used for coregistration, (b) elevation difference before coregistration, (c) elevation difference after coregistration considering all areas and (d) elevation difference after coregistration considering only the stable areas. Figure 7. Stepwise Pleiades DEM accuracy assessment: (a) the surfaces used for coregistration, (b) elevation difference before coregistration, (c) elevation difference after coregistration considering all areas and (d) elevation difference after coregistration considering only the stable areas. Figure 8. DEM error (elevation difference between the reference and source DEM) distribution for stable areas. which is expected to accumulate on the steep slopes. Except for the highest elevation band, i.e. 4800 m a.s.l., accumula- tion rates at all elevation bands show a general decreasing trend. For example, at the 3900 m a.s.l. elevation band, accu- mulation rates fell from 32 mm yr−1 from 1952 to 2001 to 28 mm yr−1 from 2001 to 2012 and further to 18 mm yr−1 between 2012 and 2019. This shows that temperatures in the MBM are increasing, while, on the other hand, accumulation on steep slopes is decreasing over time. Figure 13 shows the annual variation of the accumulation on steep slopes at dif- ferent elevations. The ﬁrst observation from this trend shows that very little precipitation accumulates on steep slopes in the winter months, while accumulation occurs almost en- tirely in the summer months. Further, the accumulation is more signiﬁcant at higher elevations (4200–4500 m a.s.l.) in the summer months than at lower elevations. At lower el- evations, accumulation is predominantly observed in pre- summer (May) and post-summer (October) months. Figure 14a presents the correlation between the ratio of the mean measured surface area at time t, S(t), to the initial area, S(t0), with the ratio of the mean modelled surface area using the GSB transformed data to the initial area for 2001 and 1952. 5.1 Accuracy of the DEM We consider the ratio of S(t)/S(t0) as an indicator Figure 8. DEM error (elevation difference between the reference and source DEM) distribution for stable areas. https://doi.org/10.5194/tc-16-4251-2022 https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4262 5.4 Inﬂuence of the local topography and other factors on the area loss of IAs Each parameter, as described in Sect. 4.2, was individually regressed with the relative area loss of IAs for the three pe- riods, and their inﬂuence was assessed by the coefﬁcient of determination (R2) and Pearson’s r value. A joint analysis of the surface area lost by the IAs and the direct solar radiation reveals a strong correlation between the values of DSR and the relative surface area loss of IAs for all the three time periods (1952–2001, 2001–2012 and 2012–2019) (Fig. 15a; Table 3). However, this is the ﬁrst ev- idence of the potential negative impact of solar radiation on small ice bodies like IAs. Previous analysis of Guillet and Ravanel, 2020, with the climate variables indicated a poten- tial relationship between the elevation and the surface area loss of IAs. This is somewhat statistically signiﬁcant from the regression analysis, as we found a negative correlation between the surface area loss and the mean elevation of the IAs (Fig. 15b; Table 3). A further comparison of the IAs (200 IAs) distribution with elevation and aspect shows that most IAs (∼77 % of the total number) exist at elevations above 3200 m a.s.l. Further, most IAs (∼56.5 %) exist in the north- ern aspects (N, NW, NE), while the E and W aspects are the least favoured (Supplement; supplementary material 2). In addition, we found a moderate positive correlation between the average MARST values and the surface area loss of IAs. The correlation observed was not very signiﬁcant compared to the previous two factors. It indicates that the effect of rock surface temperatures on the area loss of IAs is not strong on a regional scale (Fig. 15c; Table 3). However, this relation- ship needs to be examined in a more site-speciﬁc and local- ized area to understand better its impact on the surface area loss of IAs. We also observed that the correlation was higher for a more extensive observation period (1952–2001) than for shorter periods. This could suggest that the inﬂuence of rock surface temperatures potentially becomes more promi- nent with a more extensive observation period. Figure 14. Correlation between the mean normalized measured and modelled surface areas (a) with GSB data transformed to AdM data and (b) with SAFRAN reanalysis data at time t. The colour and size of the ticks represent the mean elevation of the IA. to estimate the area loss between the two time periods. S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs Figure 9. A comparison of the total surface area of all IAs (423 IAs) in the MBM over 67 years. Figure 9. A comparison of the total surface area of all IAs (423 IAs) in the MBM over 67 years. Figure 9. A comparison of the total surface area of all IAs (423 IAs) in the MBM over 67 years. Figure 9. A comparison of the total surface area of all IAs (423 IAs) in the MBM over 67 years. Figure 9. A comparison of the total surface area of all IAs (423 IAs) in the MBM over 67 years. Figure 10. The distribution of MAD values based on multiple digi- tizations of the IA area for all periods. Figure 10. The distribution of MAD values based on multiple digi- tizations of the IA area for all periods. Figure 12. Variation of the average accumulation rates on steep slopes at different elevations for each period of observation. Figure 12. Variation of the average accumulation rates on steep slopes at different elevations for each period of observation. Figure 10. The distribution of MAD values based on multiple digi- tizations of the IA area for all periods. Figure 13. Accumulation (steep slopes) trends for the year 2019 at different elevations. Figure 11. The variation of annual PDD values estimated based on monthly mean temperatures at different elevations in the MBM from 1952 to 2019. Figure 11. The variation of annual PDD values estimated based on monthly mean temperatures at different elevations in the MBM from 1952 to 2019. Figure 13. Accumulation (steep slopes) trends for the year 2019 at different elevations. The Cryosphere, 16, 4251–4271, 2022 https://doi.org/10.5194/tc-16-4251-2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4263 hik et al.: Effects of topographic and meteorological parameters on the surface area loss of IA S. Kaushik et al.: Effects of topographic and meteorologic Figure 14. Correlation between the mean normalized measured and modelled surface areas (a) with GSB data transformed to AdM data and (b) with SAFRAN reanalysis data at time t. The colour and size of the ticks represent the mean elevation of the IA. on the NE face of Col de la Brenva (∼4160 m a.s.l.), and one IA on the S face of Col du Bionnassay (∼4050 m a.s.l.). S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs As observed, all these IAs are located at elevations higher than 4000 m a.s.l. As an exception, it can be expected that a few IAs could show an increase in the surface area. However, this increase is not dramatic (∼10 % increase). The results, how- ever, reafﬁrm the proﬁciency of the proposed surface area model in predicting new IA states from the accumulation and ablation proxies. Similar results were observed for the other two time periods, i.e. 2001–2012 and 2012–2019, as seen in Table 2. 5.4 Inﬂuence of the local topography and other factors on the area loss of IAs A high ratio value (i.e. value close to 1) in the present context indicates that the relative surface area loss of IAs between the two periods is comparatively less than that of IAs whose ratio is closer to 0. A value larger than 1 indicates an increase in the surface area over time. From the results, we do not see a strong correlation (r = 0.73) between the modelled area (from GSB transformed cli- mate data) and the measured area for the 200 IAs spread across the MBM (Fig. 14a). However, the correlation im- proves signiﬁcantly (r = 0.86) when we use the SAFRAN data based on different elevations and remodel the surface area for each IA (Fig. 14b). This can be seen from the values of R2, Pearson’s r, RMSE and the p value estimates from the T test achieved from both datasets (Table 2). The best-ﬁtting line presents a slope of 1.0 and an intercept of 0.0. Both ﬁgures show that IAs at lower elevations (blue to green colour and small tick size) generally show lower ra- tio values than IAs at higher elevations (yellowish colours and bigger tick size). This implies that the elevation of the IAs potentially plays a crucial role in their response to the changing climate. Overall, the surface area of IAs decreased throughout the massif from 1952 to 2001 except for four IAs, which showed an increase in surface area. These four IAs are two IAs on the N and NW face of Rochers Rouges Inferieurs (∼4350 and 4050 m a.s.l.) near the Grand Plateau, one IA A similar analysis of IA area loss with the TRI showed a weak positive correlation (Fig. 15d; Table 3). An increase in TRI values (i.e. increase in terrain ruggedness) may result in more ice area loss on a site-speciﬁc scale, but this rela- tionship is hard to observe globally. Like the results from the https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4264 Figure 15. Scatter plots showing relationships between topographic factors and the area loss of IAs from 1952 to 2019. (a) Direct solar radiation, (b) elevation, (c) MARST, (d) TRI, (e) slope, (f) curvature and (g) size of the IAs. Figure 15. 6.1 Area loss of ice aprons and the role of the changing climate As observed from the results in Sect. 5.2, IAs have been los- ing surface area at an alarming rate. This rate of surface area loss is disconcerting because, compared to the glaciers in the MBM, the IAs are losing their area at a higher rate (∼24 % for glaciers from the end of LIA until 2008, according to Gar- dent et al., 2014, while IAs have lost ∼47 % of surface area in the last 70 years). The small size of IAs makes them more vulnerable to global warming than large glaciers. An increase in average annual temperatures and a decrease in precipita- tion rates put the existing IAs at risk of losing their mass en- tirely before the end of this century. In addition, considering that the effects of local topography are also more pronounced in the case of IAs than for large glaciers, continuous moni- toring of these critical ice bodies has become imperative. Re- sults discussed in Sect. 5.3 indicated the strong inﬂuence of temperature and precipitation on the surface area changes of IAs. The results raise further questions regarding the sensitiv- ity of the IAs to extreme weather events, like the heatwaves experienced in the study region. Unfortunately, our sampling rate does not allow us to quantify the effects of individual extreme weather events. Nevertheless, there is a strong argu- ment in favour that these events, especially in the past two decades, cause the IAs to lose mass more rapidly than in the previous decades. Further, the heatwaves occurring dur- ing winter and midsummer, when the IA surfaces are free of snow, will have the worst adverse effect. analysis with MARST, the strongest correlation was again observed for the largest study period. Further, like the TRI, we also found a weak correlation between the terrain slope and curvature with the surface area loss of IAs. We must note that our criteria for selecting IAs already limit us to areas with slope angles steeper than 40◦(Fig. 15e; Table 3). Hence it was difﬁcult to observe any signiﬁcant impact of terrain slope on the rate of area loss of IAs. Similarly, terrain cur- vature seems to have the most negligible impact (Fig. 15f; Table 3). As cited in Sect. 4.2. Table 3. Linear regression parameters and correlation metrics for each studied parameter. Variable Time period R2 Pearson’s r Direct solar radiation 1952–2001 0.64 0.79 2001–2012 0.67 0.81 2012–2019 0.51 0.72 Elevation 1952–2001 0.61 −0.78 2001–2012 0.57 −0.75 2012–2019 0.51 −0.71 MARST 1952–2001 0.40 0.63 2001–2012 0.34 0.58 2012–2019 0.27 0.52 TRI 1952–2001 0.37 0.60 2001–2012 0.30 0.55 2012–2019 0.32 0.57 Slope 1952–2001 0.29 0.54 2001–2012 0.25 0.50 2012–2019 0.21 0.46 Curvature 1952–2001 0.06 −0.26 2001–2012 0.03 −0.18 2012–2019 0.06 −0.24 Size of IA 1952–2001 0.04 −0.22 2001–2012 0.06 −0.26 2012–2019 0.04 −0.22 5.4 Inﬂuence of the local topography and other factors on the area loss of IAs Scatter plots showing relationships between topographic factors and the area loss of IAs from 1952 to 2019. (a) Direct solar radiation, (b) elevation, (c) MARST, (d) TRI, (e) slope, (f) curvature and (g) size of the IAs. The Cryosphere, 16, 4251–4271, 2022 https://doi.org/10.5194/tc-16-4251-2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4265 2. Linear regression parameters and correlation metrics for each period (a) using GSB transformed data and ysis product. (a) Time period Slope Intercept R2 Pearson’s r RMSE (km2) p value 1952–2001 0.79 0.12 0.53 0.73 0.010 < 0.001 2001–2012 0.70 0.26 0.56 0.75 0.102 < 0.001 2012–2019 0.89 0.04 0.63 0.80 0.097 < 0.001 (b) Time period Slope Intercept R2 Pearson’s r RMSE (km2) p value 1952–2001 1.01 −0.04 0.73 0.86 0.075 < 0.001 2001–2012 0.74 0.22 0.67 0.82 0.086 < 0.001 2012–2019 1.37 −0.39 0.83 0.91 0.071 < 0.001 Table 3. Linear regression parameters and correlation metrics for each studied parameter. Table 3. Linear regression parameters and correlation metrics for each studied parameter. the erosion and accumulation dynamics on steep slopes, but this is not the case for IAs in the MBM. We performed the last comparison between the relative surface area loss of IAs with their initial area. Our results were similar to the one Lopez et al. (2010) reported, as we did not ﬁnd any correla- tion between the two quantities (Fig. 15g; Table 3). 6.2 Area loss of ice aprons and the role of topographic parameters Since ice/glacier bodies within the same climate regime can also respond to climate change differently, the last part of the analysis (Sect. 5.4) was dedicated to understanding the linkages of local topographic factors to the surface area loss of IAs. As reported previously by Salerno et al. (2017), some local topographic factors inﬂuence the response of IAs to cli- mate change more signiﬁcantly than others. A ﬁrst analysis showed that IAs that receive more solar radiation from the sun throughout the year lose their surface area faster than those that receive less DSR. Similar results for other regions in mountain environments have also been reported previously by Oerlemans and Klok (2002), Mölg (2004), and Johnson and Rupper (2020). Incoming solar radiation is an essential component of all surface energy and mass balance models. But the signiﬁcance of DSR on the surface area loss of small ice bodies like IAs is reported for the ﬁrst time in our study. Kaushik et al. (2021) further showed that most IAs exist in extremely rugged terrains: 51 % of the total IAs mapped exist in the TRI’s high and very high ruggedness class, while only 8 % exist in the low ruggedness. Thus, comparing the terrain ruggedness to the area loss of IAs makes sense since the topography around the snow/ice bodies can critically in- ﬂuence their stability (Deline et al., 2015). Increasing terrain ruggedness is associated with slope instability and further ice volume loss. However, for our study, this relation was not very pronounced, showing that the topography’s ruggedness does not substantially affect the area loss of IAs. Previous analysis by Kaushik et al. (2021) also showed that most IAs in the MBM (83 %) lie at mean slopes between 40 and 65◦. Increasing slope steepness limits accumulation, while avalanches further scour away snow from the surface of the IA, thus exposing the ice directly to the sun and wind (Vionnet et al., 2012). However, the differences in slope an- gles of the IAs were not a dominant factor affecting the rates of area loss. A plausible explanation for this could be that since we limit the slope criteria to > 40◦and most IAs lie in the range of 40 to 65◦slope, the effect of terrain slope is not as well pronounced as it would be between low (< 15◦) and extreme slopes (> 65◦). Similar results were observed by Li et al. S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs 4266 coming decades, understanding the effects of climate vari- ables on the sensitivity of IAs is even more critical. face of Aiguille des Grands Charmoz (3445 m a.s.l.), which completely disappeared during the 2017 summer heatwave (Guillet and Ravanel, 2020). Further, several authors have previously also accounted for the variations in solar radiation in mass balance modelling studies (Huss et al., 2009; Thibert et al., 2018). Our results showed a strong correlation of DSR with area change, mak- ing this argument stronger. However, since the focus was to show the impact of climate variables separately, we preferred a temperature-index model as the ﬁrst approach. However, we expect solar radiation to play a signiﬁcant role in the sen- sitivity of ice aprons, and future studies on ice apron evolu- tions in the 21st century should address this question. In addition, on a local scale, some correlation between the rock surface temperatures and the area loss of IAs was observed from the analysis. Guillet et al. (2021) suggested that IAs are cold ice bodies that exist predominantly on permafrost-affected rock walls. They further reported tem- peratures < 0 ◦C at the base of the ice core taken from the IA on the north face of Triangle du Tacul (3970 m a.s.l.). Heat- ing from rock surfaces is predominantly the cause of per- mafrost degradation, which further affects mountain slope stability, leading to an increased rock mass wasting (Magnin et al., 2017). Cold surfaces demonstrate more ice cohesion with the underlying surfaces, while a rise in surface tempera- tures decreases basal cohesion, increasing the sliding process and leading to more ice ﬂow (Deline et al., 2015). Thus, it is likely that underlying permafrost conditions aid the sustain- ability of IAs in the long term, and an increase in rock sur- face temperatures around IAs could result in IAs losing mass more rapidly. 6.1 Area loss of ice aprons and the role of the changing climate previous studies may have shown that terrain curvatures could play an essential role in As suggested by Meehl and Tebaldi (2004), with an in- crease in the intensity and frequency of extreme events in the https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs inant factor in the dynamics of glacier/ice bodies, but this relation was not established for our study. – Over the study period 1952–2019, IAs have lost their surface area at a very alarming rate. The total area of IAs in MBM was 7.93 km2 in 1952, dropping to 5.91 km2 in 2001, 4.91 km2 in 2012, and 4.21 km2 in 2019 (∼47 % drop in total surface area in less than three-quarters of a century). At last, a comparison of the rate of surface area loss of IAs with the original size of the IAs was performed, and we observed no correlation between the two factors. Although previous studies by Paul et al. (2004), Jiskoot et al. (2009) and Garg et al. (2017) have shown the correlation between the size of the ice/glacier bodies with the area loss, this is not evident in our case. Unlike previous studies, which consid- ered different glaciers ranging in size from less than a square kilometre (km2) to several hundred square kilometres, IAs are small ice bodies (0.0005 to 0.1 km2). Hence, it is plau- sible that the effect of IA size on area loss rate is not pro- nounced in our case. Similar results were shown by Lopez et al. (2010), who analysed 72 glaciers in South America and reported no correlation between the glacier length and the area loss of glaciers. – The observed rate of relative area loss in the last 18 years (∼29 %) is more than the overall area loss dur- ing the 48 previous years (1952–2001; ∼26 %). – Results from the analysis of IA surface area loss and meteorological parameters (i.e. temperature and precip- itation) conclusively proved the strong impact of these parameters on the behaviour of small ice bodies like IAs. – Further analysis of IA surface area loss with different topographic parameters showed a strong correlation of IA surface area loss with the DSR and elevation. Other factors like MARST, TRI and mean terrain slope could also play an important role locally, but their effect is not signiﬁcant regionally. Terrain curvature and the size of the IAs were not found to impact the IA surface area loss signiﬁcantly. Another critical factor to consider, along with the impact of topography, is the role of avalanches in the erosion and deposition processes on the IA surface. S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs Analysis of the ice core from the N face of Triangle du Tacul showed that IAs are almost immobile cold ice bodies (Guillet et al., 2021). Hence IAs do not directly participate in feeding the larger glacier systems below them. However, the avalanches trig- gered above can bring fresh snow/debris and lead to erosion or deposition on the IA surface. We expect this factor to also play a role in the area change dynamics of the IA, which we have not considered as part of this study. Although this impact on the scale of an IA is tough to determine, future studies should focus on ascertaining this impact at least on a local site-speciﬁc scale. Looking at the melting rate of IAs and the future predictions of global climate change, it is evident that these small and critical ice bodies are most vulnerable to adverse impacts. It is hard to imagine any of the IAs surviving the next few decades with increasing temperatures at the present and fu- ture melting rates. The loss of IAs will thus be the loss of crucial glacial heritages and playgrounds for the iconic prac- tice of mountaineering. Hopefully, this study forms a basis to encourage further studies on IAs. 6.2 Area loss of ice aprons and the role of topographic parameters (2011), as they observed very slight variations in area loss for small glaciers with differences in slope. They suggested other local topographic factors could mitigate the effects of slope in the event of small ice/snow bodies. Further, the correlation between elevation and surface area loss of IAs was the second most signiﬁcant of all topo- graphic factors. IAs located at lower elevations are poten- tially subject to more intense degradation and lose their sur- face area faster than those at higher elevations. On a more local scale, other topographic factors could also play a criti- cal role in the surface area variations of IAs. However, eleva- tion seems to be a dominant causative factor on a regional scale. Elevation strongly inﬂuences meteorological condi- tions (temperature, precipitation and wind speeds) and per- mafrost; this likely strongly inﬂuences the durability of IAs in the context of changing climate. Hantel et al. (2012) sug- gested the median summer snowline for the Alps to be at 3083 ± 121 m a.s.l. (1961–2010), while Rabatel et al. (2013) documented the regional ELA at 3035 ± 120 m a.s.l. (1984– 2010). Rabatel et al. (2013) further described the rising of the ELA to 3250±135 m a.s.l. during the 2003 heatwave. Subse- quent heatwaves of 2006, 2015 and 2019 would have likely resulted in similar scenarios (Hoy et al., 2017). Since ∼77 % of the total IAs reported in this study exist at elevations above 3200 m a.s.l., the rising of the ELA in future climate scenar- ios risks more IAs towards faster degradation and disappear- ance. An example of this is the case of the IA on the north Similarly, terrain curvature also has a negligible effect on the surface area loss of IAs. As suggested by Alkhasawneh et al. (2013) and Yanuarsyah and Khairiah (2017), convex proﬁle curvature favours the erosion processes, while in lo- cations with concave curvature, the deposition process can be predominant. Over time, the terrain curvature can be a dom- https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 4267 References Alkhasawneh, M. S., Ngah, U. K., Tay, L. T., Mat Isa, N. A., and Al-batah, M. S.: Determination of Important Topographic Factors for Landslide Mapping Analysis Using MLP Network, Sci. World J., 2013, 1–12, https://doi.org/10.1155/2013/415023, 2013. 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Because of the difﬁculties associated with their monitoring and relative unimportance to mountain hydrology, no stud- ies solely based on their evolution on a regional scale have been performed before. This paper presented an analysis of 200 IAs spread throughout the MBM and existing in differ- ent topographic settings to understand their dynamics in the context of climate warming. For this purpose, we accurately mapped the IAs on very high-resolution aerial and satellite images available for 1952, 2001, 2012 and 2019. Using our extensive database of IAs, we compared the total area vari- ation of IAs for three periods. Further, we also attempted to establish a relationship between the surface area lost by IAs with meteorological parameters (i.e. temperature and precip- itation) and their associated topographic parameters. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/tc-16-4251-2022-supplement. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/tc-16-4251-2022-supplement. Author contributions. SK designed the study and drafted the paper, which all co-authors revised. LR and FM helped in data interpre- tation and analysis. YY and ET proofread the manuscript and pro- vided valuable inputs for improving the overall quality of the paper. DC processed and provided the DEM used for the study. Competing interests. The contact author has declared that none of the authors has any competing interests. Some important outcomes from the study are the follow- ing. Acknowledgements. This research is part of the USMB Couv2Glas and GPClim projects. Pleiades data were acquired within the https://doi.org/10.5194/tc-16-4251-2022 The Cryosphere, 16, 4251–4271, 2022 S. Kaushik et al.: Effects of topographic and meteorological parameters on the surface area loss of IAs S. 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https://openalex.org/W3046654351	https://digital.csic.es/bitstream/10261/232813/1/the-identity-of-calicium-corynellum-ach-ach.pdf	English	null	The identity of <i>Calicium corynellum</i> (Ach.) Ach.	Lichenologist/The Lichenologist	2,020	cc-by	2,233	The identity of Calicium corynellum (Ach.) Ach. Maria Prieto1,3 , Ibai Olariaga1 , Sergio Pérez-Ortega2 and Mats Wedin3 1Department of Biology and Geology, Physics and Inorganic Chemistry, Rey Juan Carlos University, C/ Tulipán s/n, 28933, Móstoles, Madrid, Spain; 2Real Jardín Botánico (CSIC), C/ Claudio Moyano 1, 28014 Madrid, Spain and 3Department of Botany, Swedish Museum of Natural History, P.O. Box 50007, 10405, Stockholm, Sweden (Accepted 21 April 2020) (Accepted 21 April 2020) (Accepted 21 April 2020) Fig. 1. Calicium corynellum habitus (M. Prieto C4 (ARAN-Fungi 8454)). Scale = 1 mm. In colour online. Recently, Yahr (2015) studied British populations of Calicium corynellum (Ach.) Ach. to test whether these were distinct from C. viride Pers. As C. corynellum had the highest conservation priority in Britain, and was apparently declining rather dramatic- ally, it was important to clarify its taxonomic status. Yahr (2015) used both genetic (ITS rDNA) and morphological data from two British Calicium aff. corynellum populations and could not find differences with C. viride, suggesting that the British material represented saxicolous populations of the otherwise epiphytic or lignicolous C. viride. Although this study focused on British material, it introduced serious doubts about the identity and rela- tionships of these two species in other parts of the distribution area of C. corynellum. Interestingly, the British material that Yahr (2015) investigated was morphologically very similar to C. viride, but the latter was described as differing rather substantially from C. corynellum in other parts of its distribution area. Thus, C. corynellum differs from C. viride in its short-stalked, greyish white pruinose ascomata (C. viride has long stalks and a brown pruina), distinctly narrower spores compared with C. viride, and the leprose thallus (Fig. 1) which is granular to verrucose in C. viride (Tibell 1999). Fig. 1. Calicium corynellum habitus (M. Prieto C4 (ARAN-Fungi 8454)). Scale = 1 mm. In colour online. We sampled our own recently collected material, several herbarium specimens and a selection of samples from GenBank (Table 1). Calicium salicinum Pers. was included as outgroup based on Prieto & Wedin (2017). DNA was extracted and ampli- fied (nrITS region) following Prieto & Wedin (2017). Sequences were assembled and edited using Sequencher v. 4.10.1 (Genes Codes Corporation, Ann Arbor, MI, USA) and deposited in GenBank (Table 1). Subsequently, sequences were aligned manu- ally using MacClade 4.01 (Maddison & Maddison 2001). No ambiguous regions and introns were found. ttps://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282920000250 Downloaded from https://www.cambridge.org/core. CSIC, on 04 Mar 2021 at 10:41:16, subject to the Cambridge Core terms of use, available at The identity of Calicium corynellum (Ach.) Ach. A phylogenetic analysis was carried out using maximum likelihood-based inference (ML) as implemented in RAxML v. 8.2.10 (Stamatakis 2014) run on the CIPRES Science Gateway v. 3.3 (Miller et al. 2010). The analysis was performed with a GTRGAMMA model for tree inference and GTRCAT model and 1000 replicates for bootstrapping. Calicium viride is a common and widely distributed species in temperate areas of the Northern Hemisphere and southern South America (Tibell 1999; GBIF 2019). Calicium corynellum is, how- ever, not as well understood, with scattered occurrences in Europe and North America (Sarrión et al. 1999; Tibell 1999; Pérez-Ortega 2007; GBIF 2019). Additionally, C. corynellum is currently being evaluated for the Red List of lichens of Spain and Portugal (Atienza et al. 2019), which makes it essential to assess the taxonomic status of the species. As C. corynellum is such a poorly known species and the results obtained by Yahr et al. (2015) may be interpreted to suggest that C. corynellum could be better treated as a synonym of C. viride, the status of C. corynellum should be studied further. Our aim here is to study the delimitation of these two taxa based on a larger sample, covering the whole known distribution range of C. corynellum. For that purpose, we have conducted a phylogenetic analysis based on sequences of the ITS region of the nuclear rDNA. p pp g Thin-layer chromatography was carried out following standard methods (Orange et al. 2001) and using solvent systems B and C. As data given in the literature (i.e. Tibell 1999) refer to the thal- lus and not to the ascomata, we sampled thallus parts. A total of seven sequences were newly generated for this study (Table 1). The data set consisted of 20 taxa and 486 unambigu- ously aligned sites. The ML tree with bootstrap values is shown in Fig. 2 and demonstrates that samples classified as Calicium cor- ynellum and C. viride based on morphology form two distinct © British Lichen Society 2020. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. © British Lichen Society 2020. © British Lichen Society 2020. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Maria Prieto1,3 , Ibai Olariaga1 , Sergio Pérez-Ortega2 and Mats Wedin3 1Department of Biology and Geology, Physics and Inorganic Chemistry, Rey Juan Carlos University, C/ Tulipán s/n, 28933, Móstoles, Madrid, Spain; 2Real Jardín Botánico (CSIC), C/ Claudio Moyano 1, 28014 Madrid, Spain and 3Department of Botany, Swedish Museum of Natural History, P.O. Box 50007, 10405, Stockholm, Sweden Cite this article: Prieto M, Olariaga I, Pérez-Ortega S and Wedin M (2020) The iden- tity of Calicium corynellum (Ach.) Ach. Lichenologist 52, 333–335. https://doi.org/ 10.1017/S0024282920000250 Author for correspondence: Maria Prieto. E-mail: maria.prieto@urjc.es Cite this article: Prieto M, Olariaga I, Pérez-Ortega S and Wedin M (2020) The iden- tity of Calicium corynellum (Ach.) Ach. Lichenologist 52, 333–335. https://doi.org/ 10.1017/S0024282920000250 The Lichenologist (2020), 52, 333–335 doi:10.1017/S0024282920000250 The Lichenologist (2020), 52, 333–335 doi:10.1017/S0024282920000250 The Lichenologist (2020), 52, 333–335 doi:10.1017/S0024282920000250 Author for correspondence: Maria Prieto. E-mail: maria.prieto@urjc.es The identity of Calicium corynellum (Ach.) Ach. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (htt which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 334 Maria Prieto et al. Table 1. Calicium specimens used in this study, with GenBank Accession numbers. Entries for newly obtained sequences and new chemical analyses are in bold. Specimen data are given for newly produced sequences with collection number and location of voucher. Chemistry: R = rhizocarpic acid, U = usnic acid, E = epanorin, C = calycin. Codes enclosed by parentheses indicate trace amounts. Data from specimens not analyzed here come from Yahr (2015). Species Code/Voucher Locality GenBank Acc. no. Chemistry Calicium corynellum 1 S. Pérez-Ortega S103 Madrid, Spain MT068633 not tested C. corynellum 2 Thor 23134 (UPS) Södermanland, Sweden MT068630 R C. corynellum 3 M. Prieto C5 (ARAN-Fungi 8329) Madrid, Spain MT068634 R, U C. corynellum 4 Svensson 940 (UPS) Småland, Sweden MT068635 R C. corynellum 5 M. Prieto C4 (ARAN-Fungi 8454) Madrid, Spain MT068632 R, (C?) C. corynellum 6 M. Prieto CC1 (S) Cabañeros, Spain KX512908 C. corynellum 7 Spribille 11865 (UPS) Montana, USA MT068631 R C. viride 1 (as C. aff. corynellum in Yahr 2015) Yahr 5398 Whitfield, England KF991222 R C. viride 2 Yahr 5404 Whitfield, England KF991224 R C. viride 3 (as C. aff. corynellum in Yahr 2015) Yahr 5400 Whitfield, England KF991227 R, (U), (E) C. viride 4 - England FR799144 - C. viride 5 - England FR799143 - C. viride 6 - England FR799142 - C. viride 7 AFTOL348 - HQ650703 - C. viride 8 M. Wedin 8283 (S) Södermanland, Sweden MT068629 R C. viride 9 - Whitfield, England KF991223 - C. viride 10 Wedin 24/4 2000 - AY143393 - C. viride 11 Tibell 17553 (UPS) Argentina DQ812142 - C. salicinum 1 Tibell 23270 (UPS) India DQ812133 - C. salicinum 2 Tibell 23193 (UPS) India DQ812131 - Fig. 2. Phylogenetic tree (best maximum likelihood (ML) tree) of Calicium corynellum and Calicium viride resulting from RAxML analysis of nrITS sequences. Bootstrap supports (ML-BS) are shown above branches and supported clades (ML-BS ≥70) are marked with thicker black branches. Calicium salicinum is used to root the tree. ://doi.org/10.1017/S0024282920000250 nloaded from https://www.cambridge.org/core. CSIC, on 04 Mar 2021 at 10:41:16, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. Fig. 2. References Atienza V, Fos S, Burgaz AR, López de Silanes ME, Millanes A, Olariaga I, Paz-Bermúdez G, Pérez-Vargas I, Prieto M and Pérez-Ortega S (2019) Contribution to assessment of threatened species in red list of lichen- forming and lichenicolous fungi in Spain and Portugal. In Book of Abstracts of the XXII Symposium of Cryptogamic Botany, 24–26 July 2019, Lisbon, Portugal, p. 146. p y y g The two species also apparently differ chemically, as C. viride contains rhizocarpic acid and epanorin, and C. corynellum con- tains rhizocarpic and usnic acids (Tibell 1999; Yahr 2015). This was not evident in the study by Yahr (2015). Of the samples col- lected by Yahr (C. aff. corynellum) that grew on rock, only three out of seven chemically tested specimens contained usnic acid. From the five herbarium specimens chemically studied by Yahr (probably representing the real C. corynellum), one did not have usnic acid. The C. corynellum specimens sampled here con- tained only rhizocarpic acid, except one sample that also contained usnic acid (Table 1). Similar to Yahr, we did not detect epanorin in the C. viride specimen studied. Our results further support that the amounts of usnic acid in C. corynellum and epanorin in C. viride are at least very variable (and that usnic acid may also occur in saxicolous C. viride) and difficult to use to identify these species, just as Yahr (2015) suggested. GBIF (2019) Calicium corynellum (Ach.) Ach. [WWW resource] URL https:// www.gbif.org/species/2609257 [Accessed 2019-04-11]. Maddison WP and Maddison DR (2001) MacClade: Analysis of Phylogeny and Character Evolution, version 4.01. Sunderland, Massachusetts: Sinauer Associates. Miller MA, Pfeiffer W and Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In Proceedings of the Gateway Computing Environments Workshop (GCE), 14 November 2010, New Orleans, Louisiana, pp. 1–8. Orange A, James PW and White FJ (2001) Microchemical Methods for the Identification of Lichens. London: British Lichen Society. Pérez-Ortega S (2007) Contribución al conocimiento de los líquenes y hongos liquenícolas de Castilla y León. Botanica Complutensis 31, 13–22. The studied specimens of Calicium corynellum were not liche- nicolous and produced well-developed thalli (Fig. 1). The Spanish specimens were all found in a very typical but easily overlooked habitat: overhanging siliceous rocks in shaded and humid situa- tions. The identity of Calicium corynellum (Ach.) Ach. Phylogenetic tree (best maximum likelihood (ML) tree) of Calicium corynellum and Calicium viride resulting from RAxML analysis of nrITS sequences. Bootstrap supports (ML-BS) are shown above branches and supported clades (ML-BS ≥70) are marked with thicker black branches. Calicium salicinum is used to root the tree. s://doi org/10 1017/S0024282920000250 nloaded from https://www.cambridge.org/core. CSIC, on 04 Mar 2021 at 10:41:16, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. Fig. 2. Phylogenetic tree (best maximum likelihood (ML) tree) of Calicium corynellum and Calicium viride resulting from RAxML analysis of nrITS sequences. Bootstrap supports (ML-BS) are shown above branches and supported clades (ML-BS ≥70) are marked with thicker black branches. Calicium salicinum is used to root the tree. https://doi.org/10.1017/S0024282920000250 Downloaded from https://www.cambridge.org/core. CSIC, on 04 Mar 2021 at 10:41:16, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. The Lichenologist 335 monophyletic groups, which we interpret here as different species. While there is some genetic variation within Calicium viride, C. corynellum has very little (Fig. 2). Acknowledgements. This project was funded by the Swedish Research Council (VR 2016-03589). Acknowledgements. This project was funded by the Swedish Research Council (VR 2016-03589). Author ORCIDs. Maria Prieto, 0000-0002-1692-9821; Ibai Olariaga, 0000- 0002-0334-7750; Sergio Pérez-Ortega, 0000-0002-0334-7750; Mats Wedin, 0000-0002-8295-5198. y y g Our results suggest that Calicium corynellum is a distinct species which can be distinguished from C. viride by both morphological and genetic characters. Our results also support that the two British populations studied by Yahr (2015) correspond to speci- mens of C. viride growing on rocks. However, this does not exclude the possibility that the real C. corynellum might also exist in Britain. https://doi.org/10.1017/S0024282920000250 Downloaded from https://www.cambridge.org/core. CSIC, on 04 Mar 2021 at 10:41:16, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. References The Spanish distribution is mainly along the Central Mountains but, as we have recently found a number of new local- ities, we believe that the species is probably very overlooked in these areas, as in the rest of the world. Prieto M and Wedin M (2017) Phylogeny, taxonomy and diversification events in the Caliciaceae. Fungal Diversity 82, 221–238. Sarrión F, Aragón G and Burgaz AR (1999) Studies on mazaediate lichens and calicioid fungi of the Iberian Peninsula. Mycotaxon 71, 169–198. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313. Tibell L (1999) Calicioid lichens and fungi. In Ahti T, Jørgensen PM, Kristinsson H, Moberg R, Søchting U and Thor G (eds), Nordic Lichen Flora, Volume 1. Uddevalla: Nordic Lichen Society, pp. 20–94. We can conclude that Calicium corynellum is a species distinct from C. viride and thus should be treated separately in future con- servation activities and Red List assessments. Yahr R (2015) The status of the conservation priority species Calicium cory- nellum in the British Isles. Lichenologist 47, 205–214.
https://openalex.org/W4231151736	https://brill.com/downloadpdf/journals/nwig/94/1-2/article-p82_4.pdf	English	null	Bookshelf 2019	NWIG, New West Indian guide/NWIG	2,020	cc-by	15,226	nwig nwig New West Indian Guide 94 (2020) 82–111 New West Indian Guide 94 (2020) 82–111 brill.com/nwig Bookshelf 2019 Richard Price and Sally Price Coquina Key, Florida, USA rixsal@gmail.com; www.richandsally.net Richard Price and Sally Price Coquina Key, Florida, USA rixsal@gmail.com; www.richandsally.net This year, once again, we begin by expressing our gratitude to all the reviewers who have, collectively, provided such a rich resource for keeping up with writ- ing on the region. At the same time, we must lament the fact that some of the people who’ve accepted a book and promised to review it have, despite gentle reminders over a year or two, never shared their assessment with NWIG read- ers. With apologies to the authors of books that have therefore not been given their due in these pages, we simply list them here. ColonialPhantoms:BelongingandRefusalintheDominicanAmericas,fromthe19thCen- tury to the Present, by Dixa Ramírez (New York: NYU Press, 2018, paper US$30.00) (Six scholars declined to review this book before one said yes. After sending three reminders to that person, we finally gave up hope.) ColonialPhantoms:BelongingandRefusalintheDominicanAmericas,fromthe19thCen- tury to the Present, by Dixa Ramírez (New York: NYU Press, 2018, paper US$30.00) (Six scholars declined to review this book before one said yes. After sending three reminders to that person, we finally gave up hope.) ColonialPhantoms:BelongingandRefusalintheDominicanAmericas,fromthe19thCen- tury to the Present, by Dixa Ramírez (New York: NYU Press, 2018, paper US$30.00) (Six scholars declined to review this book before one said yes. After sending three reminders to that person, we finally gave up hope.) Voices from Mariel: Oral Histories of the 1980 Cuban Boatlift, by José Manuel García (Gainesville: University Press of Florida, 2018, cloth US$24.95) Voices from Mariel: Oral Histories of the 1980 Cuban Boatlift, by José Manuel García (Gainesville: University Press of Florida, 2018, cloth US$24.95) Voices from Mariel: Oral Histories of the 1980 Cuban Boatlift, by José Manuel García (Gainesville: University Press of Florida, 2018, cloth US$24.95) Island Historical Ecology: Socionatural Landscapes of the Eastern and Southern Carib- bean, edited by Peter E. Siegel (New York: Berghahn, 2018, cloth US$130.00) Island Historical Ecology: Socionatural Landscapes of the Eastern and Southern Carib- bean, edited by Peter E. Siegel (New York: Berghahn, 2018, cloth US$130.00) BeyondCubanWaters:Africa,LaYuma,andtheIsland’sGlobalImagination,byPaulRyer (Nashville TN: Vanderbilt University Press, 2016, paper US$27.95) BeyondCubanWaters:Africa,LaYuma,andtheIsland’sGlobalImagination,byPaulRyer (Nashville TN: Vanderbilt University Press, 2016, paper US$27.95) Crime, Violence, and Security in the Caribbean, edited by M. Raymond Izarali (London: Routledge, 2017, cloth US$145.00) Crime, Violence, and Security in the Caribbean, edited by M. © richard price and sally price, 2020 \| doi:10.1163/22134360-09401049 This is an open access article distributed under the terms of the CC BY-NC 4.0 license. Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ © richard price and sally price, 2020 \| doi:10.1163/22134360-09401049 This is an open access article distributed under the terms of the CC BY-NC 4.0 license. Downloaded via Open Access. This is an open © richard price and sally price, 2020 \| doi:10.1163/22134360-09401049 This is an open access article distributed under the terms of the CC BY-NC 4.0 license. Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. Bookshelf 2019 Raymond Izarali (London: Routledge, 2017, cloth US$145.00) Practices of Resistance in the Caribbean: Narratives, Aesthetics, and Politics, edited by Wiebke Beushausen, Miriam Brandel, Joseph T. Farquharson, Marius Littschwager, Annika McPherson & Julia Roth (London: Routledge, 2018, cloth US$149.95) Practices of Resistance in the Caribbean: Narratives, Aesthetics, and Politics, edited by Wiebke Beushausen, Miriam Brandel, Joseph T. Farquharson, Marius Littschwager, Annika McPherson & Julia Roth (London: Routledge, 2018, cloth US$149.95) Borderline Citizens:The United States, Puerto Rico, and the Politics of Colonial Migration, by Robert C. McGreevey (Ithaca NY: Cornell University Press, 2018, cloth US$45.00) Borderline Citizens:The United States, Puerto Rico, and the Politics of Colonial Migration, by Robert C. McGreevey (Ithaca NY: Cornell University Press, 2018, cloth US$45.00) Population,Migration,andSocioeconomicOutcomesamongIslandandMainlandPuerto Ricans: La Crisis Boricua, by Marie T. Mora, Alberto Dávila & Havidán Rodríguez Population,Migration,andSocioeconomicOutcomesamongIslandandMainlandPuerto Ricans: La Crisis Boricua, by Marie T. Mora, Alberto Dávila & Havidán Rodríguez (Lanham MD: Lexington Books, 2017, cloth US$95.00) AfricanKingsandBlackSlaves:SovereigntyandDispossessionintheEarlyModernAtlan- tic, by Herman L. Bennett (Philadelphia: University of Pennsylvania Press, 2018, cloth US$24.95) review articles 83 We asked seven reviewers to tackle the following two books together but none were willing, so we merely list the titles: Patriot or Traitor: The Life and Death of SirWalter Ralegh, by Anna Bee (London: Oneworld Publications, 2018, cloth US$27.95), and Walter Ralegh: Architect of Empire, by Alan Gallay (New York: Basic Books, 2019, cloth US$40.00). After reading Crossroads of Colonial Cultures: Caribbean Literatures in the Age of Revolution, by Gesine Müller (Berlin: Mouton De Gruyter, 2018, paper US$45.99), our reviewer wrote that she found it impossible to review the book because it was “very disorganized and also lacking in coherence, such that whatever merits there may be in its many arguments were difficult to judge.” We regret not being able to publish a review. Our readers may also be interested in the following exchange, concerning Decolonial Puerto Rican Women’s Writings: Subversion in this Flesh, by Roberta Hurtado (Basingstoke, U.K.: Palgrave Macmillan, 2019, cloth US$74.99), which speaks to the state of publishing these days. The Book Review Coordinator of Springer Nature (which now handles book review requests for Palgrave Macmillan, a major publisher on the Caribbean) wrote to our reviewer offering to provide an eBook instead of a print copy since, she said, in order to imple- ment a more sustainable and environmentally friendly system, they were pro- moting the use of digital copies of their books wherever possible. Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 This is an open access article distributed under the ter of the CC BY-NC 4.0 licen https://creativecommons.org/licenses/by-nc/4 New West Indian Guide 94 (2020) 82–111 New West Indian Guide 94 (2020) 82–111 review articles 84 The Defining Sea, by Robert Friedman (Mechanicsburg PA: Brown Posey Press, 2019, paper US$14.95) AnInquiryintoChoteo,byJorgeMañach,translatedandwithanintroductionbyJacque- line Loss (Barcelona: Linkgua Ediciones, 2019, paper US$14.00) On Being Committed to a Small Place, by Annalee Davis (San José, Costa Rica: TEOR/ éTica, 2019, paper US$20.00) Girl, Woman, Other, by Bernadine Evaristo (London: Hamish, Hamilton, 2019, paper Girl, Woman, Other, by Bernadine Evaristo (London: Hamish, Hamilton, 2019, paper US$1 00) [Winner of the Booker Pri e] US$17.00) [Winner of the Booker Prize] Unraveling, by Karen Lord (New York: DAW, 2019, cloth US$26.00) US$17.00) [Winner of the Booker Prize] Unraveling, by Karen Lord (New York: DAW, 2019, cloth US$26.00) The Golden Hour: A Novel, by Beatriz Williams (New York: William Morrow, 2019, cloth, US$26.99) Waiting for the Revolution, Cuba: The Unfinished Journey, by Gustavo Gac-Artigas (New Jersey: Ediciones Nuevo Espacio, 2019, paper US$9.99) Waiting for the Revolution, Cuba: The Unfinished Journey, by Gustavo Gac-Artigas (New Waiting for the Revolution, Cuba: The Unfinished Journey, by Gustavo Gac-Artigas (New Jersey: Ediciones Nuevo Espacio, 2019, paper US$9.99) When the Sky Fell: Hurricane Maria and the United States in Puerto Rico, by Michael Jersey: Ediciones Nuevo Espacio, 2019, paper US$9.99) When the Sky Fell: Hurricane Maria and the United States in Puerto Rico, by Michael Jersey: Ediciones Nuevo Espacio, 2019, paper US$9.99) When the Sky Fell: Hurricane Maria and the United States in Puerto Rico, by Michael Deibert (New York: Apollo Publishers, 2019, cloth US$24.99) When the Sky Fell: Hurricane Maria and the United States in Puerto Rico, by Michael Deibert (New York: Apollo Publishers, 2019, cloth US$24.99) Deibert (New York: Apollo Publishers, 2019, cloth US$24.99) My Time Among the Whites: Notes from an Unfinished Education, by Jennine Capó Crucet (New York: Picador, 2019, paper US$17.00) My Time Among the Whites: Notes from an Unfinished Education, by Jennine Capó Crucet (New York: Picador, 2019, paper US$17.00) Black Leopard, Red Wolf (The Dark Star Trilogy Book 1), by Marlon James (New York: Riverhead, 2019, cloth US$30.00) GrabaSnakebytheTail:AMurderinHavana’sChinatown,byLeonardoPadura(London: Bitter Lemon, 2019, paper US$14.95) Le théâtre d’Aimé Césaire, by Gérard Cogez (Lausanne: Ides et Calendes, 2018, paper €10.00) Le théâtre d’Aimé Césaire, by Gérard Cogez (Lausanne: Ides et Calendes, 2018, paper €10.00) We begin our mini-reviews, as usual, with novels. Bookshelf 2019 The reviewer replied,“Ihavecarpalandcubitaltunnelinbothhandsandtherefore,aneBook will be very hard to handle. I really understand the importance of promoting environmental friendly policies, but due to my medical condition I really hope we can make an exception and work something out.” Springer’s coordinator wrote back: “Thank you for your e-mail and I am sorry for your health prob- lems. Unfortunately, I can’t make an exception in this case.” If this policy continues, we may well need to stop providing reviews of Pal- grave books for the several thousand readers of the NWIG. How short-sighted on the part of Springer/Palgrave and what a shame for Caribbeanists! One needn’t be overly cynical to wonder whether environmental concerns might be a less important motivation for Palgrave than the cost of sending review copiesthroughthemail.(SeveralotherEuropeanpublishershavebeguntogive reviewers a choice between receiving a PDF or a book, not citing the environ- ment but rather the high price of international book mailing.) We next list books that we requested for review in Bookshelf but which the publisher, for whatever reason, did not send. Kazal: Memories of a Massacre under Duvalier—a Photographic Approach, by KOLEK- TIF2D, edited under the direction of Nicola Lo Calzo (Roquevaire, France: André Frère Éditions, 2019, cloth €29.00) New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08A via Open Access. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 We begin our mini-reviews, as usual, with novels. Around Harvard Square, by Jamaica-born novelist and journalist C.J. Far- ley (Brooklyn NY: Akashic, 2019, paper US$14.95) explores issues of race and class among a small cluster of Ivy-League freshmen as they learn the intri- cacies of Cambridge life in the 1990s. An African American student-athlete, an Asian American who sells dope, a child-star Bollywood actress, and a girl from rural Jamaica are swept up in the world of final clubs, the Crimson, and the Lampoon, all the while finding out who they are. It’s chock-full of (unreferenced) bits and pieces of literary works (from Sylvia Plath to Claude McKay),esotericaof Harvardhistory(aconversationaboutPickering’sHarem), and more—not every reader will pick up on the same set (though some, such as the discussion of Willie DuBois and the theory of double uncon- sciousness, will be easy for any NWIG reader). For anyone who likes satire, this quick-witted tale, which sometimes borders on action-comix narration, catches a bundle of truths about a very particular and powerful corner of our world. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 85 Patsy (New York: Liveright, 2019, cloth US$26.95), Jamaican Brooklynite Nicole Dennis-Benn’s second novel, is, like her earlier Here comes the Sun, a page-turner that explores the lives of women who love women, anxieties about race, color, and sexuality (this time in New York as well as Jamaica), and family relations, especially between mothers and daughters. Patsy immigrates (guiltily leaving her daughter behind) and works (without papers) as a bath- room attendant in a faux-Jamaican restaurant and as a nanny, while her daugh- ter,broughtupinherfather’shousehold,navigatesadolescenceandheartbreak in Kingston. In its depiction of the rough and lonely life of undocumented aliens in New York, it brings to mind Évelyne Trouillot’s Absences sans fron- tiers (2013), in which it is a father who devotes his life to working at mean jobs so his daughter back home can have a brighter future, but here it is the guilt of separation and abandonment that dominates. As we noted about Dennis- Benn’s debut novel, Patsy neatly captures the complex pathos of contemporary Caribbean realities. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Downloaded from Brill.com 10/24/2024 04:12:08AM Bookshelf 2019 Curdella Forbes’s haunting, intriguing, and absorbing fifth novel, A Tall His- tory of Sugar (Brooklyn NY: Akashic, 2019, cloth US$28.95), takes us from the ruralJamaicaof the1950stoKingstontoBrixtonandback,tracingthelovestory of a strange-looking pale-skinned boy, found as a baby in a basket by the sea, and the book’s narrator, a beautiful black girl who grows into a political activist. Fairy-tale or myth-like, with colonialism, race, sugar, and slavery always in the background, the story unfolds with frequent use of Jamaican Creole and allu- sions to Caribbean literature. A memorable read. Drafts of a Suicide Note (Raleigh NC: Regal House Publishing, 2019, paper US$19.95), by Mandy-Suzanne Wong (according to the publisher’s website, “a born Bermudian of Jamaican parentage and confusing Afro-Chino-Cuban her- itage”), is a long and dark debut novel centered on a mysterious woman who disappeared, leaving ten cryptic suicide notes. The main protagonists are elite Bermudans involved in the murky world of risk management, wealth manage- ment,reinsurance,documentshredding,anddesignerdrugs,butit’salsoastory of love, mistrust, obsession, and betrayal. The author clearly has a love of liter- ature as well as a lively imagination, and she weaves a memorable, complex tale. Queen of Bones (A Havana Mystery), by Teresa Dovalpage (New York: Soho, 2019, cloth US$26.95), is fast-paced crime fiction by a Cuban-born, New Mex- ico-resident writer, who loads lots of local color into her plot twists. Santeros, detectives, and mortuary employees mix with cross-dressers and American tourists in this telenovela-like tale. Plastered in Pretty (Philipsburg, St. Maarten: House of Nehesi, 2018, paper US$20.00), a novella by Vincentian writer and teacher Natasha C. Marks, New West Indian Guide 94 (2020) 82–111 86 review articles describesanislandworldinwhichsocialmediarulesandwherepeoplelookup from their cellphones only for the briefest of sexual or workplace encounters andareobsessedwithonlinebuying,posing,selfies,andseekingdigitalnirvana in the form of “likes” and happy emojis. The genre is satire and the tone chick- lit, but it depicts a Caribbean dystopia, sometimes comic, yet disturbingly close enough to a possible near future to foster thought and unease. Dominicana (New York: Flatiron, 2019, paper US$26.99), Angie Cruz’s third novel, is based on her mother’s coming-of-age experience as a fifteen-year-old country girl whose parents marry her off to a much older businessman, who quickly whisks her away to the Washington Heights of the 1960s. Unflinching initsportrayalof maritalviolenceandpatriarchalnorms,itpaintsathoroughly believable picture of Dominican immigrant life, both growing up surrounded by canefields and living in a squeezed apartment across from the Audibon Ball- room. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Relationships and events grab you and stir the emotions; it’s hard to put down. On its initial publication in Cuba, Marcial Gala’s La catedral de los negros (2012) was awarded the Alejo Carpentier Prize and the Premio de la Crítica Literaria. Now published as The Black Cathedral (New York: Farrar, Straus & Giroux, 2020, cloth US$26.00) in a fine translation by Anna Kushner, it is indeed a memorable work, narrated in a series of one- or two-page first-person fragments by a myriad of colorful inhabitants of a run-down neighborhood of Cienfuegos. Painting, architecture, and poetry rub shoulders with a serial killer, cannibalism, sex, violence, racism, and love, as provincial Cubans stay in place (or prison), flee to the United States and Barcelona, and deal with a never-finished overblown Protestant cathedral. The writing is finely honed, sometimes darkly comic, as the protagonists pick up and interrupt each other’s stories. A dystopian yet heart-rending vision not only of contemporary Cuba but also of the disappointments of the Obama-era United Sates of Amer- ica. Two novels of questionable origin. The latest iteration of William Williams’s The Journal of Penrose, Seaman (newly subtitled: The New Robinson Crusoe and the First American Novel), edited by Welsh author Terry Breverton (Car- marthenshire, Wales: Cambria Books, 2014, paper £20.00), is a modernized version of the ca. 1775 manuscript, which was first published in a bowdlerized edition in 1815 and then in a full transcription in 1969 (by Indiana U.P.), relat- ing the author’s (alleged) experience of being marooned on the Miskito coast among the Rama Indians. Joining it is the probably fictional/forged Hayti (Car- marthenshire, Wales: Cambria Books, 2017, paper US$17.99), with the author listed as Kurtis Sunday (a living novelist) and a preface claiming that this “is probably the first work of fiction written in a European language in the New New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 87 World,” and that the original manuscript was discovered in Granada in 1904 (or, alternatively, in France ca. 1800) and then destroyed during the Spanish Civil War, but meanwhile published in 1924 in Barcelona in Catalan, then in 1939 in London in English, and finally in 1969 in Mexico in Spanish. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 (The “translator” claims to have relied on all editions.) The apparently fictional text is said to have been written by a Hieronymite nun named Lucrezia di Marchionni and depicts Spanish atrocities, conversations with the Tainos, maroon resistance, and other happenings around 1515. Raphaël Confiant has added a 350-page thriller to his dozens of published books: L’enlèvement du mardi-gras, enquête sur une disparition (Paris: Éditions Écriture, 2019, paper €20.00). At once ostentatiously vulgar and ironic, this comic roman à clef centers on Martiniquan university life, with corrupt and inept police vying with corrupt and inept faculty and elected officials. With his usual joyful vengeance, Confiant takes on what he sees as the underbelly of Martinique and its inhabitants, reveling in the everyday racism and concu- piscence, the greed and selfishness, the violence and humor. You either put it down quickly (our tendency) or, perhaps, you laugh along with the clever but often facile turns of phrase. Tercer Mundo (San Juan: Zemi Books, 2019, paper US$24.95), by prolific Puerto Rican novelist/science fiction writer Pedro Cabiya (who lives in the Dominican Republic), begins with the crash in Santurce of a spaceship con- taining treasure. Mixing genres and registers, it explores the shenanigans of the bureaucrats who administer “la República Borikwá” and compete to get ahold of it, creating a phantasmagoric counterpart to current Puerto Rican political and existential realties. The Restless, by Gerty Dambury (New York: The Feminist Press at CUNY, 2018, paper US$16.95)—Judith G. Miller’s excellent translation of the 2014 French original, Les rétifs—tells the story of the 1967 labor violence in Guade- loupe, where the French forces de l’ordre killed or seriously wounded more than 100 protesters and bystanders. Narrated in syncopated first-person chap- ters in the form of a Caribbean quadrille, with a nine-year-old girl whose schoolteacher (accused of being a subversive) has disappeared leading the way, most of the other tale-tellers are already dead and include Queen Nono (anear-centenarianwithacut-off leg),Hilaire(“MademoisellePansy”),asome- time cross-dressing teacher who hanged himself after a snitch falsely de- nounced him as a pedophile, an honest construction worker, and others. Issues of class,race,andcolonialismunderliethisdeceptivelysimple,sweetbutbrutal tale. TheindefatigableMaryseCondé,aidedbyherlong-timetranslator(andhus- band) Richard Philcox, offers readers yet another in a string of recent auto- New West Indian Guide 94 (2020) 82–111 review articles 88 biographical meanderings: Of Morsels and Marvels (Calcutta: Seagull Books, 2020, cloth US$27.50 [French original 2015]). Bookshelf 2019 At the outset, she claims that “My enduring crime of treason is the subject of this book,” by which she refers to her penchant for “comparing cooking with literature … mixing sheep with goats, jute with silk.” In this characteristically irreverent look at past travels (from West Africa to India, from Berkeley to New York, from Guade- loupe to Paris, and beyond), emphasizing the dishes she has been served and her varied reactions to them, she minces few words. In an almost stream-of- consciousness style, she opines (often caustically) on places, people, and foods she has encountered during her long life, sparing few. Indeed, we felt some- thing like relief, having been dinner guests in her Paris apartment and having hosted her to meals in both Martinique and Paris, to have been left out of her account. Quince Duncan’s Weathered Men and The Four Mirrors: Two Novels of Afro-Costa Rican Identity (Basingstoke, U.K.: Palgrave Macmillan, 2019, cloth US$84.99), translated and with a helpful introduction by Dorothy E. Moseby, seem to be the first novels in Spanish (published in the early 1970s) by an author of West Indian heritage who writes about Jamaican immigrants to Cen- tral America. Born and raised in Limón, Duncan, who has published five other novels as well as short stories, sets out to contradict traditional Costa Rican nationalist identity centered on whiteness, Europeanness, et cetera. These are, as the translator argues, “novels of Afro-Costa Rican identity.” Days by Moonlight (Toronto: Coach House Books, 2019, paper US$17.95), by prize-winning Canadian novelist André Alexis (born in Trinidad), is firmly focused on Canada rather than the Caribbean. The narrator travels through a series of small towns in Southern Ontario, including one where black people are not allowed to talk during daylight hours, and others in which he attends bizarre patriotic rituals and parades, takes a guided tour of the Museum of Canadian Sexuality, and has mind-bending conversations, but throughout dis- playing a love of language, poetry, and the absurd. It’s a real trip, filled with fantastical, sometimes bitter, and often ironic imaginings. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 In “Bookshelf 2011,” we praised Bivouac (2010), by multiple-prize-winning poet and novelist Kwame Dawes, as “a dark novel about death, politics, fam- ily, and sex in a Jamaica that has a ‘scarcely understood sense of temporariness and dislocation,’ with dialogue that puts you right onto the streets of Kingston.” Reissued now by Akashic (2019, paper US$15.95), it reads better than ever, a dreamlike work about the island in the 1980s. Another welcome reissue: Jamaica-born Patricia Powell’s captivating, dialect-rich, debut novel, Me Dying Trial (Boston: Beacon Press, 2019, cloth US$16.00), first published by Heinemann in England in 1993, reissued by Bea- New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 89 con in 2003, and now graced with an admiring introduction by Edwidge Dan- ticat, who calls Powell “one of the most exciting writers living and writing on the island that is the Caribbean-American hyphen.” And, finally, a resurrected novel, Claude McKay’s RomanceinMarseille (New York: Penguin Classics, 2020, paper US$20.00), which has languished as an unfinished manuscript in Yale’s Beineke Library and, in more finished but still unpublished form, in the Schomburg Center, since some time after its ini- tial creation in the 1920s. An introduction and “note on the text”—together, more than 50 dense pages—by editors Gary Edward Holcomb and William J. Maxwell, set the scene for this strange Lost Generation novel by the Jamaica- born intrepid traveler and gay icon of the Harlem Renaissance. Originally intended as a follow-up to Banjo (1929), also sited in Marseille, this tale of whores, pimps, itinerant seamen, and waterfront dive bars, loosely based on a mixture of contemporary news stories about black stowaways on steamships and McKay’s own seedy life in the multicultural port city, is as interesting for its insights into McKay’s thoughts (for example, he fictionalizes W.E.B. Du Bois’s NAACP into an organization he calls The Christian Unity of Negro Tribes a.k.a. C.U.N.T.) as for the characters or plot of the novel, though they too are often memorable. Whether viewed as one of the earliest overtly queer fictions in the Black Atlantic canon or for its views on black nationalism, the publication of this work is to be celebrated. Now, on to short stories. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Downloaded from Brill.com 10/24/2024 04:12:08AM Bookshelf 2019 Edwidge Danticat’s writing—crystalline, spare, every-word-mattering, and consistently heartrending—puts her short stories in a different league from all others. Her latest collection, Everything Inside (New York: Knopf, 2019, cloth US$25.95), consists of eight stories previously published in such venues as Callaloo, Ms., and The New Yorker. Most of them concern Haitians who live in the Diaspora but the family relationships she describes so poignantly speak universally. Truly memorable. Grand Union (New York: Penguin Press, 2019, cloth, US$27.00), by award- winning novelist and NYU professor Zadie Smith, is her first book of short stories. Raunchy or tender, gritty or ethereal, foreboding or comical, each one is told with a stunning gift for getting into the skin of diverse characters in varied situations. You’ll surely like some better than others, but by the time you get to thefinalstory—aconversationbetweenthenarratorandherdeadmotherwho has become Nanny of the Maroons—you’re likely to be sorry that this wonder- ful collection has come to its end. WhereThere are Monsters (Leeds, U.K.: Peepal Tree Press, 2019, paper £9.99) is Trinidadian Breanne Mc Ivor’s first collection of short stories (several of which have been previously published and won prizes). Crisp tales with mem- New West Indian Guide 94 (2020) 82–111 90 review articles orable characters—men as well as women—who hold varying positions in the race/class structure, set in contemporaryTrinidad, but with periodic irruptions of folkloric beliefs from other eras. A brisk and enjoyable read. j y Hazel D. Campbell’s Jamaica on My Mind: New and Collected Stories (Leeds, U.K.: Peepal Tree Press, 2019, paper £14.99), comprised of several dozen previ- ously published short stories plus eight new ones, takes readers into a different Caribbean from that of Mc Ivor or Plastered in Pretty: a world of the 1960s, 70s, and 80s, where many people speak Creole and where the pace of life and the concerns and values are those of the grandparents of the younger writers. Even in the new stories, where cell phones and dancehall appear, it’s the human rela- tions of an earlier Jamaica, and the ever-presence of churches, that provide constancy and—through explorations of well-drawn characters—bring con- siderable pleasure to the reader. Bookshelf 2019 Now / After: Stories (Leeds, U.K.: Peepal Tree Press, 2019, paper £9.99), by Anton Nimblett, a Trinidadian living and writing in Brooklyn, is a different ket- tleof short-storyfish.Wildlyvariedinsubject,location,andtone,itmovesfrom a Haiku-like visit to Dia and its Serra rust-tinged steels to gay re-imaginings of fragments from Moby Dick, Gulliver’s Travels, In the Castle of my Skin, and A House for Mr. Biswas, to a drag-queen drama set in Jersey City, and a wake in the Haitian countryside or conversations in rural Trinidad. Often intriguing, always interesting, we recommend it. Love War Stories, by Ivelisse Rodriguez (New York: The Feminist Press at CUNY, 2018, paper US$16.95), presents a searing and frighteningly persua- sive look into the world of Puerto Rican teenage girls, mostly in New York (but also on the island and in Holyoke, Massachusetts), questing after true love despite their mothers’ or aunts’ marital betrayals and deceptions. The teenage and college boys who are the object of their longings appear to con- firm the pan-Caribbean saying, “men are dogs.” The protagonists vary from a fifteen-year-old small-town island girl; a pack of high-schoolers in Holyoke; a rejected college girl who becomes a stalker; and even some gay boys—all believable and sympathetic. The stories are tough, smart, and eminently read- able. Next, this year’s crop of poetry. Kei Miller’s In Nearby Bushes (London: Carcanet, 2019, paper US$14.99), a magnificent collection meant to be read cover-to-cover at one sitting, dis- plays a lilting love of language, a love of Jamaican words and places and smells alongside everyday violence and singular events, like the escape dur- ing a 1988 hurricane of a troop of traveling reindeer into the hills of Port- land, where they seem destined to multiply forever, despite hunters, in nearby bushes. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. Bookshelf 2019 https://creativecommons.org/licenses/by-nc/4.0/ review articles 91 Crossfire, A Litany for Survival: Poems 1998–2019 (Chicago: Haymarket, 2019, paper US$19.95), by Staceyann Chin—New York-based performance artist, actor,andactivist,andauthorof a2010memoirof growingup(andcomingout) in Jamaica (The Other Side of Paradise)—begins with a preface briefly recount- ing experiences with her teachers (Derek Walcott, Mervyn Morris, Edward Baugh), and then moves on to a collection of poems that cry out to be read aloud. Poems about love, gender, desire, and many kinds of sex, but also family, motherhood, Jamerica, rape, racism, U.S. and international politics, and being a black and Asian lesbian poet. She’s a storyteller and these poems are direct and topical, overflowing with life. The Perseverance (London: Penned in the Margins, 2018, paper £9.99) is Raymond Antrobus’s first collection since To Sweeten Bitter (2017), which we very much liked. Unlike the earlier collection, these new poems by the British Jamaican writer focus on his congenital deafness and its effects on his ways of being in the world. Reading Antrobus opens eyes as well as ears to lyrical and moving words, sounds, and experiences. The book has won two major poetry prizes and we recommend it highly. Translations from Memory (Manchester, U.K.: Carcanet, 2018, paper £12.99) is Guyanese-British Fred D’Aguiar’s eighth published collection, some 88 mag- nificent poems that rewrite and comment on world history, philosophy, and literature with a mixture of irony, humor, and passion. The inspirations range from Gilgamesh, Homer, and a slew of ancient Greeks and Romans through St. Thomas Aquinas, the Franciscans and Machiavelli, and on into Rousseau, the Enlightenment, and the Romantics, through twentieth-century French theo- rists (Fanon, Barthes, and Lévi-Strauss) and on to W.E.B. DuBois, Mal- colm, M.L.K., Bearden,Wilson Harris, Aimé Césaire, Martin Carter,Walter Rod- ney,Walcott, Brathwaite, and “Our King James” (C.L.R.).These irreverentpieces highlight the lasting legacies of colonialism and racism while asking broadly humanistic questions about redemption and hope. The combination of light- ness and deep feeling is magical. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Nothing we read this year stretches the mind quite like Vahni Capildeo’s poems in Skin Can Hold (London: Carcanet, 2019, paper US$14.99), her eighth published collection, which ranges from encounters with Shakespeare and a homage to fellow poet Zaffar Kunial to various carnivalesque Trinidadian allu- sions before settling on a description and analysis of her staging of an exper- imental theater version of Martin Carter’s “I am no Soldier.” This last evoked to us a precious moment in the 1990s, watching twilight fall with George Lam- ming on the porch of our house in Martinique, looking out at the sea, while he reminisced about traipsing from one Georgetown rumshop to the next with Martin, as they talked about Walter Rodney and what might have been. New West Indian Guide 94 (2020) 82–111 92 review articles In Enemy Luck (Leeds, U.K.: Peepal Tree Press, 2019, paper £10.99), Trinida- dian literary editor and impresario Nicholas Laughlin’s second collection of poetry, we are treated to a remarkably varied set of forms and puzzles and entertainments, always forcing thought, asking readers to search memories of a lifetime of reading. Found fragments, evocations of scenery, and people from around the world combine to create compelling mysteries and tales. This is a book that rewards. Honeyfish (Kalamazoo MI: New Issues Poetry and Prose, 2019, paper US$16.00), winner of the 2018 Green Rose Prize, presents Trinidad-born-and- raised Lauren K. Alleyne’s second collection of poems—elegiac and beautiful, some evoking her own island and one in Greece, yet most expressing relentless horror at the unending U.S. White-on-Black murders and oppression: Trayvon, Sandra Bland, Tamir Rice, Charleston, Charlottesville, and burning crosses in Iowa, as well as neo-Nazis marching in Leipzig. Extraordinarily moving poetic testimony. Poet and performer Roger Robinson, who lives between Brixton and Trini- dad, displays his full storytelling skills in his latest collection, A Portable Par- adise (Leeds, U.K.: Peepal Tree Press, 2019, US$12.48), winner of the prestigious T.S. Eliot Poetry Prize. From its first moving section on the Grenfell Tower dis- aster to poems of slavery and modern police brutality to ironic takes on race in Britain, these poems pull you in, some making you laugh, others almost weep. Direct and important. In I’lltradeyouthisisland byCindyJiménez-Vera(San Juan:Aguadulce,2018, n.p.), translator Guillermo Rebollo-Gil presents his English versions on pages facing the Spanish originals. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Ironic, dark, raw, filled with the small material things of life, and agnostic about the future, these poems evoke a particular vision of Puerto Rico: “There’s no bread in my house / but the bananas are ripe /and wehave blackcoffee,/ nosugar.”Or,“I’lltradeyouthisisland/ foramatch- box / and four candles / to light / out on the corners.” I Offer My Heart as a Target / Ofrezco mi corazón como una Diana, by Puerto Rico-born Johanny Vázquez Paz (Brooklyn NY: Akashic, paper US$15.95), is a prize-winning dual-language collection, with English translations by Lawrence Schimel. Many of the poems speak of violence, misogyny, and assaults on dig- nity, others of the bitterness of migration and exile, but difficulties are met throughout with a steely-eyed perseverance. Lasana M. Sekou’s latest book of poetry, Hurricane Protocol (Philipsburg, St. Maarten: House of Nehesi, 2019, paper US$15.00), chronicles in his inimitable style the devastating passage of Irma and Maria through his beloved island in 2017. Many pages are illustrated with hieroglyphs from the Dresden Codex, depictingMayan(standingforindigenousAmerican)astronomicalpredictions and calculations. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the term of the CC BY-NC 4.0 license https://creativecommons.org/licenses/by-nc/4.0 New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 93 Sara Florian’s Caribbean Counterpoint:The Aesthetics of Salt in Lasana Sekou (Philipsburg, St. Maarten: House of Nehesi, 2019, paper US$20.00) discusses variousthemes(salt,sugar,maroons,andmore)intheworkof thepoet,activist, and publisher (House of Nehesi), who has labored tirelessly for several decades for the independence and sovereignty of St. Maarten/St. Martin as a unified island. Salt, the island’s major colonial export (in contrast to the rest of the Caribbean’s sugar) is a central metaphor in his work; Florian’s brief book is an attempt to situate Sekou’s work in its broader Caribbean context. Nomad (Philipsburg, St. Maarten: House of Nehesi, 2019, paper US$20.00) is veteran writer, actress, and teacher Yvonne Weekes’s first collection of poetry. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 https://creativecommons org/licenses/by-nc/4 0/ 94 review articles present, with international travelers touching down at Hewanorra and leaving in taxis and rental cars to the land of all-inclusives and disappearing man- groves. g A richly informative 16-page foreword by translator (and University of Chi- cago professor) Haun Saussy introducesWhen the Pipirite Sings: Selected Poems (Evanston IL: Northwestern University Press, 2019, paper US$18.95), by the late Haitian writer and neurologist Jean Métellus.Written in Parisian exile from the Duvaliers, in French, these poems are nevertheless Haitian to the core, indeli- bly evoking the Jacmel of the writer’s youth. Sometimes Homeric, sometimes Césairian in tone and reference, they engage with the lwas, the land, the sun, and the sea, with the Middle Passage and the great figures of Haitian history always lurking in the background. A richly informative 16-page foreword by translator (and University of Chi- cago professor) Haun Saussy introducesWhen the Pipirite Sings: Selected Poems We welcome the paperback reprint of The Collected Poems of Édouard Glis- sant, translated by Jeff Humphries & Melissa Manolas and edited (with an introduction) by Jeff Humphries (Minneapolis: University of Minnesota Press, 2019, paper US$22.95), which originally appeared in cloth from that publisher in 2005. The introduction could have been updated to reflect changes in post- colonial scholarship during the past decade and a half, but the singular poems now permit Glissant to speak from the grave, as his literary influence continues to increase. We also welcome the first English-language translation, by Nathanaël, of Glissant’s earliest published work, Soleil de la conscience (1956) as Sun of Con- sciousness (NewYork: Nightboat Books, 2020, paper US$14.95), which recounts in poetic prose (and occasional straight poetry) his complex feelings upon arriving to study in 1946 Paris, where he remained for eight years. His thoughts range widely, from racism and colonialism to universalism and poetry: it is “a time of chaotic opening … prose, chaos, measure, knowledge and poetry being signs of my experience, seen from inside. Bookshelf 2019 Accessible, matter-of-fact, often surprising, these autobiographical poems recount her uncomfortable upbringing in England, her joyful return to her par- ent’s birthplace, Monserrat (where she founded a theater company and served as the island’s first Director of Culture) and her exile, after the devastating Soufrière volcano eruption in 1996, to Barbados, where she has since lived and worked, now with grandchildren. Kendel Hippolyte’sWordplanting (Leeds, U.K.: PeepalTree Press, 2019, paper £9.99) bring us poems about what might have been, the world that was once hoped for and dreamed of and fought for but never came to pass: “i woke one morning and the Caribbean was gone.” He seeks her, first by the sea, then in the town, then in the market, then at a crossroads, and finds only occasional glimpses of her presence “in a far hillside district … or a glint of zinc from a housechanginghalf of itsroof onaSaturdayhalf-day,giventoakoudmen,lend- hand, gayap, koumbit, fajina, jollification, maroon, gotong rojong … Harder to find now.” So, a sense of loss in a modernizing, globalizing St. Lucia, but also small pleasures in familiar domestic acts—morning coffee, making a bed with a loved one. In his seventh published collection of poems, Hippolyte’s lan- guorous rhythms draw us in: mature, thoughtful work that rewards. St. Lucian poet, journalist, and librarian John Robert Lee has done the Caribbean literary world a great service by conceptualizing and editing Saint LucianWriters andWriting: An Author Index of PublishedWorks of Poetry, Prose, and Drama (London and Trafalgar, Dominica: Papillote Press, 2019, paper £9.99). The book, which includes not only writers of S.L. origin but also those who have written about the island, is divided by genres: poetry, short fiction, novels, drama, literary periodicals, and so forth, and it includes unpublished dissertations and theses about the island as well. A labor of love, filled with surprises, that can be enjoyed by many. A Lesson on Wings (Vieux Fort, St. Lucia: Jako Books, 2019, paper US$21.95), by senior St. Lucian poet Modeste Downes, is best when—in a nostalgic mode—he recounts the storied past of his beloved Vieux Fort and rues its New West Indian Guide 94 (2020) 82–111 New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Outside, it is the French truth oppos- ing mine.” Or again, “And here I am freezing between these two oceans; the true and immortal abyss of the sea, on the one hand, that exiles me from myself (frommyreality,frommyrootsinthegroundplantedlikesomanypitchforksof truth); then the other, the enormous Wave besides that rolls here, Parisian … I know of a sudden its secret: and it is that Paris is an island, that intercepts from all sides and diffracts forthwith.” Twenty-five years before the publication of Le discours antillais, this early work contains only hints of that Antillean mani- festo but leads more directly into L’intentionpoétique and perhaps even toward the idea of le Tout-Monde. And, finally, works of nonfiction not otherwise reviewed in the NWIG: Recently retired Yale professor Hazel V. Carby has gifted us a remarkable book, Imperial Intimacies (London: Verso, 2019, cloth US$29.95), part memoir, New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed unde of the CC BY-NC https://creativecommons.org/licenses review articles 95 part historical study, and deeply personal throughout. Exploring family history, beginning with her birth in England of a Jamaica-born, World War II RAF pilot and a Welsh mother, she traces better than any book we can remember the ways that “Black British” is truly an oxymoron, as well as the broader conse- quences of race and empire. Following oral, and eventually archival, genealogy, she carries us to eighteenth-century Jamaica and Bristol and slowly brings us up to the present, all the way examining how the terrible violence of colo- nialism and empire has shaped, and continues to shape, ordinary people’s lives. In Voices of theWindrush Generation:The Real StoryTold by the PeopleThem- selves (London: Blink, 2019, cloth £18.99), journalist David Matthews (who, born in Hackney of British Guiana immigrant parents, styles himself a mem- ber of the Windrush 2.0 generation) interviews two handfuls of Windrush-era West Indians living in the United Kingdom, reminiscing about their (British) West Indian childhoods, their frightening and discrimination-filled first years inthemothercountry(andcontinuingraceprejudicetilltoday),andtheiradult lives as nurses, seamstresses, teachers, musicians, and parents. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Clumsier than the oral history collections by his model Studs Terkel, this one nonetheless lets people whose whole lives have been under the thumb of empire speak out, each in their own voice. It makes the Windrush scandal of recent years, which is barely touched on in the book, all the more heartbreaking. Journalist Ross Kenneth Urken, a self-described Jewish boy from New Jer- sey who speaks with a Jamaican accent, offers a memoir, Another Mother (Kingston: Ian Randle Press, 2019, cloth US$18.00), devoted to his Jamaican nanny Dezna Sanderson, who not only raised him but also served as peace- maker in his parents’ dysfunctional marriage. Moving back and forth between talesof hischildhoodandhisyoungadultquesttolearnaboutDezna’spre-USA life in Jamaica, through trips and interviews with her scattered family mem- bers, this somewhat self-indulgent book captures the realities of thousands of late-twentieth-century Caribbean women immigrants and serves as a loving homage to their sacrifices. If you wish to know the remarkable woman that is Christiane Taubira, her most recent memoir, Nuit d’épine (Paris: Plon, 2019, paper €16.90), is an excel- lent start.Vignettes of her life, from details of growing up in Guyane all the way to dealing, as France’s minister of justice, with the bombing of the Bataclan, are spiced with lyrics from the songs she loves (Brazilian, Cuban, West African, French, and American) and excerpts from her favorite poets (who range from Césaire, Damas, and Monchouachi to Countee Cullen, Pablo Neruda, and Ossip Mandelstam). She invitesus toreadover hershoulder as shediscoversthework of Maryse Condé, Steve Biko, and others, and as she lays bare her life expe- New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 96 review articles rience, she constantly enriches her reminiscences with references to authors and works in a variety of world literatures (and cinemas). In short, she opens a window, if only partially, into how she became one of the most interesting and, for many, admirable public figures of our time. Bookshelf 2019 Aftershocks of Disaster: Puerto Rico Before and After the Storm (Chicago: Hay- market Books, September 2019, paper US$17.00), edited by Yarimar Bonilla & Marisol LeBrón, is a truly important anthology—essays, poems, and photos that grapple with the island’s long-standing colonial problems and post-Maria dilemmas.AsArcadioDíaz-Quiñoneswritesinhisforeword,“thethreatof total collapse continues to raise major themes of discussion in a global context: colonialcapitalism,humanrights,genderequality,democracy,unpayabledebt, climate change, migration and citizenship, environmental policies, education, and health care.” More than thirty interventions by specialists of all stripes are deftly woven together by the editors to bring readers a vivid picture of the cur- rent imbroglio as well as useful pointers toward building a sustainable future for the island’s people. A Girl Named Lovely, by Catherine Porter (NewYork: Simon & Schuster, 2019, paper US$17.99), is a Canadian journalist’s story of how she first discovered Port-au-Prince whilereportingonlydaysafter the2010earthquakeandbecame involved in financially supporting a two-year-old girl miraculously pulled from the rubble and later much of her poverty-stricken family as well as a whole school. It recounts the ethical conundrums and private joys and frustrations she experienced as she witnessed and participated in the international aid effort in the years following the disaster.Well-meaning, honest, somewhat self- indulgent, and sometimes slightly breathless in tone, it stands as one woman’s personal reaction to the complex realities of inequality faced by almost any outsider in Haiti. Agua por todas partes (Barcelona: Tusquets, 2019, paper €19.00) is a collec- tion of Leonardo Padura’s essays/reflections on becoming and being Cuba’s best-known writer internationally. The book’s first part concerns his feelings of rootedness in the barrio of Mantilla, on Havana’s southern edge, and why he could never leave. But there is also much about contemporary Cuba, his irritation at the widespread love of reggaetón, descriptions of the daily lucha of his friends and neighbors, his thoughts about exile, and dialogues with the work of other writers, particularly Alejo Carpentier. The rest of the essays are loosely autobiographical but necessarily speak about evolving Cuban realities since the 1970s. The final chapters are more explicitly literary, engaging the form of the novel and the act of writing, with Cuban preoccupations, from baseball to the persecution of homosexuals, always in the background. In addi- tion to Carpentier, Padura highlights the work of José Lezama Lima and Vir- New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 97 gilio Piñera (who wrote, in 1943, of “la maldita circonstancia del agua por todas partes”) and displays a wide range of reading—novelists, poets, and literary critics. Goodbye, My Havana: The Life and Times of a Gringa in Revolutionary Cuba (Stanford CA: Redwood Press, 2019, paper US$24.00) is an autobiographical account by Anna Veltfort, presented in “graphic memoir” form, of her early life in Berkeley, California, her left-leaning family’s 1962 move to Cuba when she was of high-school age, her sexual (gay) awakenings, her life at the University of Havana (filled with revolutionary ardor as well as persecution), and her final departure for the United States in 1972. The graphic panels depict a number of her experiences during her Cuban decade, from meeting Fidel and Che and workinginsugarharvestsandpeasanteducationdrivestowatchingpurgesand show trials. The tone is frank and cheery, the story zips right along, as both the author and the Revolution come of age. In North of Havana: The Untold Story of Dirty Politics, Secret Diplomacy, and theTrialof theCubanFive (NewYork:The New Press, 2019, cloth US$26.99), vet- eran trial lawyer Martin Garbus, who earlier represented prominent dissidents including Daniel Ellsberg, Cesar Chavez, and Cuban poet Heberto Padilla, tells the gripping story of his role in this Kafkaesque case of international intrigue. The highly charged political world of 1990 Miami’s Little Havana comes alive, as we see the role of elite Cuban exiles in influencing everything from the appointment of U.S. attorneys to national elections. From the original show trial of the five Cubans (who were, indeed, Fidel’s spies, though innocent of the trumped-up murder charges that they were convicted for), conducted in the immediate wake of the Elián González affair, all the way through the Obama- Raúl-negotiated prisoner exchange that concluded the matter a decade and a half later, the author lays bare the working of the U.S. court and prison system, emphasizing its dangerous political vulnerabilities. Garbus wrote the book, he says, “to show how our government can subvert the press and inter- fere with our jury system. It chronicles an unprecedented pollution of the American legal system in order to advance a political cause.” Though chron- icling the recent past, its relevance for the present and immediate future is acute. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 A to Z of Caribbean Art, edited by Melanie Archer & Mariel Brown (Port of Spain: Robert & Christopher Publishers, 2019, paper US$44.05), offers single- page introductions to almost 300 twentieth- and twenty-first-century artists with ties to the Caribbean by birth, parentage, residence, adoptive home, or other links, many of whom maintain relationships both in and out of the region. Flipping through its pages, which include a color image of work by each artist, one cannot but be struck by the richness of media—painting, sculpture, New West Indian Guide 94 (2020) 82–111 98 review articles photography, engraving, video, collage, and glass to works made from fishing line and mosquito netting or a cement mixer and sunscreen. (Not surprisingly, there were difficult choices; for example, Martiniquan photographer Robert Charlotte is included, but well-known Martiniquan photographer Jean-Luc de Laguarigue is not.) All in all, a valuable resource for anyone interested in art of the Caribbean. CommittedtoMemory:TheArtof theSlaveShipIcon,byCherylFinley(Prince- ton NJ: Princeton University Press, 2018, cloth US$49.50), began as a 2002 Yale University dissertation but now benefits from considerable further research and travel. Generously illustrated in color, the book explores the initial (1788) impact and continuing afterlife of the iconic woodcut of the lower deck of the Liverpudlian slaver Brookes. A remarkable range of artists and artworks are engaged, from the eighteenth century to the present, tracing the icon’s role in multiple movements of political protest and even in modern-day roots tourism. Having coauthored a book (with Sidney W. Mintz) that used the icon on its jacket, I (RP) learned here of uses I’d never have imagined—it is indeed the central image in what Finley calls the “mnemonic aesthetics” of transat- lantic slavery, a foundational, if terrifying and persisting, symbol of the Black Atlantic. AntonioMartorellisthegiftthatkeepsongiving…mostrecentlyacollection of memories and reflections that offer yet more of the visual creativity, poetic sensitivity, and human warmth we’ve come to know from his earlier work. Pier- dencuentra (San Juan: Gaviota, 2019, paper n.p.), another tactually and visually beautiful object, offers touching word-portraits of his family (grandfather don Antonio who lost his lefthand pinkie, auntie Lucy who cooked up a fantastic goulash, aunt Consuelo who lost her name, one letter at a time …) with side trips to other things on his mind (“how words and illustrations found them- selves on one of my pages,” etc.). New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 He has said it is about things lost and found, without always beingable toknowwhichis which.The volumeisaccompanied by a CD of el maestro reading some of his word portraits. Another very beautiful (though in a more conventional sense) book focus- ing on an artist living and working in Santurce, Puerto Rico, is an exhibition catalog on the work of Zilia Sánchez organized by the Phillips Collection in Washington DC. Zilia Sánchez Soy Isla (New Haven CT: Yale University Press, 2018, cloth US$50.00) recounts Sánchez’s life from her early years in Cuba to travels in Europe and a decade in New York before a return to the Caribbean in 1971. Essays reflecting on her work, which “resists traditional categorization and floats in between” like an island, are accompanied by hundreds of full- color images, from sensuous erotic topologies, shaped canvasses, and “tattooed works” to the acrylics on stretched canvas that dominated the exhibition. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 99 L’Art à Cuba, by Gilbert Brownstone, with photographs by Camilo Guevara (Paris: Flammarion, 2019, cloth €39.90), is both elegantly produced and fasci- nating to read. An opening essay by Graziella Pogolotti recounts the history of the national art school, which began in an abandoned golf course; Guevara— son of “le Che”—reflects on his father’s interest in photography; and Brown- stone offers a substantive introduction to the place of art in Cuban culture. But it’s the 32 artists’ statements (from interviews by Brownstone), each sev- eral pages long and beautifully illustrated by Guevara, that constitute the heart of the volume. (That said, we have to assume that if the artists had been French rather than Cuban, Flammarion would have taken a bit more care in the half- pagepromotionaltextthatcomeswiththebook,whichmisspellsWifredoLam, Gilbert Brownstone, and Camilo Guevara [twice].) Picturing Cuba: Art, Culture, and Identity on the Island and in the Diaspora, edited by Jorge Duany (Gainesville: University Press of Florida, 2019, cloth US $80.00), is based on a 2017 conference held at the Frost Art Museum, part of Florida International University where Duany teaches and directs the Cuban Research Institute. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Fifteen contributors—academics and art collectors, all ap- parently living in the United States—highlight relations between Cuban and Cuban-American art (painting, photography, architecture) and cultural iden- tity from the seventeenth century to the present. The bulk of the book is de- voted to the twentieth and twenty-first centuries, and Miami-based artists and institutions often take pride of place. The 48 color plates and over a dozen black and white images remind us of the vibrancy of Cuba’s artistic output and helpfully illustrate the essays, which cover topics from colonial prints, national identity, and the role of women artists, through Concretism, to art in the Dias- pora. Hew Locke: Here’s theThing is the visually stunning catalog for an exhibition of works in various media by Guyanese/British artist Hew Locke, who styles himself part of the “post-Windrush generation.” Essays by historian Richard Drayton and curator Diana Tuite comment usefully on the way Locke has played with sources from ships and houses to colonial statues and share certifi- cates to produce a rich and imaginative evocation of the Caribbean past and present, with a special focus on voyages. The catalog (paper n.p.) is a 2019 co- publication of the Ikon Gallery in Birmingham, the Kemper Museum in Kansas City,andtheColbyCollegeMuseumof ArtinWatervilleMaine,wheretheexhi- bition was shown. In Born Ya: The Life and Loves of a Jamaican Painter (London: Beattie Books, 2019,paperUS$15.99),JudyAnnMacMillanoffersaforthrightaccountof grow- ing up as a light-skinned girl in a privileged family in Kingston, her art school years in Scotland, her return home and short-lived marriage to an American New West Indian Guide 94 (2020) 82–111 100 review articles engineer who took her to Ohio, and her settling back into the Jamaica of the Manleyswhere,despitegenderstereotypes,shefinallyfindsherself asapainter. The book brims with salient observations about her countrymen, both rural and urban, and her love of, and curiosity about, the island. AnAmericanOdyssey:TheLifeandWorkof RomareBearden,byMarySchmidt Campbell (New York: Oxford University Press, 2018, cloth US$34.95), almost completelyignorestheCaribbeanworkof themastercollagistandpainter,with hardly a word about his hundreds of Caribbean watercolors—the thirty or so Martinique paintings, the several hundred referring to St. Martin, the Carnival series, or the astounding Rituals of the Obeah. For anyone interested in his life in the Caribbean and its influence on his art, our own Romare Bearden: The Caribbean Dimension (or its French translation) remains the touchstone. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 With an over-the-top foreword by Ishmael Reed, Cimarrón: Freedom and Masquerade (London: Thames & Hudson, 2019, cloth US$40.00) consists of art photographer Charles Freger’s glossy, vividly colorful, dramatically cos- tumed, carefully posed carnavelesque images from around the African dias- pora: Guadeloupe, Martinique, Guyane, Antigua, Monserrat, Saint Croix, Haiti, theDominicanRepublic,Cuba,Brazil,Colombia,Mexico,Belize,Panama,Peru, and New Orleans. The two dozen pages of explanation at the book’s end, describing the carnival groups and the characters depicted, are less inaccurate than one might expect from this sort of project. The Haiti Reader: History, Culture, Politics, edited by Laurent Dubois, Kaiama L. Glover, Nadève Ménard, Millery Polyné & Chantalle F. Verna (Durham NC: Duke University Press, 2020, paper US$29.95), presents well over 100 texts in more than 500 lovingly assembled pages—everything from fiction, poetry, songs, and folktales to scholarly essays and political tracts, beginning with the Tainos, slavery, and the Revolution, moving on through the U.S. occupation, the Duvalier years and their aftermath, and continuing right up to the present. Much of the volume, which is intended to highlight Haitian points of view, offers small tastes of longer works, whetting the reader’s appetite for more.The English translations from French and Creole are fluid and welcome.The collec- tion introduces many of Haiti’s major writers and thinkers and will be a boon to college teachers, a solid basis for a course on the first independent nation of the Caribbean. The Cuba Reader: History, Culture, Politics, edited by Aviva Chomsky, Barry Carr, Alfredo Prieto & Pamela Maria Smorkaloff (Durham NC: Duke University Press, 2019, paper US$32.95), is an expanded second edition of the 2003 work we praised in Bookshelf 2004.With more than twenty new selections exploring the post-Fidel years, this thick volume continues to be an excellent introduc- tion to the island and its people. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 101 United States Reconstruction across the Americas, edited by William A. Link (Gainesville:UniversityPressof Florida,2019,clothUS$34.95),consistsof three chapters, the first concerning the United States and Brazil, the second the United States and Mexico, and the third, by Edward B. Bookshelf 2019 Rugemer, the effects of newsaboutJamaica’sMorantBayRebellionof 1865onRadicalRepublicanjour- nalistsandlegislatorsintheUnitedStates,astheydealtwithwhitesupremacist efforts to undermine the recently enacted Emancipation. Beryl McBurnie (Kingston: University of the West Indies Press, 2018, cloth US$25.00) is Trinidadian journalist Judy Raymond’s brief, dutiful, but affec- tionate portrait of the anticolonialist dancer who championed her country’s Afro traditions (as well as those of the rest of the Caribbean, from the “talking drums” of Suriname to Martinique’s “belé” and beyond) and founded the Lit- tle Carib Theatre in Port of Spain (which also housed Derek Walcott’s Theatre Workshop), and whose work and influence spanned the whole second half of the twentieth century. y In Sons of the Soil: The Maingots and French Creoles in Trinidad History (CreateSpace, 2019, paper US$19.95), prolific Caribbeanist Anthony P. Main- got delves deep into his family’s history, beginning with the 1776 voyage of an ancestor from Bordeaux to Martinique and, ten years later, to SpanishTrinidad. The English conquest and the growth of slave plantations led, in the early nine- teenth century, to “a French culture tinged withWest African influences … [and a] French Creole identity known for its congenial life style, its music, cuisine and piquant sense of humor. So was Carnival and Calypso born.” Conceived as a gift to his family, the book contains a wealth of local history (understandably, a bit French Creole-centric). Afro-Catholic Festivals in the Americas: Performance, Representation, and the Making of Black AtlanticTradition (University Park: Pennsylvania State Univer- sity Press, 2019, cloth US$89.95), edited by art historian Cécile Fromont, con- sists of eight chapters that range from New Orleans to Brazil (with half on the latter) and only one on the Caribbean. Dianne M. Stewart’s dense chapter, “The Orisa House that Afro-Catholics built: African Antecedents toYoruba Religious Formation in Trinidad,” will be of interest to all scholars of Caribbean religion, as it criticizes the assumptions and methods of both historians and anthropol- ogists and raises questions that stretch well beyond Trinidad or Shango. My Father Is No Longer There (Vieux Fort, St-Lucia: Jako Books, 2019, paper US$24.95) is a memoir by St. Lucian Anderson Reynolds centered on the recent death of his father, a respected Adventist elder and beekeeper. About Reynolds’s 2017 novel, The Stall Keeper, we wrote that “the strong very local color somewhat makes up for the prosaic writing.” This time, there’s less local color and more inner torment. Unfortunately, the writing remains rather flat. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 New West Indian Guide 94 (2020) 82–111 New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons org/licenses/by-nc/4 0/ 102 review articles Zwazo, récit de vie d’un prêtre hindou commandeur d’habitation à la Mar- tinique, by Gerry L’Étang & Victorien Permal (Paris: HC Éditions, 2018, paper €12.50), is a wonderful little book that begins with a lyrical homage by anthro- pologist Jean Benoist, who first met Zwazo in 1957 and, with his help, made a documentary film about Hindu religious practice in Martinique. Becoming the go-to interlocutor on all things Indian, and the last real Tamil-speaker of the island (nineteenth-century, immigrant version), Antoine Tangamen a.k.a. Zwazo (1902–92) even made a cameo appearance in Naipaul’s The Middle Passage. Martiniquan anthropologist Gerry L’Étang met him in 1980, while doing doctoral research on the Hindu population of Martinique and, between 1986 and 1990, conducted the interviews (along with Guadeloupean Victorien Permal) that make up the bulk of this work. The book is presented as a first- person, chronological life history, beginning with the departure of the nar- rator’s grandmother from India. (After the abolition of slavery, some 25,000 Indians—90 percent Tamils, with only 15 percent Muslims—were transported to Martinique as indentured laborers, the majority through the French port of Pondichéry,withmostendingupinthenorthof theisland.)Zwazopaintsapic- tureof plantationlifeduringthewholecourseof thetwentiethcentury—cane- cutting (he eventually became a commandeur [“overseer” or “foreman”] in the fields), labor strikes and trials, and his life as a Hindu priest who sang and spoke with the ancient (now creolized) gods. Extensive historical and ethnographic notes enhance the text, which is followed by annexes that add comparative materials as well as relevant historical texts. Madeleine Jouye de Grandmaison has chronicled her life as a Martiniquan activist in Une Voix pour le Nord: Le Nord mon terroir, La Martinique mon pays (Saint-Denis La Plaine, France: Seed Publications, 2018, paper €27.00). Back in 1987, as we were preparing to move to Martinique from Paris, Michel Leiris told us that the one person we should be sure to meet was Madeleine, then a cru- cial player in Aimé Césaire’s political world. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 For anyone curious about what it takes to promote a region—culturally, politically, touristically—, this is a rich archive (225 oversized pages plus a USB key), brimming with evidence of the manydimensionsthatpromotionalactivismdemands,fromletters,maps,peti- tions, protests, speeches, and quiet diplomacy to archival research and, above all, a caring, passionate commitment. Serving in political positions as diverse as long-time vice-president of the Conseil Régional, president of the Comité Martiniquais du Tourisme, and Députée Européenne, she has worked tirelessly for her beloved island. In La forge de Zobel: Textes et reportages parus dans Le Sportif de Fort-de- France de 1938 à 1959 (Paris: Scitep, 2019, paper €22.00), literary critic Charles W. Scheel has gathered together some 200 pages of little-known writings first New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ 103 review articles published in a sports-oriented Martiniquan newspaper that had the courage to print these often-fascinating literary works by this friend and student of Césaire, better known for such novels as La Rue Cases-Nègres and Diab’-là. Prefaced with an interesting personal reflection by Zobel’s daughter Jenny, these stories, journalistic reports, and memoirs add measurably to the Zobel archive, as well as to an understanding of the continuing meaning of Mar- tinique to members of Zobel’s generation who, like him, moved on to the metropole. Élie Stephenson (Paris: Les Éditions du Manguier, 2018, paper €12.00), by Lydie Ho-Fong-Choy Choucoutou & Monique Dorcy, is an oversized, 56-page publication in the Collection Orénoque, which is intended to introduce French Guianeseauthors.ThisonefollowsothersdevotedtoLéon-GontranDamasand Alfred Parépou, effectively situating Stephenson—poet, playwright, novelist, and media commentator—within the literary and political history of post- WorldWar II Guyane, and includes several pages of interviews with the author, as well as numerous period illustrations. TheIslandof Lace:DrawnThreadworkonSabaintheDutchCaribbean,byEric A. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Eliason with photographs by Scott Squire (Jackson: University of Mississippi Press, 2019, cloth, US$40.00), is a beautiful, extended homage to the women responsible for making Saba known as “The Island of Lace.” First clarifying the essential difference between lace made elsewhere (built up from threads) and on Saba (made by removing threads from cloth), it offers a rich history of the art (including its commercial aspects since its beginnings in the early twenti- eth century), illustrates hundreds of named patterns, and ends with essays on individual “Saba Lace Ladies” working today. In Schoolboy: Memories of My Early Years in Wartime (Philipsburg, St. Maar- ten: House of Nehesi, 2018, paper, US$15.00), Gerard van Veen—a former priest who came to the Caribbean in 1961, living in Aruba and St. Maarten, where he wrote newspaper articles and served as probation officer and univer- sity lecturer—recounts his Dutch childhood, living in German-occupied Alk- maar. Encountering—Retracing—Mapping: The Ethnographic Legacy of Heinrich Harrer and Peter Aufschnaiter, edited by Mareile Flitsch, Maike Powroznik & Martina Wernsdörfer (Zurich, Switzerland: Ethnographic Museum, Univer- sity of Zurich/Stuttgart, Germany: Arnoldsche Art Publishers, 2018, US$70.00), includesa30-pagechapterbycuratorMaikePowroznik(withacontributionby Saamaka-born Vinije Haabo) about Harrer’s recently rediscovered collection of hundreds of Saamaka woodcarvings, vacuumed up during an 11-day stay in 1966. Famed in Germany and Austria as a mountain climber and explorer (par- ticularly in Tibet), Harrer, who once received a medal from Hitler, somehow New West Indian Guide 94 (2020) 82–111 104 review articles managed to acquire these objects—food stirrers, paddles, peanut-grinding boards, combs, and drums—during his very brief stay, accompanied by cam- eraman Herbert Raditschnig (who was filming for German television), Belgium KingLeopoldIII,andvariousDutchcolonialofficials.Thechapterdiscussesthe 1966 visit, includes excellent color photos of many of the pieces, and describes the ongoing collaboration between the Zurich museum and the fledgling Saa- maka Maroon Museum in Pikiseei, on the Suriname River. Shadows in Suriname, by Margaretta Pos (Lindisfarne, Tasmania: Forty South, 2016, paper, AUD 25.00), consists of vignettes—mostly by the author (a Tasmanian writer/journalist), but a couple by her more famous father Hugo Pos (the late Suriname-born writer and jurist)—that trace their complex fam- ily relations as well as her own brief visits to Suriname as an adult to explore her Jewish ancestry. Despite the emotional meaning to the author, her account of the country remains at travelog level. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 What the Oceans Remember: Searching for Belonging and Home, by Sonja Boon (Waterloo ON: Wilfrid Laurier University Press, 2019, cloth US$27.99), is another highly personal (often intimate) memoir but a far more interest- ing one. The author, formally a professional flautist but now a feminist uni- versity professor in Canada, tells us that her father’s family came from Bra- bant but, speaking of her mother’s, that “Sometime around 1935 [in Suriname], my Catholic grandmother with her Chinese name and her Hindu indentured ancestors met my grandfather, the child of a German man and his Creole con- cubine and the descendant of enslaved Africans, and fell in love.” Her book recounts her efforts to delve deeply into her Suriname roots, which began with nineteenth-century African plantation slavery and Chinese and Indian indentured labor. Sophisticated musings about research in multiples archives, walking the streets on several continents, interviewing far-flung relatives, and engaging in constant self-questioning about identity, race, origins, and belong- ing make this a rewarding read, as Boon is unafraid to write about what the experiences of diverse ancestors might have been like, even while questioning her right to attempt this act of historical imagination. After two decades in New York working as a cinematographer, Milton Kam returned to his native Suriname to photograph its indigenous peoples. The resulting book, Points of Recognition: Suriname’s Indigenous Peoples in the 21st Century (Dedemsvaart, the Netherlands: Kapelka Books, 2019, paper €19.99), presents beautiful color photos with informative captions. People are identi- fied by name, the many villages visited are briefly discussed, and contempo- rary issues (such as struggles with the national government for land rights and education) are made clear. Individuals are shown in their complex present- day realities, whether gardening or celebrating festivals or fishing in mercury- New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ 105 review articles polluted rivers or studying at boarding schools in the capital far from their families. Published with the cooperation and collaboration of VIDS, the offi- cial organization of indigenous peoples in Suriname, this is a modest, but admirable work. Calling this next book shameless plagiarism might be too kind. Sur les traces de Boni: Histoires de marronnages. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Suivi de l’émergence d’un peuple, by Collec- tif Mama Bobi (Matoury, Guyane: Ibis Rouge, 2019, paper €28.00), claims to present Aluku history from an Aluku perspective. The bibliography lists some twenty works but the footnotes refer to many dozen more sources (in par- tial form, such as “Genovese 1975”) that are nowhere listed or spelled out—we noticed, for example, frequent cryptic references to Price 1976, 1982, 1983 and 1990 in the footnotes but the only “Price” reference in the bibliography is “1973” (Maroon Societies). More troubling is the very close reproduction/translation of work by Wim Hoogbergen (particularly, The Boni Maroon Wars in Suriname [orig. 1990]), which makes up the bulk of the book, generally using the old trick of citing the archival sources from Hoogbergen’s writing but not the fact that they were taken from him. (And he is not the only scholar quietly plagiarized.) This hotchpotch of a text may offer some young Alukus a taste, in French, of theirpeople’searlyhistory,butitisashamethatthismustsubstituteforhonest, scholarly work. (The collective that produced this book received considerable funds some years ago from the State to produce a French translation of Hoog- bergen’s book, which was never accomplished; this publication is hardly what the grantors would have expected.) In Les Bushinengué de Guyane: Rites et croyances autour du textile (Paris: L’Harmattan, 2017, paper €20.00), Gabonese Landri Ekomie-Obame makes a well-meaning attempt to explain the life of Aluku Maroons, with whom he spent four years as a schoolteacher. Placing traditional clothing (especially what he calls the “nay pangi” [decorated skirt-cloth]) at the center of every- thing from childbirth, menstruation, marriage, funeral rites, and relations with ancestors,hisanalysissuffersfromfancifuletymologiesandconfusedcitations, as well as a feeble grasp of the most basic elements of Maroon textile arts, such as the distinction between embroidery and patchwork or the fact that Maroon kamisas are loincloths, not shirts or capes. Jean Moomou’s preface is politely on-target in suggesting that the book would have benefitted from more care with ethnographic facts and a clearer historical perspective—Ekomie-Obame proposes, largely on the basis of visual resemblances, that most of Maroon cul- ture derives directly from that of West and Central Africa. A Global History of Runaways: Workers, Mobility, and Capitalism, 1600–1850, edited by Marcus Rediker, Titas Chakraborty & Matthias van Rossum (Berke- ley: University of California Press,2019,paper US$34.95), is a curiouscollection New West Indian Guide 94 (2020) 82–111 New West Indian Guide 94 (2020) 82–111 via Open Access. Bookshelf 2019 This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons org/licenses/by nc/4 0/ 106 review articles of papers first presented at 2015/2016 conferences in Amsterdam and Pitts- burgh.Theaimislaudable:bringingtogetheranalysesof oneformof resistance (“running away”) by “workers of all kinds—slaves, indentured servants, con- victs, domestic workers, soldiers, and sailors … during global capitalism’s long ascent from 1600 to 1850.” But only three of the eleven chapters concern the Caribbean and they are strictly circumscribed (the Danish West Indies, 1672– 87; the Leeward Archipelago, 1627–1727; and the British Caribbean, 1808–28), with the rest on Bengal, Australia, the Cape of Good Hope, and more. Com- pletely unmentioned are Suriname Maroons, Jamaican Maroons, and the other important cases one might expect in such a collection (though the cover of the book does feature J.G. Stedman’s “rebel Negro armed & on his Guard”). We welcome a new edition of Michèle-Baj Strobel’s Lesgensdel’or:mémoire des orpailleurs créoles du Maroni (Guyane) (Paris: Plon, 2019, paper €27.00), now in the prestigious Terre Humaine collection. Peter Redfield reviewed its initial publication two decades ago in NWIG 74(3&4); the expanded edition has just won the Prix du livre d’histoire des outre-mer 2020. This remarkable work about the final generation of Creole goldminers from St. Lucia and neigh- boring islands who made their lives in the hinterlands of Guyane has a new introduction, bringing the situation up to the present, as well as the original preface by Richard Price. Once again, Rosemarijn Hoefte has provided an overview of recent (mostly—) Dutch-language books that may be of interest to our readers: Starting with Suriname, IndianenvanSuriname:Bruggenbouwersenerfgoed- dragers van een vergeten volk, text by Petra Nelstein, photographs by Diederik van Goethem (Volendam, the Netherlands: LM Publishers, 2019, paper €19.50), aims at a Dutch general audience. In 21 short chapters, readers get a poorly- written crash course on the history, culture, society, and spirituality of indige- nous people in the Para district. The book is based on a recent expedition that was inspired by the discovery of the manuscript of the Penard brothers (Frederik Paul and Arthur Philip) during the renovation of the Dutch National Museum of Ethnology in Leiden in 2010 (according to the Museum’s website) or 2011 (according to the book). The link to the Penard manuscript is weak at best. Enjoy the book for the photographs. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 The source publication Ten exempel van anderen: De processen tegen opvarenden van de piratenschepen Trompeuse en Resolution in Suriname en op St.Thomasin1684 (Zutphen, the Netherlands:WalburgPers, 2019, cloth €27.50) details the voyages of these pirate ships, the conditions on board, and the con- stantly changing composition of its crews, as well as the four court cases (three in Paramaribo, one on St. Thomas). In the substantial introduction, the edi- tors Karwan Fatah-Black and Aart Ruijter argue that the Trompeuse’s captain, New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 107 Jean Hamlin, initiated a new era, as he was one of the first to raid outside the Caribbean by sailing to West Africa and North America. De mensen van Vossenburg en Wayampibo: Twee Surinaamse plantages in de slaventijd (Hilversum, the Netherlands: Verloren, 2019, paper €25.00), by Bert Koene, is a history of two plantations in Suriname in the eighteenth and nine- teenth centuries, based on documents by Dutch owners Gerard de Vree and the Brantsen family. It is an interesting addition to the publications on the eco- nomic history of plantation Vossenburg by Humphrey Lamur, published in the 1980s and 1990s. It includes lists of the enslaved population of Vossenburg in 1705, 1728, 1744, 1760, 1784, 1852, and 1863 and of Wayampibo in 1727, 1760, and 1784. A small gem: Tenen van de leguaan: Verhalen uit de wereld van Surinaamse leprapatiënten. Over lepra, slavernij, kolonialisme en de Surinaamse ecologie (Volendam: LM Publishers, 2019, paper €24.50), by Henk Menke,Toine Pieters, Melinda Reyme & Jack Menke, is a truly interdisciplinary study of leprosy. Dividedintofourparts,thevolumediscussesthehistoryof colonialism,slavery, and the fear of leprosy, followed by ten oral histories of leprosy sufferers. The two final parts analyze stigmas, taboos, discrimination, and (religious) treat- ments among the different population groups as well as the current approach to the disease. This richly illustrated book offers a non-European perspective on the evil that locals called boasie; it includes short summaries in English, Por- tuguese, and Spanish. Moving fully to the twentieth century. Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Op zoek naar Papa Koenders: Over een strijder voor emancipatie in het koloniale Suriname (Volendam: LM Pub- lishers, 2019, cloth €19.50), by Jules Rijssen, Roy Wijks & Andre Reeder, is slightly uneven but includes some fascinating chapters (3, 6, 7) with reminis- cences by people who were acquainted with the teacher Julius “Papa” Koenders (1886–1957). Koenders was an advocate for Afro-Surinamese culture in general and Sranan Tongo in particular. His life and activities, especially his editor- ship of the influential magazine Foetoe-boi, are highlighted in the volume’s first half. The interviews with people who knew him give a lively sketch of Afro- Surinamese sociocultural life in Paramaribo in the 1950s as well as the cultural and political nationalist movement in the 1950s and 1960s. The book includes a CD with texts and songs performed by Randy Kersout-Gunning. After 50 years away, sociologist Herman Vuijsje returned to Suriname where he did his MA research on multireligiosity. God zij met ons Suriname: Religie als vloek en zegen (Zutphen, the Netherlands: WalburgPers, 2019, paper €19.99) focuses on the pervasiveness of both religion and fear in the country. This fast-paced, journalistic account zooms in on new religious developments; the author is especially fascinated by the emancipation of Winti. New West Indian Guide 94 (2020) 82–111 108 review articles Bruynzeelwoningen in Suriname, met herinneringen van Kees Tempelaar, by Karolien Janssens, Marte Wierenga & Dirk Laporte (Volendam: LM Publish- ers, 2019, cloth €24.50), details the history of the iconic, modern Bruynzeel homes made from Suriname timber between 1955 and 1990. These precut pre- fab houses were also exported, for example to Grenada after Hurricane Janet in 1955.This richly illustrated volume includes the extensive memories of a for- mer Dutch employee, Kees Tempelaar, and thus doubles as a company history of Bruynzeel in the postwar era. For specialists. In Suriname there seems to be a mini-boom in autobiographical and family histories. K.R. Sing’s Uit de klei van Saramacca: Een Surinaamse familiegeschie- denis (Amsterdam: Boom, 2019, paper €20.00) is one of the better of the crop. Thiswell-writtenstoryof emancipation,wartsandall,featurestheauthor’spar- ents Nandoe and Trees Sing or Raghoebarsing. It is based on archival research plus the family archives including letters (not all of them very interesting), photos, and official documents. The family history of teacher Trees and tailor Nandoe also gives a good impression of socioeconomomic and political devel- opments in Suriname since the abolition of Indian indenture. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 On to fiction: Arend van Dam’s De reis van Syntax Bosselman: Verhalen over de slavernij, with illustrations by Alex de Wolf (Amsterdam: Van Holkema & Warendorf, 2018, cloth €16.99), is a “factional” book on Dutch colonialism, slav- ery, and abolition in Suriname, and the exposition of 28 Surinamers at the World Expo in Amsterdam in 1883. The clever design of this prize-winning children’s book allows it to be read at different levels. It includes the author’s personal notes about his exploration of these difficult histories. AstridRoemer’sGebrokenWit (Amsterdam:Prometheus,2019,paper€19.99) is a family history situated between World War II and Suriname’s indepen- dence. Alternating between Paramaribo and the Netherlands, this complex saga of three generations, featuring the female line, shows how the two coun- tries are inextricably linked through personal ties. Despite the fact that the spelling and punctation are sometimes peculiar, this novel grew on me. Read- ing it while watching sunrises on the Suriname River may have helped. Ruben Gowricharn’s De prijs van geluk (Soesterberg, the Netherlands: Uit- geverij Aspekt 2019, paper €22.50) is a novel about Hindostani migration from Suriname to The Hague and the integration of the protagonists in Dutch soci- ety. “Migration is a permanent social amputation.” In the end, the socioeco- nomic success of the couple’s three children cannot compensate for the empti- nesstheyfelt.Inconveyingthismessage,thevoiceof thesociologistsometimes overshadows that of the novelist. Mireille Geus’s Madame Jeanette (Amsterdam: Nw Amsterdam, 2019, paper €20.00) is a semi-autobiographical novel on abandonment and Surinamese cooking as therapy. A quick read. New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ 109 review articles This year we have received only three books on Curaçao. Landhuizen van Curacao: Juwelen uit het verleden (Willemstad: Stichting Curaçao Style/Volen- dam: LM Publishers 2019, cloth €45.00) is a magnificent full color coffee table book presenting 78 plantation houses on the island. Michael A. Newton dis- cusses their history and architecture, Carel de Haseth records memories of (previous) inhabitants, and François van der Hoeven looks at the ruins of plan- tation houses. The main text, describing the history of all surviving mansions, is by Jeannette van Ditshuizen. Also available in English: Plantation Houses of Curaçao: Jewels from the Past. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Three Caribbean Artists: José Maria Capricorne, Nelson Carrilho, Philippe Zanolino, by Marianne de Tolentino, Susan Wilczak & Monique van Herksen (Volendam: LM Publishers, 2019, paper €29.50), begins with a brief introduc- tion to Caribbean art and the “Dutch” Caribbean islands and then discusses the magical reality of Capricorne (b. 1932), the art and vision of Carrilho (b. 1953), and the spirituality of Zanolino (b. 1960). Capricorne and Carrilho were born in Curaçao, while French-born Zanolino migrated to the island in 1986. The book includes some 100 illustrations in color. Eddy Wegman’s Photographs of post war Curaçao, 1945–1963 (Volendam: LM Publishers, 2019, cloth €17.50) contains a selection of more than 80 b/w pho- tos by this amateur photographer, who was born in the Netherlands and left for Curaçao in 1945, to work for the Shell Oil Company. The portraits in particular are forceful. Andfinally,onebookonthenon-DutchCaribbean.MarcelCatsburg’s,Grond zonderrust:BreuklijneninHaitiaansebodem(Soesterberg,theNetherlands:Uit- geverij Aspekt, 2019, paper €28.95) is a history of Haiti, inspired by the 2010 earthquake. This well-written account is based on extensive study of literature published in French and English as well as the author’s intimate knowledge of the country, where he has lived and worked for five years. We end this year’s Bookshelf by listing information on titles that we have noticed but neither examined nor requested for review—in some cases be- cause their Caribbean content is restricted to a chapter or two, in others because they didn’t seem sufficiently compelling given NWIG space limita- tions, or for a variety of other reasons.Together, they testify to the large number of books being published that at least touch on the Caribbean. CreoleComposition:AcademicWritingandRhetoricintheAnglophoneCaribbean,edited by Vivette Milson-Whyte, Raymond Oenbring & Brianne Jaquette (Anderson SC: Parlor Press, 2019, paper US$36.00) Approaches to Teaching the Works of Edwidge Danticat, edited by Celucien L. Joseph, New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 110 review articles Suchismita Banerjee, Marvin E. Hobson & Danny M. Hoey Jr. (London: Routledge, cloth US$140.00) Suchismita Banerjee, Marvin E. Hobson & Danny M. Hoey Jr. (London: Routledge, cloth US$140.00) Suchismita Banerjee, Marvin E. Hobson & Danny M. Hoey Jr. Bookshelf 2019 (London: Routledge, cloth US$140.00) Chula, by Amanda Alcantara (self-published, 2019, paper US$14.99) [“an imaginative bilingual collection of intimate poems, short stories, memories and vignettes about the life of a Dominicana before and after moving to the United States”] Operación Funámbula: Antología personal (1973–2018), by Áurea María Sotomayor (Madrid: Amargord Ediciones, 2019, n.p.) [poetry] Operación Funámbula: Antología personal (1973–2018), by Áurea María Sotomayor (Madrid: Amargord Ediciones, 2019, n.p.) [poetry] Three Hundred Years of Decadence: New Orleans Literature and the Transatlantic World, by Robert Azzarello (Baton Rouge: LSU Press, 2019, cloth US$45.00) Three Hundred Years of Decadence: New Orleans Literature and the Transatlantic World, by Robert Azzarello (Baton Rouge: LSU Press, 2019, cloth US$45.00) The Dictator Novel: Writers and Politics in the Global South, by Magalí Armillas-Tiseyra (Evanston IL: Northwestern University Press, 2019, paper US$34.95) La tinta roja: Estudios sobre literatura de Puerto Rico, by Miguel Ángel Náter (San Juan: Editorial Tiempo Nuevo, 2019, paper US$40.00) Hijosdelsilencio:Ensayos,byJoséAlcántaraAlmánzar(SanJuan:IslaNegra,2018,paper n.p.) Discourses from Latin America and the Caribbean: Current Concepts and Challenges, editedbyEleonoraEsposito,CarolinaPérez-Arredondo&JoséManuelFerreiro(Bas- ingstoke, U.K.: Palgrave Macmillan, 2019, cloth US$149.99) News Media Coverage of Environmental Challenges in Latin America and the Caribbean: MediatingDemand,DegradationandDevelopment,editedbyBrunoTakahashi,Juliet Pinto, Manuel Chavez & Mercedes Vigón (Basingstoke, U.K.: Palgrave Macmillan, 2019, paper US$84.99) From the Tricontinental to the Global South: Race, Radicalism, and Transnational Soli- darity, by Anne Garland Mahler (Durham NC: Duke University Press, 2018, paper US$27.95) Caribbean Legion: And Its Mercenary Air Force, 1947–1950, by Dan Hagedorn & Mario Caribbean Legion: And Its Mercenary Air Force, 1947–1950, by Dan Hagedorn & Mario Overall (Warwick, U.K.: Helion and Company, 2019, paper US$29.99) Overall (Warwick, U.K.: Helion and Company, 2019, paper US$29.99) Overall (Warwick, U.K.: Helion and Company, 2019, paper US$29.99) Disciplining Coolies: An Archival Footprint of Trinidad, 1846, by Amar Wahab (Berlin: Peter Lang, 2018, paper US$52.94) Disciplining Coolies: An Archival Footprint of Trinidad, 1846, by Amar Wahab (Berlin: Peter Lang, 2018, paper US$52.94) MotherCountry:RealStoriesof theWindrushChildren, by Charlie Brinkhurst-Cuff (Lon- don: Headline, 2019, cloth US$26.99) Fundador de la República: Federico Pérez Carbó y sus combates por la independencia de Cuba (1855–1901), by José L. Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Bolívar Fresneda & Rafael Ángel Simón Arce (n.p.: Cre- ateSpace, 2017, paper US$29.99) The Urban Gardens of Havana: Seeking Revolutionary Plants in Ideologized Spaces, by Ola Plonska & Younes Saramifar (Basingstoke, U.K.: Palgrave Pivot, 2019, cloth US$59.99) WakinginHavana:AMemoirof AIDSandHealinginCuba,byElenaSchwolsky(Phoenix AZ: She Writes Press, 2019, paper US$16.95) Last Seasons in Havana: The Castro Revolution and the End of Professional Baseball New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ New West Indian Guide 94 (2020) 82–111 via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ review articles 111 in Cuba, by César Brioso (Lincoln: University of Nebraska Press, 2019, cloth US$ 29.95) in Cuba, by César Brioso (Lincoln: University of Nebraska Press, 2019, cloth US$ 29.95) Cuba Talks: A New Perspective on Cuban Art Now, edited by Laura Salas Redondo & Jérôme Sans (New York: Rizzoli, 2019, cloth US$80.00) Artifacts of the Spanish Colonies of Florida and the Caribbean, 1500–1800: Volume 2: Portable Personal Possessions, by Kathleen Deagan (Washington DC: Smithsonian Books, 2019, cloth US$60.00) TheCaribsof St.Vincent,byFr.AdrienleBretonS.J.(apparentlypublishedinKingstown, St. Vincent, paper EC$20.00) [translation of the 1982 French edition published by St. New West Indian Guide 94 (2020) 82–111 Downloaded from Brill.com 10/24/2024 04:12:08AM via Open Access. This is an open access article distributed under the terms of the CC BY-NC 4.0 license. https://creativecommons.org/licenses/by-nc/4.0/ Bookshelf 2019 Vincent, paper EC$20.00) [translation of the 1982 French edition published by the Historical Society of Martinique] “In theVortex of the Cyclone”: SelectedPoems by Excilia Saldaña (Gainesville: University the Historical Society of Martinique] “In theVortex of the Cyclone”: SelectedPoems, by Excilia Saldaña (Gainesville: University Press of Florida, 2019, paper US$19.95) “In theVortex of the Cyclone”: SelectedPoems, by Excilia Saldaña (Gainesville: University Press of Florida, 2019, paper US$19.95) You Can Cross the Massacre on Foot, by Freddy Prestol Castillo (Durham NC: Duke Uni- versity Press, 2019, paper US$22.95) You Can Cross the Massacre on Foot, by Freddy Prestol Castillo (Durham NC: Duke Uni- versity Press, 2019, paper US$22.95) Civil Society Organisations, Governance and the Caribbean Community, by Kristina Hinds (Basingstoke, U.K.: Palgrave Macmillan, 2019, cloth US$84.99) Civil Society Organisations, Governance and the Caribbean Community, by Kristina Hinds (Basingstoke, U.K.: Palgrave Macmillan, 2019, cloth US$84.99) Cacicazgos en el Caribe y el continente americano, by Francisco Moscoso (San Juan: Edi- ciones Puerto, 2019, paper US$11.95) Development, Political, and Economic Difficulties in the Caribbean, edited by Ann Marie Bissessar (Basingstoke, U.K.: Palgrave Macmillan, 2019, cloth US$119.99) [diverse subjects, with seven chapters on Trinidad & Tobago, the others on various islands] Memoir of a Cocoa Farmer’s Daughter: From Caribbean Rural Development Activist to Rural Entrepreneur, by Regina Dumas (Kingston: Arawak, 2018, paper US$35.00) Reducing Disaster Risks: Progress and Challenges in the Caribbean Region, edited by Ian Davis,SteveBender,FredKrimgold&FranklinMcDonald(London:Routledge,2020, cloth US$130.00) Trésors cachés et patrimoine naturel de la Martinique vue du ciel, by Anne Chopin (pho- tos) & Patrick Chamoiseau (text) (Paris: HC Editions, 2019, cloth €28.50) [new edi- tion of a 2007 photo book] Land, Labour, and Power in a Colonial Catholic Mission in Trinidad, by Maximilian C. Forte (Alert Press [self-published], 2019) New West Indian Guide 94 (2020) 82–111
https://openalex.org/W3007293658	https://europepmc.org/articles/pmc7033215?pdf=render	English	null	Transcriptome analysis of ankylosed primary molars with infraocclusion	International Journal of Oral Science/International journal of oral science	2,020	cc-by	9,031	INTRODUCTION calciﬁcation of infancy (GACI) found increased cementogenesis and reported histories of infraocclusion and ankylosis.15 On the other hand, in a staining study, bone from ankylosed human primary molars demonstrated no difference in the expression of NADH-diaphorase, acid phosphatase, and alkaline phosphatase.16 Dental ankylosis is deﬁned as a fusion of cementum or dentine with alveolar bone.1 Primary molar ankylosis can cause severe clinical consequences in the growing child, including:1–4 (1) Tooth infraocclusion and vertical bone defect, (2) Tipping of adjacent teeth into the space of infraocclusion, causing loss of arch space, dental asymmetry, midline deviation,4 and impaction of the ankylosed tooth and its successor, (3) Supra-eruption of opposing teeth, and (4) Deﬂected path of eruption of successors, with displacement in the form of tipping and ectopic eruption of successors. The above studies suggest that animal models can provide useful insights into the functions of selected proteins and pathways, but are not representative of the ankylotic process in humans, which is unique in that it is not induced by trauma, is seemingly idiopathic and spontaneous, and has high prevalence (e.g. 22% in Finland17 and 38% in Israel18) that cannot be explained by rare genetic mutations. So while these candidate gene and protein approaches can be informative, they are hampered by scale and scarcity of established candidates. Thus, it is timely to approach the problem using human transcriptome-wide analysis. In this study, RNA sequencing (RNA-seq) was used to characterize and compare the transcriptome proﬁles of primary molars with and without infraocclusion. Following analysis, a small number of differentially expressed (DE) genes were examined for their protein distribution in developing tooth germs. Despite the abundance of clinical and epidemiological data, little is known about the molecular correlates of primary molar ankylosis. Genetic associations have been proposed, mostly based on epidemiological data from familial, ethnicity, and dental anomaly pattern studies.5–10 However, despite compelling evi- dence for strong familial and ethnic associations, no candidate genes or molecular pathways have been identiﬁed. A number of studies have tested hypotheses of molecular pathways of ankylosis. Prime among them is the dysregulation of proteins involved in hard tissue turnover. International Journal of Oral Science International Journal of Oral Science International Journal of Oral Science www.nature.com/ijos ARTICLE OPEN Transcriptome analysis of ankylosed primary molars with infraocclusion Annie Tong 1, Yuh-Lit Chow2, Katie Xu1, Rita Hardiman1, Paul Schneider1 and Seong-Seng Tan2 Primary molar ankylosis with infraocclusion can retard dental arch development and cause dental asymmetry. Despite its widespread prevalence, little is known about its molecular etiology and pathogenesis. To address this, RNA sequencing was used to generate transcriptomes of furcal bone from infraoccluded (n = 7) and non-infraoccluded (n = 9) primary second molars, all without succeeding biscuspids. Of the 18 529 expressed genes, 432 (2.3%) genes were differentially expressed between the two groups (false discovery rate < 0.05). Hierarchical clustering and principal component analysis showed clear separation in gene expression between infraoccluded and non-infraoccluded samples. Pathway analyses indicated that molar ankylosis is associated with the expression of genes consistent with the cellular inﬂammatory response and epithelial cell turnover. Independent validation using six expressed genes by immunohistochemical analysis demonstrated that the corresponding proteins are strongly expressed in the developing molar tooth germ, in particular the dental follicle and inner enamel epithelium. The descendants of these structures include the periodontal ligament, cementum, bone and epithelial rests of Malassez; tissues that are central to the ankylotic process. We therefore propose that ankylosis involves an increased inﬂammatory response associated with disruptions to the developmental remnants of the dental follicle and epithelial rests of Malassez. ; https://doi.org/10.1038/s41368-019-0070-1 International Journal of Oral Science (2020) 12:7 ; https://doi.org/10.1038/s41368-019-0070-1 calciﬁcation of infancy (GACI) found increased cementogenesis and reported histories of infraocclusion and ankylosis.15 On the other hand, in a staining study, bone from ankylosed human primary molars demonstrated no difference in the expression of NADH-diaphorase, acid phosphatase, and alkaline phosphatase.16 The above studies suggest that animal models can provide useful insights into the functions of selected proteins and pathways, but are not representative of the ankylotic process in humans, which is unique in that it is not induced by trauma, is seemingly idiopathic and spontaneous, and has high prevalence (e.g. 22% in Finland17 and 38% in Israel18) that cannot be explained by rare genetic mutations. So while these candidate gene and protein approaches can be informative, they are hampered by scale and scarcity of established candidates. Thus, it is timely to approach the problem using human transcriptome-wide analysis. In this study, RNA sequencing (RNA-seq) was used to characterize and compare the transcriptome proﬁles of primary molars with and without infraocclusion. Following analysis, a small number of differentially expressed (DE) genes were examined for 1Melbourne Dental School, The University of Melbourne, Melbourne, Australia and 2Florey Institute of Neuroscience, The University of Melbourne, Melbourne, Australia Correspondence: Annie Tong (annie.c.tong@gmail.com) Received: 20 September 2019 Revised: 27 November 2019 Accepted: 15 December 2019 INTRODUCTION For example, descriptive studies of ﬁxed tissues suggest altered expression of osteoprote- gerin (OPG), receptor activator of nuclear factor kappa-β (RANK), and RANK ligand (RANKL) in a rat model of ankylosis induced by thermal trauma using dry ice.11 PDL space mineralization and dental ankylosis have been observed in animals with altered bone metabolism, for example in mutant mice with elevated Wnt signalling,12 in osteopetrotic mutant rabbits with reduced osteoclast-mediated bone resorption,13 and in mice injected with bisphosphonate.14 A study of ﬁve human subjects with loss-of- function mutations in ENPP1 associated with generalized arterial 1Melbourne Dental School, The University of Melbourne, Melbourne, Australia and 2Florey Institute of Neuroscience, The University of Melbourne, Melbourne, Australia C d A i T ( i t @ il ) RESULTS DE genes in bone tissue DE genes in bone tissue At cut-offs of FDR <0.05 (marked by green dotted e) and logFC of ≤−1 or ≥1 (marked by blue dotted lines), 356 genes were differentially expressed (marked by red dots): 253 genes had reased expression (red dots right of blue dotted line), and 103 genes had decreased expression (red dots left of blue dotted line) in th aoccluded group compared to the non-infraoccluded group. b Hierarchical clustering and heatmap of 432 DE genes (FDR < 0.05 aoccluded (D1–D7) and non-infraoccluded (C1–C9) samples clustered into their respective groups. Each row in the heatmap denotes on ne, with the expression level ranging from low (blue) to high (yellow); hence each gene’s consistency of expression across all samples can visualized. Gene clusters 1 and 2 denote the genes that had decreased and increased expression, respectively, in the infraoccluded group mpared to the non-infraoccluded group. c Principal component analysis was performed across all 18 529 identiﬁed genes. All infraoccluded –D7, coloured red) and non-infraoccluded (C1–C9, coloured blue) samples separated into their respective groups at approximately −0.1 in ncipal component 2 (PC2). Greater variation in the infraoccluded group compared to the non-infraoccluded group is evidenced by greate tter of infraoccluded samples on the PCA plot Decreased expression a 4 3 FDR=0.05 FDR<0.05 2 -2 0 2 4 6 8 -log10 FDR log2 fold change 1 5 0 -1 log–fold +1 log–fold Increased expression b 1 Infraoccluded Non-infraoccluded Color key and histogram Count 300 0 -2 0 Value 2 FDR<0.05 c 0.4 0.2 0.0 -0.2 D2 D7 D1 D6 D5 D3 D4 C8 C2 C3 C1 C6 C7 C5 C4 C9 -0.2 0.0 0.2 0.4 PC 1 (25.78%) 0.6 0.8 -0.4 PC 2(15.04%) C1 C2 C3 C9 C5 C4 C6 C7 C8 D1 D3 D6 D4 D2 D7 D5 C1 C2 C3 C9 C5 C4 C6 C7 C8 D1 D3 D6 D4 D2 D7 D5 Fig. 1 Visualization of gene expression using volcano plot, hierarchical clustering, and principal component analysis. a All 18 529 genes were plotted as individual dots on a graph of log2 fold change (logFC) versus negative log10 FDR. DE genes in bone tissue After excluding genes with low counts (see the “Materials and methods” section), 18 529 genes were retained for further analysis, from which 432 genes (2.3%) were found to be differentially expressed between the two groups (false discovery rate (FDR) < 0.05) (Supplementary Table 1). Of the 432 DE genes, all except two had log2 fold change (logFC) of ≤−0.59 or ≥0.59 (i.e. exhibiting 1.5-fold Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. 2 change or more), and 356 genes had logFC of ≤−1 or ≥1 (i.e. twofold change or more) (Fig. 1a). Hierarchical clustering and heatmap analysis clustered all samples into their respective experimental and control groups, indicating clear differences between the two groups (Fig. 1b). Furthermore, there was greater variation between samples in the infraoccluded group compared to the non-infraoccluded group. This was evidenced by infraoccluded samples clustering at higher points on the dendrogram (the higher up the linkage between clusters occurs, the greater the difference between these clusters). The greater variation in the infraoccluded group was also demonstrated by Principal component analysis (PCA). The ﬁrst two principal components, which accounted for 40.82% of the total variance of the data, separated all samples into their respective two groups (Fig. 1c). p y p g g p g Decreased expression a b c 4 3 FDR=0.05 FDR<0.05 2 -2 0.4 0.2 0.0 -0.2 D2 D7 D1 D6 D5 D3 D4 C8 C2 C3 C1 C6 C7 C5 C4 C9 -0.2 0.0 0.2 0.4 PC 1 (25.78%) 0.6 0.8 2 1 Infraoccluded Non-infraoccluded Color key and histogram Count 300 0 -2 0 Value 2 -0.4 0 2 4 6 8 -log10 FDR PC 2(15.04%) log2 fold change 1 5 0 -1 log–fold +1 log–fold Increased expression C1 C2 C3 C9 C5 C4 C6 C7 C8 D1 D3 D6 D4 D2 D7 D5 1 Visualization of gene expression using volcano plot, hierarchical clustering, and principal component analysis. a All 18 529 genes wer tted as individual dots on a graph of log2 fold change (logFC) versus negative log10 FDR. DE genes in bone tissue Each node represents a signiﬁcantly enriched pathway (FDR < 0.05). Red nodes represent positive enrichment in infraoccluded samples (i.e. increased expression in infraocclusion), while blue nodes represent negative enrichment in infraoccluded samples (i.e. decreased expression in infraocclusion). Size of node represents number of genes enriching the pathway. Edges represent pathway overlap (i.e. genes common to both pathways), and width of edges represents size of overlap (i.e. number of genes common to both pathways) (overlap coefﬁcient > 0.25) Innate immune response Granulocyte migration Response to type I interferon Positive regulation of inflammatory response Inflammatory response Myeloid leukocyte migration Cell chemotaxis Leukocyte chemotaxis DNA packaging complex Positive regulation of response to wounding Defense response to bacterium Humoral immune response Inflammatory response Monosaccharide transport Protein DNA complex Defense response to other organism Central nervous system neuron differentiation Nuclear nucleosome Epithelial cell development Epithelial cell development Epithelial cell differentiation Keratinocyte differentiation Peptide cross linking Epidermis development Epidermal cell differentiation Intermediate filament cytoskeleton Desmosome Intermediate filament Structural constitute of cytoskeleton Cell cell adherens junction Cell cell junction Heterotypic cell cell adhesion Keratinization Homotypic cell cell adhesion Cell cell junction Heterotypic cell cell adhesion Cell cell adherens junction Myeloid leu migrati Keratinization Epidermal cell differentiation ss linking Epidermis de Response to type I interferon cyte ation Enzyme activation Enzyme activation Activation of cysteine type endopeptidase activity Zymogen activation Regulation of peptidase activity Oxidoreductase activity acting on the aldehyde or oxo group of donors Oxidoreductase activity acting on ch oh group of donors Regulation of peptidase activity Monosaccharide transport Protein DNA vation Fig. 2 Enrichment map of signiﬁcant GO pathways created using Cytoscape. Two main pathway clusters were found: (1) Epithelial cell differentiation, keratin expression, and cell junction activity, and (2) Inﬂammatory response. A third cluster related to non-speciﬁc enzyme activation. Each node represents a signiﬁcantly enriched pathway (FDR < 0.05). Red nodes represent positive enrichment in infraoccluded samples (i.e. increased expression in infraocclusion), while blue nodes represent negative enrichment in infraoccluded samples (i.e. decreased expression in infraocclusion). Size of node represents number of genes enriching the pathway. Edges represent pathway overlap (i.e. genes common to both pathways), and width of edges represents size of overlap (i.e. number of genes common to both pathways) (overlap coefﬁcient > 0.25) The results (summarized in Table 1) showed that the dental follicle was the most common site of protein expression, with ﬁve out of the six selected proteins involved. DE genes in bone tissue At cut-offs of FDR <0.05 (marked by green dotted line) and logFC of ≤−1 or ≥1 (marked by blue dotted lines), 356 genes were differentially expressed (marked by red dots): 253 genes had increased expression (red dots right of blue dotted line), and 103 genes had decreased expression (red dots left of blue dotted line) in the infraoccluded group compared to the non-infraoccluded group. b Hierarchical clustering and heatmap of 432 DE genes (FDR < 0.05). Infraoccluded (D1–D7) and non-infraoccluded (C1–C9) samples clustered into their respective groups. Each row in the heatmap denotes one gene, with the expression level ranging from low (blue) to high (yellow); hence each gene’s consistency of expression across all samples can be visualized. Gene clusters 1 and 2 denote the genes that had decreased and increased expression, respectively, in the infraoccluded group compared to the non-infraoccluded group. c Principal component analysis was performed across all 18 529 identiﬁed genes. All infraoccluded (D1–D7, coloured red) and non-infraoccluded (C1–C9, coloured blue) samples separated into their respective groups at approximately −0.1 in principal component 2 (PC2). Greater variation in the infraoccluded group compared to the non-infraoccluded group is evidenced by greater scatter of infraoccluded samples on the PCA plot International Journal of Oral Science (2020) 12:7 Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. 3 Epithelial cell differentiation Keratinocyte differentiation Peptide cross linking Innate immune response Epidermis development Epidermal cell differentiation Intermediate filament cytoskeleton Granulocyte migration Response to type I interferon Desmosome Intermediate filament Structural constitute of cytoskeleton Cell cell adherens junction Cell cell junction Positive regulation of inflammatory response Heterotypic cell cell adhesion Epithelial cell development Enzyme activation Inflammatory response Keratinization Myeloid leukocyte migration Homotypic cell cell adhesion Cell chemotaxis Leukocyte chemotaxis DNA packaging complex Positive regulation of response to wounding Defense response to bacterium Humoral immune response Inflammatory response Activation of cysteine type endopeptidase activity Zymogen activation Regulation of peptidase activity Monosaccharide transport Protein DNA complex Oxidoreductase activity acting on the aldehyde or oxo group of donors Defense response to other organism Oxidoreductase activity acting on ch oh group of donors Central nervous system neuron differentiation Nuclear nucleosome Fig. 2 Enrichment map of signiﬁcant GO pathways created using Cytoscape. Two main pathway clusters were found: (1) Epithelial cell differentiation, keratin expression, and cell junction activity, and (2) Inﬂammatory response. A third cluster related to non-speciﬁc enzyme activation. DE genes in bone tissue Three proteins (CK14, DSG3, TIMP2) were from the infraoccluded group and two proteins (POSTN and MMP2) from the control group. CK14 (over-expressed in infraocclusion) was strongly stained in the dental follicle (Fig. 4b) as well as in the inner enamel epithelium, stratum intermedium, stellate reticulum and ameloblast layer (Fig. 4c). DSG3 (over- expressed in infraocclusion) was also strongly stained in the dental follicle as well as in the ameloblasts layer (Fig. 4d). PKP1 (over- expresssed in infraocclusion), while strongly expressed in the dental follicle, was also expressed in the inner enamel epithelium and ameloblasts (Fig. 4e). In contrast, POSTN (under-expressed in infraocclusion) was stained solely in the dental follicle and not elsewhere in the dental organ (Fig. 4f). MMP2 (under-expressed in infraocclusion) was similarly active predominantly in the dental follicle but also in the stellate reticulum (Fig. 4g). Of the six proteins, only TIMP2 (under-expressed in infraocclusion) failed to be found in the dental follicle, being localized only to ameloblasts and odontoblasts (Fig. 4h). To identify speciﬁc biological pathways that may be linked to infraocclusion, Gene set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) were performed. GSEA produced 36 sig- niﬁcantly enriched gene ontology (GO) pathways (FDR < 0.05). Pathway size (i.e. number of genes in dataset enriching each pathway), overlap (i.e. number of genes in dataset common to two pathways), and positive/negative enrichment were separately mapped (Fig. 2). Two principal clusters were found, relating to (1) Epithelial cell differentiation, keratin expression, and cell junction activity, and (2) Inﬂammatory response. y y p IPA produced 20 signiﬁcantly activated or inhibited down- stream functions (z-score ≥1.7 or ≤−1.7) (Supplementary Table 2). The functions could be categorised into two broader functions: (1) Activation of inﬂammatory response and recruit- ment of leukocytes (Fig. 3a); and (2) Activation of epithelial cell proliferation and differentiation, and inhibition of epithelial cell movement (Fig. 3b). Immunohistochemical detection of proteins encoded by DE genes in tooth germs Immunohistochemical detection of proteins encoded by DE genes in tooth germs Immunohistochemistry was conducted to validate the spatial expression of DE genes in tooth structures. Of the six genes under consideration, three were over-expressed, and three were under- expressed, in infraocclusion. These genes were selected based on the criterion that antibodies to their proteins were commercially available, not because they exhibited the greatest fold-change or any other variable. DISCUSSION d h 4 Cytokine/growth factor Legend Enzyme G-protein coupled receptor Ligand-dependent nuclear receptor Peptidase Transmembrane receptor Transporter Other Function Gene involved in function (unclear effect) Differential expression leads to activation of function Differential expression inconsistent with activation of function Function likely activated Very increased differential expression Increased differential expression Decreased differential expression a 4 Legend Enzyme G-protein coupled receptor Gene involved in function (unclear effect) Differential expression leads to activation of function Differential expression leads to inhibition of function Differential expression inconsistent with predicted activation/ihhibition of function Very increased differential expression Increased differential expression Decreased differential expression Cytokine/growth factor Growth factor Peptidase Transcription regulator Transporter Other Function Function likely activated Function likely inhibited b b Legend Enzyme G-protein coupled receptor Gene involved in function (unclear effect) Differential expression leads to activation of function Differential expression leads to inhibition of function Differential expression inconsistent with predicted activation/ihhibition of function Very increased differential expression Increased differential expression Decreased differential expression Cytokine/growth factor Growth factor Peptidase Transcription regulator Transporter Other Function Function likely activated Function likely inhibited b Fig. 3 IPA found DE genes in infraocclusion. They were associated with a increased activation of inﬂammatory response and recruitment of leukocytes, and b increased activation of epithelial cell proliferation and differentiation, and inhibition of epithelial cell movement. Due to the hierarchical nature of classifying pathways, only the higher-level pathways are illustrated (lower pathways that are subsets of higher pathways are not shown separately) Fig. 3 IPA found DE genes in infraocclusion. They were associated with a increased activation of inﬂammatory response and recruitment of leukocytes, and b increased activation of epithelial cell proliferation and differentiation, and inhibition of epithelial cell movement. Due to the hierarchical nature of classifying pathways, only the higher-level pathways are illustrated (lower pathways that are subsets of higher pathways are not shown separately) From these genes, biological pathways relating to inﬂammatory response and epithelial cell turnover were predicted to be affected in ankylosis. In contrast with animal models of ankylosis, these ﬁndings open up new gateways for studying the pathogenesis of human molar ankylosis. From these genes, biological pathways relating to inﬂammatory response and epithelial cell turnover were predicted to be affected in ankylosis. In contrast with animal models of ankylosis, these ﬁndings open up new gateways for studying the pathogenesis of human molar ankylosis. International Journal of Oral Science (2020) 12:7 DISCUSSION d h To date there are no published studies on the transcriptome characteristics of primary molar ankylosis. The present study addresses this knowledge gap and has uncovered 432 DE genes, representing 2.3% of the total genes expressed by the furcal bone. International Journal of Oral Science (2020) 12:7 From these genes, biological pathways relating to inﬂammatory response and epithelial cell turnover were predicted to be affected in ankylosis. In contrast with animal models of ankylosis, these ﬁndings open up new gateways for studying the pathogenesis of human molar ankylosis sensitivity for discovering low copy transcripts.19 Most im tantly, RNA-seq is highly accurate for quantifying trans abundance across technical and biological replicates, wit the need for subsequent veriﬁcation using quantitative PCR. To maximize signal strength the experimental design nee Cytokine/growth factor Legend Enzyme G-protein coupled receptor Ligand-dependent nuclear receptor Peptidase Transmembrane receptor Transporter Other Function Gene involved in function (unclear effect) Differential expression leads to activation of function Differential expression inconsistent with activation of function Function likely activated Very increased differential expression Increased differential expression Decreased differential expression a Legend Enzyme G-protein coupled receptor Gene involved in function (unclear effect) Differential expression leads to activation of function Differential expression leads to inhibition of function Differential expression inconsistent with predicted activation/ihhibition of function Very increased differential expression Increased differential expression Decreased differential expression Cytokine/growth factor Growth factor Peptidase Transcription regulator Transporter Other Function Function likely activated Function likely inhibited b Fig. 3 IPA found DE genes in infraocclusion. They were associated with a increased activation of inﬂammatory response and recruitme leukocytes, and b increased activation of epithelial cell proliferation and differentiation, and inhibition of epithelial cell movement. Due t hierarchical nature of classifying pathways, only the higher-level pathways are illustrated (lower pathways that are subsets of higher path are not shown separately) Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. 4 Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. DISCUSSION d h Subsequently, regeneration of ERM 10 weeks after denervation corresponded with widening of the PDL space.27 Other studies have observed that absence of the ERM in regenerated PDL is associated with narrower PDL space,28 and ERM are always present in the vital PDL areas of replanted teeth.29 These ﬁndings suggest that normal, so-called “quiescent” ERM may be involved in the maintenance of PDL space, and consequently, prevention of ankylosis. Therefore, ERM dysregulation may result in loss of its ability to prevent ankylosis. Over-expressed genes (indicated by up arrows) were all present in the dental follicle as were under-expressed genes (indicated by down arrows), with the exception of TIMP2 The dental follicle was the most over-represented structure to be stained without presumption of ankylosis. This is because studies have demonstrated ankylosis to be invariably present in infraoccluded primary molars with missing successors,16 but the same cannot be said for non-infraoccluded primary molars. Although the vast majority of the latter are non-ankylosed, histological ankylosis has occasionally been found.21 Therefore, non-infraoccluded teeth cannot be assumed to be non-ankylosed, especially since normal percussion sound and normality mobility do not reliably exclude ankylosis.22 In the current study, tissue scarcity precluded a parallel histological analysis, although even this would be of limited value given that histology cannot detect transcriptional activity leading to future clinical ankylosis. The results showed that primary molar infraocclusion may be regarded as a phenotype with a molecular correlate involving at least 432 DE genes. However, since non-infraocclusion may represent a temporary absence of ankylosis in a dynamic disease, the differential gene outputs between the two groups may be viewed as a continuum, rather than as a distinct separation, between two different states of the pathology. Familial studies suggest that ankylosis is not a monogenic disease.5 Likewise, the present study did not identify a dominant gene for ankylosis. A pathological condition such as ankylosis requires the coordinated expression of multiple genes, often linked across multiple signalling pathways, as revealed in the current study. For this reason, the differential expression of one gene alone would be neither expected nor meaningful. On the other hand, the differential expression of multiple genes involved in a common biological pathway provides conﬁdence that this pathway is being affected in infraocclusion. Pathway analysis using GSEA showed that certain pathways, in particular those associated with epithelial cell development and the inﬂammatory response, were over-represented in the infraoccluded group. DISCUSSION d h Summary of immunostained proteins in the P1 molar tooth germ for the six genes under consideration Tooth structure Protein expression POSTN ↓TIMP2 ↓MMP2 ↓CK14 ↑DSG3 ↑PKP1 ↑ Dental follicle + − + + + + Stellate reticulum − − + + − − Stratum intermedium − − − + − − Inner enamel epithelium − − − + − + Ameloblast − + − + + + Enamel − − − − − − Dentine − − − − − − Odontoblast − + − − − − Pulp − − − − − − Over-expressed genes (indicated by up arrows) were all present in the dental follicle as were under-expressed genes (indicated by down arrows), with the exception of TIMP2 The dental follicle was the most over-represented structure to be stained Table 1. Summary of immunostained proteins in the P1 molar tooth germ for the six genes under consideration p yp Tooth ankylosis is a disruption in tooth eruption associated with loss of periodontal ligament (PDL) integrity. Given that genes causing disease are likely to be ontogenetically active during organ development,25 we reasoned that DE genes should also be expressed in developing tooth structures. Using immunohistochemical validation, we showed that all selected proteins were expressed in developing tooth structures. Although the spatial distribution of these proteins cannot speak to their etiology in tooth ankylosis, it would reafﬁrm the notion that pathological conditions frequently recapitulate their onto- genetic history during development. Of special note is the dental follicle (immunostained in ﬁve out of six proteins) that is comprised of pluripotent ectomesenchymal cells that subse- quently give rise to the periodontal ligament, bone and cementum; all of which are involved in the ankylotic process. The detection of CK14 and PKP1 in the inner enamel epithelium deserves special mention given its developmental role leading to the epithelial rests of Malassez (ERM), a structure that has been implicated in ankylotic pathogenesis. The ERM play an important role in maintaining PDL space to prevent ankylosis.26 For example, experimental reduction of ERM distribution in rat teeth via denervation of the inferior alveolar nerve resulted in narrowing of the PDL space and ankylosis after 6 weeks. DISCUSSION d h sensitivity for discovering low copy transcripts.19 Most impor- tantly, RNA-seq is highly accurate for quantifying transcript abundance across technical and biological replicates, without the need for subsequent veriﬁcation using quantitative PCR.20 sensitivity for discovering low copy transcripts.19 Most impor- tantly, RNA-seq is highly accurate for quantifying transcript abundance across technical and biological replicates, without the need for subsequent veriﬁcation using quantitative PCR.20 To maximize signal strength, the experimental design needed to minimize clinical differences between the two sample groups. For this reason, molars with successors were excluded to prevent contaminating genes originating from the erupting tooth follicle. The two sample groups were designed to be as similar as possible, differentiated only by the presence or absence of infraocclusion To maximize signal strength, the experimental design needed to minimize clinical differences between the two sample groups. For this reason, molars with successors were excluded to prevent contaminating genes originating from the erupting tooth follicle. The two sample groups were designed to be as similar as possible, differentiated only by the presence or absence of infraocclusion With respect to methodology, RNA-seq was selected over microarrays for its advantage in providing direct digital readout of expressed genes without reliance on pre-existing gene knowl- edge. Compared to hybridization approaches, RNA-seq has low background signals (from DNA contamination) and greater Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. 5 outputs.23 In contrast, exposure of a single cell type (Acinetobacter baumannii) to a single provocation (ethanol) elicited only 2% DE genes with RNA-seq.24 This supports the view that the current experimental design has maximized biological similarities between the sample groups, with the main difference being the presence, or not, of infraocclusion. It follows that the DE genes are likely to represent gene outputs associated with the infraocclusion phenotype. Table 1. International Journal of Oral Science (2020) 12:7 DISCUSSION d h Parallel analysis using IPA conﬁrmed an increased activation of the inﬂammatory response and increased epithelial cell proliferation and differentiation in the infraoccluded group. The above caveat notwithstanding, the pattern of gene expression was clearly different between the two groups, as demonstrated by hierarchical clustering (Fig. 1b) and PCA (Fig. 1c). Not only was there a clear separation between infraocclusion and non-infraocclusion, but both hierarchical clustering and PCA showed greater variation in the infraoccluded group compared to the non-infraoccluded group, which showed tighter clustering. This variation is a common ﬁnding in pathological conditions, since the pathology is often due to variable dysregulation of normally tightly regulated biological functions. For this reason, infraoccluded samples that looked like outliers (e.g. sample D5 in Figs. 1b, c) were not excluded, as they may represent the biological variability of ankylosis. On closer inspection of the genes supporting epithelial change, keratins in particular were among the most highly expressed genes. Keratins form the intermediate ﬁlaments of epithelial cells and are known to reﬂect the tissue type as well as changes in differentiation states. The keratins in this study were consistent with those expressed in human ERM, and in particular, ERM undergoing proliferation and differentiation in response to inﬂammation30–33 (Table 2). The increased expression of desmo- somal cadherins, desmoglein 2 (DSG2) and desmocollin 2 (DSC2), and the cytoplasmic desmosome-associated protein, plakophilin 1 (PKP1), are likewise consistent with human ERM.32 Collectively, the results suggest that there is a change in the ERM state during y y The number of DE genes (432) between the two groups represented only a small proportion (2.3%) of the total number of genes detected by RNA-seq. This small number appeared to reﬂect a strong degree of biological and molecular homogeneity between the two conditions (i.e. infraoccluded versus non- infraoccluded). In other transcriptome-wide studies using RNA- seq, for example comparing various types of cancers, the percentage of DE genes usually ranged from 30–70%, which reﬂects divergent tissue types with heterogeneous gene Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. DISCUSSION d h d DSG3 was strongly stained in the dental follicle and to a lesser extent in the ameloblast layer. e PKP1 was strongly stained in the dental follicle, as well as the inner enamel epithelium and the apical aspect of the ameloblast layer. f POSTN was immunostained only in the dental follicle. g MMP2 was expressed only in the dental follicle and stellate reticulum. h TIMP2 was the only protein in this sample not expressed in the dental follicle, but was abundantly present in the ameloblast and odontoblast layers. Scale bars are 100 μm Fig. 4 Immunohistochemical staining of proteins encoded by over-expressed (CK14, DSG3, PKP1) and under-expressed (POSTN, TIMP2, MMP2) genes in infraocclusion. a Hematoxylin and Eosin staining of P1 mouse molar tooth germ in the sagittal plane with various nascent structures within the dental follicle. b CK14 was strongly stained in the dental follicle. c Higher magniﬁcation of boxed area showed CK14 immunostaining in the stellate reticulum, stratum intermedium, inner enamel epithelium and ameloblast layer. d DSG3 was strongly stained in the dental follicle and to a lesser extent in the ameloblast layer. e PKP1 was strongly stained in the dental follicle, as well as the inner enamel epithelium and the apical aspect of the ameloblast layer. f POSTN was immunostained only in the dental follicle. g MMP2 was expressed only in the dental follicle and stellate reticulum. h TIMP2 was the only protein in this sample not expressed in the dental follicle, but was abundantly present in the ameloblast and odontoblast layers. Scale bars are 100 μm ankylosis, which may be associated with an increased inﬂamma- tory response. ankylosis, which may be associated with an increased inﬂamma- tory response. The ebb and ﬂow of the ankylotic process is reﬂected in multiple niches of hard tissue resorption and remineralization. Thus it was interesting to note that several mineralization- associated genes were found to be differentially expressed. These include decreased expression of bone-degrading genes (e.g. MMP2, MMP1439), increased expression of pro-mineralization genes (e.g. C4orf26,40 ODAM,41,42 BMP5, ASPN43), and decreased expression of anti-mineralization genes (POSTN,44 AXIN2,45 NFATC246). DISCUSSION d h ankylosis, which may be associated with an increased inﬂamma- tory response The ebb and ﬂow of the ankylotic process is reﬂe multiple niches of hard tissue resorption and reminera Ameloblasts Pulp Odontoblasts Dentine Enamel Inner enamel epithelium Stratum intermedium Stellate reticulum Outer enamel epithelium Dental follicle a CK14 DAPI DF SI SR IEE b A CK14 DAPI c A IEE SI SR DSG3 DAPI d DF A e PKP1 DAPI DF A IEE POSTN DAPI f DF MMP2 DAPI DF SR g h TIMP2 DAPI A OD Fig. 4 Immunohistochemical staining of proteins encoded by over-expressed (CK14, DSG3, PKP1) and under-expressed (POSTN MMP2) genes in infraocclusion. a Hematoxylin and Eosin staining of P1 mouse molar tooth germ in the sagittal plane with various structures within the dental follicle. b CK14 was strongly stained in the dental follicle. c Higher magniﬁcation of boxed area show immunostaining in the stellate reticulum, stratum intermedium, inner enamel epithelium and ameloblast layer. d DSG3 was strongly st the dental follicle and to a lesser extent in the ameloblast layer. e PKP1 was strongly stained in the dental follicle, as well as the inner epithelium and the apical aspect of the ameloblast layer. f POSTN was immunostained only in the dental follicle. g MMP2 was expres in the dental follicle and stellate reticulum. h TIMP2 was the only protein in this sample not expressed in the dental follicle, abundantly present in the ameloblast and odontoblast layers. Scale bars are 100 μm Tong et al. 6 Ameloblasts Pulp Odontoblasts Dentine Enamel Inner enamel epithelium Stratum intermedium Stellate reticulum Outer enamel epithelium Dental follicle a CK14 DAPI c A IEE SI SR CK14 DAPI DF SI SR IEE b A b CK14 DAPI c DSG3 DAPI d DF A e PKP1 DAPI DF A IEE POSTN DAPI f DF d f e e h TIMP2 DAPI A OD MMP2 DAPI DF SR g g h Fig. 4 Immunohistochemical staining of proteins encoded by over-expressed (CK14, DSG3, PKP1) and under-expressed (POSTN, TIMP2, MMP2) genes in infraocclusion. a Hematoxylin and Eosin staining of P1 mouse molar tooth germ in the sagittal plane with various nascent structures within the dental follicle. b CK14 was strongly stained in the dental follicle. c Higher magniﬁcation of boxed area showed CK14 immunostaining in the stellate reticulum, stratum intermedium, inner enamel epithelium and ameloblast layer. DISCUSSION d h Samples were homogenized using a Bullet Blender® (Next Advance, USA) and 1.5 mL Navy Eppendorf® Bead Lysis microtubes (Next Advance, USA) ﬁlled with six additional 3.5 mm stainless steel UFO beads (Next Advance, USA), following the protocol described by Carter, et al.,47 with a modiﬁcation to increase the blending time to 8 min at speed 12. β-catenin degradation, similarly acts to increase Wnt signalling.45 Given that the differential expression pattern of these mineralization-associated genes seems to indicate a net anabolic effect, ankylosis may be associated with dysregulation of mineralization, as has been observed in animal models.12–14 , Due to the case-control nature of this study, the results can only demonstrate an association, not a causal relationship, between ankylosis and the list of DE genes. The DE genes may well be the result, rather than the cause, of ankylosis, given that the condition was pre-existing before tissue sampling. Despite this limitation, the results of the present study would be valuable to future studies. This is because the behaviour of human primary molar ankylosis is currently not replicable in animal models, and any histological or transcriptomic analysis of human teeth precludes that tooth from longitudinal follow-up. Therefore, the aim of the study was to establish the differences in gene expression of ankylosed teeth, from which to build future experiments to explore a causal relationship, which could involve testing candidate genes in animal models, or modulat- ing pathways highlighted to be important in ankylosis. g p Total RNA was isolated using the RNeasy™Mini Kit (Qiagen, Switzerland) following manufacturer’s instructions. The RNA yield, RNA quality, and salt contamination of all samples were measured using the Bioanalyzer (Agilent Technologies, USA) and NanoDrop™ spectrophotometer (Thermo Fisher Scientiﬁc, USA). Library prepara- tion involved removal of rRNA using Ribo-Zero™Gold (Illumina, USA) and conversion to cDNA. Raw 100 bp single-end RNA-seq data was produced using the Illumina HiSeq 2500 system (Illumina, USA). Bioinformatic analysis The sequence reads were aligned against the human genome (Build version HG38) using the Tophat aligner (v.2.0.14),48 utilizing the reference annotation based transcript (RABT) assembly option. Transcripts were assembled with the Stringtie tool (v1.2.4).49 In conclusion, this case-control study found that primary molar ankylosis with infraocclusion was characterized by the differential expression of genes consistent with an increased inﬂammatory response and increased epithelial cell proliferation and differentia- tion. DISCUSSION d h A study of mutant mice bred for elevated Wnt signalling via β-catenin stabilization in osteocytes and cemento- cytes showed PDL space mineralization, failure of eruption, and dental ankylosis.12 In this study, it is possible that the decreased expression of AXIN2, which negatively regulates Wnt signalling via The above is concordant with established evidence that ankylosis is highly associated with root resorption, which is driven by pro-inﬂammatory mediators.34,35 Ankylosis in primary molars appears as an area of previous root resorption repaired by bone extending from the surrounding alveolus.16,21 In this way, ankylotic sites are constantly remodelling and relocating, closely following the progression of root resorption in an apical to cervical direction.16 Interestingly, root resorption, and presumably the accompanying inﬂammation, is greater in ankylosed, compared to non-ankylosed, primary teeth,36–38 further highlighting an associa- tion between ankylosis and inﬂammation. International Journal of Oral Science (2020) 12:7 Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. 7 mean age of 11.5 years (range 7.9–3.8 years), providing nine non- infraoccluded mandibular primary second molars. This study followed the STROBE guidelines for human observational studies. Table 2. Differentially expressed keratins and their associated ERM states Keratin LogFC ERM state 16 7.61 Normal30 6A 7.17 Normal30 13 6.06 Inﬂammatory33 14 5.94 Normal30,32 Increased when inﬂammatory33 5 5.88 Normal30,32,33 15 4.65 Normal32 19 3.86 Normal30,32,33 1 2.89 Normal30 4 N/A Inﬂammatory (lower expression than Keratin 13)33 Deﬁnition of infraocclusion In this study, a tooth was deemed to be infraoccluded when the entire occlusal surface of the primary second molar was positioned at least 1 mm below the occlusal surface of the adjacent non-infraoccluded permanent molar, as judged by clinical and radiographic examination.1 Non-infraocclusion was deﬁned as the entire occlusal surface of the primary second molar being level with the occlusal surface of the adjacent non- infraoccluded permanent molar. Additionally, there must be no vertical deﬁciency of the interdental alveolar bone on the panoramic radiograph. Sample preparation and RNA-seq The steps of the experimental protocol are illustrated in Supplementary Fig. 1. Bone was harvested at time of extraction from the bifurcation area of mandibular primary second molars, quickly rinsed in sterile phosphate-buffered saline, then immedi- ately stored in RNAlater® (Qiagen, Switzerland) following manu- facturer’s instructions. DISCUSSION d h Independent validation of six DE genes showed their protein expression in developing tooth structures, especially the dental follicle. A hypothesis was generated that an increased inﬂamma- tory response and ERM dysregulation form part of the pathogen- esis of primary molar ankylosis. This provides the foundation for hypothesis testing in future experimental models. The read counts were used to estimate gene expression and identify DE genes using the R package (R version 3.2.0) ‘edgeR’ (v.3.10.5).50 Genes with >10 counts in at least seven samples (i.e. number of biological replicates) were retained for further analysis. The normalization method was RUV (remove unwanted varia- tion).51 All genes were included to compute the factor of unwanted variation using the function RUVr. The p-value was adjusted for multiple testing by using the Benjamini-Hochberg correction to obtain the FDR. International Journal of Oral Science (2020) 12:7 MATERIALS AND METHODS Sample recruitment Hierarchical clustering and the resulting heatmap were computed using all 432 DE genes with FDR < 0.05. The normalized read counts were scaled (mean-centred and divided by standard deviation), and the distance metric was Euclidean. PCA was computed using all identiﬁed genes to visualize transcriptome variation between samples. p Ethics approval was obtained from the University of Melbourne Human Research Ethics Committee (HREC ID: 1442486.1). Samples were donated, after written informed consent, by parents of children attending orthodontic clinics in Melbourne, Australia between December 2015 and January 2017. The inclusion criteria required that (1) all subjects were 15 years or under, medically healthy, and had not commenced orthodontic treatment to avoid confounders related to age, medical conditions, and orthodontic tooth movement, and (2) all primary molars were missing successors to avoid confounders emanating from erupting tooth follicles. Pathway analysis DE genes were further studied to deﬁne their cellular and biological signiﬁcance. GSEA was performed against all GO gene sets in the molecular signatures database (v6.0).52 Next, to map signiﬁcant gene sets (FDR < 0.05) and their overlap (overlap coefﬁcient > 0.25), we used Cytoscape (v3.5.1) software53 with the EnrichmentMap plugin.54 The infraocclusion group consisted of ﬁve subjects (three females and two males) with a mean age of 13.5 years (range 11.8–15.0 years), providing seven infraoccluded mandibular primary second molars. The non-infraocclusion group (controls) consisted of eight subjects (six females and two males) with a The input list of DE genes for IPA (Qiagen, Switzerland) (accessed May 2017) had signiﬁcance cut-offs of FDR < 0.05 and logFC ≤−0.59 or ≥0.59 (equivalent to a 1.5-fold change or more). Transcriptome analysis of ankylosed primary molars with infraocclusion Tong et al. Tong et al. 8 Immunohistochemistry Immunohistochemistry was carried out on coronal sections of mandibles containing developing mouse tooth germs from postnatal day 1 (P1) mice and immersed in 4% paraformaldehyde at 4 °C for 24 h. Following embedding in parafﬁn, serial sections (5 µm thick) were obtained for immunohistochemistry and staining with haematoxylin and eosin. For immunoﬂuorescence staining, sections were deparafﬁnised with xylene, followed by rehydration through graded alcohols and distilled water. Antigen retrieval was carried out by boiling the sections in sodium citrate buffer for 10 min. Sections were then washed with 0.1% PBS with Triton-X and blocked with 10% normal horse serum for 1 h at room temperature. MATERIALS AND METHODS Sample recruitment Sections were incubated with primary antibodies overnight followed by appropriate secondary anti- bodies for 1 h at room temperature, or stained with DAPI (1:10 000, Dako, Carpinteria, CA) before mounting under glass cover slips with anti-fade mounting reagent. 3. Kurol, J. & Thilander, B. Infraocclusion of primary molars with aplasia of the permanent successor: a longitudinal study. Angle Orthod. 54, 283–294 (1984). ( ) 4. Becker, A. & Karnel-R’em, R. M. The effects of infraocclusion: Part 1. Tilting of the 4. Becker, A. & Karnel-R’em, R. M. The effects of infraocclusion: Part 1. Tilting of the adjacent teeth and local space loss. Am. J. Orthod. Dentofac. Orthopedics 102, 256–264 (1992). adjacent teeth and local space loss. Am. J. Orthod. Dentofac. Orthopedics 102, 256–264 (1992). 5. Kurol, J. Infraocclusion of primary molars: an epidemiologic and familial study. Community Dent. Oral. Epidemiol. 9, 94–102 (1981). 6. Via, W. F. Submerged deciduous molars: familial tendencies. J. Am. Dent. Assoc. 69, 127–129 (1964). 7. Koyoumdjisky-Kaye, E. & Steigman, S. Ethnic variability in the prevalence of submerged primary molars. J. Dent. Res. 61, 1401–1404 (1982). 8. Shalish, M., Peck, S., Wasserstein, A. & Peck, L. Increased occurrence of dental anomalies associated with infraocclusion of deciduous molars. Angle Orthod. 80, 440–445 (2010). 9. Baccetti, T. & Tollaro, I. in Proc. of 10th International Symposium on Dental Mor- phology (eds Radlanski, R. J. & Renz, H.) 428–432 (C&M Brünne Gbr., Berlin, 1995). 10. Odeh, R. et al. Infraocclusion: dental development and associated dental varia- tions in singletons and twins. Arch. oral. Biol. 60, 1394–1402 (2015). Primary antibodies were: rabbit polyclonal anti-periostin (POSTN) (1:100, LifeSpan Biosciences, Seattle), mouse monoclonal anti-tissue inhibitor of metalloproteinases 2 (TIMP2) (1:400, Millipore, Teme- cula), rabbit polyclonal anti-matrix metalloproteinase 2 (MMP2) (1:50, Millipore, Temecula), mouse monoclonal anti-cytokeratin 14 (CK14) (1:200, Thermo Fisher Scientiﬁc, Rockford), mouse monoclonal anti- desmoglein 3 (DSG3) (1:100, Thermo Fisher Scientiﬁc, Rockford) and mouse monoclonal anti-plakophilin 1 (PKP1) (1:50, Abcam, Cam- bridge). Secondary antibodies were: Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500, Molecular Probes) and Alexa Fluor 488- conjugated goat anti-rabbit IgG (1:500, Molecular Probes). Imaging for immunoﬂuorescence was performed on a Zeiss 780 Confocal Microscope. 11. Curl, L., Tan, C. W., Dreyer, C. W. & Sampson, W. A qualitative investigation of RANKL, RANK and OPG in a rat model of transient ankylosis. Aust. orthodontic J. 30, 143–151 (2014). 12. 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The online version of this article (https://doi.org/10.1038/s41368-019-0070-1) contains supplementary material, which is available to authorized users. 30. Sculean, A. et al. Patterns of cytokeratin expression in monkey and human per- iodontium following regenerative and conventional periodontal surgery. J. Peri- odontal Res. 36, 260–268 (2001). Competing interests: The authors declare no competing interests 31. Berkovitz, B. K. B., Whatling, R., Barrett, A. W. & Omar, S. S. The structure of bovine periodontal ligament with special reference to the epithelial cell rests. J. Peri- odontol. 68, 905–913 (1997). ACKNOWLEDGEMENTS 21. Darling, A. I. & Levers, B. G. H. Submerged human deciduous molars and anky- losis. Arch. Oral. Biol. 18, 1021–IN1013 (1973). This study was supported by a grant from the Australian Society of Orthodontists Foundation for Research and Education (ASOFRE). We thank the Foundation for its generous support. We also thank the staff at the private orthodontic clinics of S.T. and P.S. for their help in organising the logistics of sample collection. 22. Andersson, L., Blomlöf, L., Lindskog, S., Feiglin, B. & Hammarström, L. Tooth ankylosis: clinical, radiographic and histological assessments. International. J. Oral. Surg. 13, 423–431 (1984). 23. Peng, L. et al. Large-scale RNA-seq transcriptome analysis of 4043 cancers and 548 normal tissue controls across 12 TCGA cancer types. Sci. Rep. 5, 13413 (2015). DATA AVAILABILITY The dataset generated by this study is publicly archived in the NIH Sequence Read Archive (accession number PRJNA558843). 19. Wang, Z., Gerstein, M. & Snyder, M. RNA-seq: a revolutionary tool for tran- scriptomics. Nat. Rev. Genet. 10, 57–63 (2009). 20. Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome deﬁned by RNA sequencing. Science 320, 1344–1349 (2008). MATERIALS AND METHODS Sample recruitment Aberrantly elevated Wnt signaling is responsible for cementum overgrowth and dental ankylosis. Bone 122, 176–183 (2019). 13. Popoff, S. N. & Marks, S. C. Jr. Relationship of abnormalities in dental and skeletal development in the osteopetrotic (os) rabbit. J. Oral. Pathol. Med. 19, 5–12 (1990). 14. Wesselink, P. R. & Beertsen, W. Ankylosis of the mouse molar after systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP). J. Clin. Peri- odontol. 21, 465–471 (1994). 15. Thumbigere-Math, V. et al. Hypercementosis associated with ENPP1 mutations and GACI. J. Dent. Res. 97, 432–441 (2018). 16. Kurol, J. & Magnusson, B. C. Infraocclusion of primary molars: a histologic study. Eur. J. Oral. Sci. 92, 564–576 (1984). 17. Odeh, R. et al. Prevalence of infraocclusion of primary molars determined using a new 2D image analysis methodology. Aust. Dent. J. 61, 183–189 (2016). 18. Steigman, S., Koyoumdjisky-Kaye, E. & Matrai, Y. 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https://openalex.org/W3041260879	https://www.epj-conferences.org/10.1051/epjconf/202023703007/pdf	English	null	Characterization of Complex Water Vapour Field Structures and their Genesis Based on the Combined use of Raman Lidar Measurements and MESO-NH Model Simulations	EPJ web of conferences	2,020	cc-by	2,589	EPJ Web Conferences 237, 03007 (2020) ILRC 29 EPJ Web Conferences 237, 03007 (2020) ILRC 29 EPJ Web Conferences 237, 03007 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023703007 ABSTRACT layer are found to overpass Algeria, descending from an elevation of 3.5-5 km. layer are found to overpass Algeria, descending from an elevation of 3.5-5 km. As part of the Cevennes-Vivarais site, the University of Basilicata Raman lidar system (BASIL) was deployed in Candillargues throughout the duration of HyMeX-SOP 1 (September-November 2012), providing high- resolution and accurate measurements, both in daytime and night-time, of atmospheric temperature, water vapour mixing ratio and particle backscattering and extinction coefficient at three wavelengths. Measurements carried out by BASIL on 28 September 2012 reveal a quite complex vertical structure of the water vapor field. Reported Raman lidar measurements were run in the time interval between two consecutive heavy precipitation events, from 15:30 UTC on 28 September to 03:30 UTC on 29 September 2012. Throughout most of this observation period, lidar measurements reveal the presence of four distinct humidity layers. 1. HyMeX-SOP1 HyMeX was conceived with the goal of improving the comprehension of the water cycle in the Mediterranean [1]. Within this program a major field campaign, named Special Observation Period 1 (SOP1), took place in the period September–November 2012 over the northwestern Mediterranean Sea and its surrounding coastal regions in France, Italy and Spain, with a specific focus on the study of heavy precipitation and flashflood events [2,3]. As part of the Cévennes-Vivarais observational site, a set of research instruments was installed in an atmospheric ‘supersite’ located in Candillargues (43o37’N, 4o04’E, elevation: 1m), near the coast and south of the Massif Central. Among these instruments, a wind profiler, a radiometer, a radiosonde launching facility and a variety of radars and lidars (BASIL being one of these) were present. The present research effort aims at assessing the origin of the different humidity filaments observed by BASIL on this day. The analysis relies on the comparisons between Raman lidar MESO-NH model simulations. Back-trajectory analyses from the model reveal that air masses ending in Candillargues at different altitudes levels are coming from different geographical regions. Specifically, the analysis reveals that air masses within the surface humidity layer were originated over the Atlantic Ocean, while air masses within the elevated filamentary humidity layer, also coming from the Atlantic Ocean, overpassed the sea stretch North of Spain and Southern France at an altitude of ~1 km. In addition, air masses within the lower of the two upper layers are found to overpass Southern Spain and Marocco, descending from an elevation of 2- 3.5 km, while air masses within the uppermost © 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/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4.0/). 3.1 Lidar measurements Raman lidar measurements considered in this research effort were run in the time interval between two consecutive heavy precipitation events, from 15:30 UTC on 28 September to 03:30 UTC on 29 September 2012. Figure 1 illustrates the time evolution of the range- corrected signal at 1064 nm as measured by BASIL over this time interval. The figure reveals the presence throughout most of the observational period of stratiform clouds around 4 km, with a very limited vertical extent (~0.5 km). Stratiform rain events are found to take place between 18:30 and 20:00 UTC on 28 September and between 00:30 and 03:30 UTC on 29 September 2019. One of these events, between 15:30 and 16:30 UTC on 28 September, appears in the form of Virga, with precipitating particles sublimating before reaching the ground. In this time interval, a lidar dark band is also observed at ~3.4 km, this phenomenon being associated with the variable scattering properties of melting hydrometeors in the snow flake to rain conversion process [10,11,12]. Figure 1: Time evolution of the range-corrected signal at 1064 nm (expressed in arbitrary units) as measured by BASIL over this time interval from 15:30 UTC on 28 September to 03:30 UTC on 29 September 2012. The red ellipses indicate the stratiform clouds, the red ellipses indicate the stratiform rain events, while the black arrow on the left portion of the figure reveal the presence of a dark band event. 8 Sep e be o 03:30 U C o 9 Sep e be 0 . The red ellipses indicate the stratiform clouds, the red ellipses indicate the stratiform rain events, while the black arrow on the left portion of the figure reveal the presence of a dark band event. Figure 2 illustrates the time evolution of water vapour mixing ratio, mH2O, as measured by BASIL over the same time period considered in Figure 1. This figure reveals the presence throughout most of the observation period of four distinct humidity layers: a surface layer extending up to 0.4-0.8 km a.s.l.; a filamentary structure, first observed around 17:30 UTC, with a progressively descending trend from ~1 km down to ~0.5 km and a variable vertical extent within the range 0.1- 0.4 km; two upper humid layers, one extending from 1.3-1.5 km to 2.4-2.8 km, with values of mH2O up to 7.5 g kg-1, and one above extending up to approx. 3.1 Lidar measurements 4 km, with values of mH2O not exceeding 6 g kg-1. Relatively high values of mH2O are observed throughout most part of the troposphere, with a residual value of 3 g kg-1 around 6 km. Figure 2: Time evolution of mH2O as measured by BASIL over the same time period considered in Fig. 1. g H2O y BASIL over the same time period considered in Fig. 1. Thus, back-trajectories can be extended back in time by 48 hours. Figure 3 illustrates the comparison between BASIL measurements and MESO-NH simulations in the time interval from 15:30-23:30 UTC on 28 September 2012. The four different humidity layers present in the Raman lidar measurements are well reproduced by Meso-NH, both in terms of timing of occurrence and mixing ratio values, with the only exception of the mixing ratio values within the elevated filamentary structure, with measured values not exceeding 7 g kg-1 and simulated values being as large as 8-9 g kg-1. 2. BASIL BASIL operated from 5 September to 5 November 2012, collecting more than 600 h of measurements, distributed over 51 measurement days and 19 Intensive Observation Periods (IOPs). BASIL uses a tripled Nd:YAG laser (single pulse energy power at 355 nm: 500 mJ, pulse repetition frequency: 20Hz, average power: 10 W) and a Newtonian telescope (primary mirror diameter: 45 cm, f/2.1). The major feature of BASIL is represented by its capability to perform high- resolution and accurate measurements of atmospheric temperature and water vapour, both in daytime and night-time, based on the application of the rotational and vibrational Raman lidar techniques in the ultraviolet [3-6]. Besides temperature and water vapour, BASIL is EPJ Web Conferences 237, 03007 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023703007 also capable of providing measurements of particle backscatter at 355, 532 and 1064 nm, particle extinction at 355 and 532 nm, and particle depolarization at 355 and 532 nm [7-10]). was run over a 1446 x 1778 km2 domain (35°-48° N, 8° W-16° E), with a horizontal resolution of 2.5 km. Atmospheric forcing are taken from AROME-WMED. The considered MESO-NH simulation started at 00:00 UTC on 27 September and ended at 00:00 UTC on 29 September 2012. 3.1 Model simulations and back-trajectory analysis For the purpose of this analysis the non- hydrostatic numerical research model MESO-NH 2 2 EPJ Web Conferences 237, 03007 (2020) ILRC 29 EPJ Web Conferences 237, 03007 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023703007 Figure 3: Time evolution of mH2O as measured by BASIL (upper panel) and simulated by MESO-NH (lower panel). of 2-3.5 km, while air masses within the uppermost layer overpassed Algeria, descending from an elevation of 3.5-5 km. Figure 3: Time evolution of mH2O as measured by BASIL (upper panel) and simulated by MESO-NH (lower panel). An assessment of the origin of the different humidity layers observed by the Raman lidar on this day is also carried out based on the use of MESO-NH simulated data. Back-trajectory analyses from this model allow revealing that air masses reaching Candillargues at different altitudes levels are coming from different geographical regions. Figure 4 illustrates the 48h- backtrajectory analysis for the air masses ending in Candillargues at 15:30 UTC on 28 September 2012. The trajectory colors identify the altitude experienced by the air masses during their transport to Candillargues, while the numbers at the beginning of each trajectory identify the air masses ending altitude in Candillargues. The red ellipse in the figure highlights the back- trajectories of the air masses ending in Candillargues within the surface humidity layer. The analysis reveals that air masses within this layer were originated over the Atlantic Ocean. Figure 5 illustrates the 48h-backtrajectory analysis of the air masses ending in Candillargues at 18:00 UTC on 28 September 2012. The red ellipses identify the back-trajectories ending at the altitude of elevated filamentary humidity layer and within the two upper layers. The figure reveals that air masses within the elevated filamentary humidity layer are also coming from the Atlantic Ocean, overpassing the sea stretch North of Spain and Southern France at an altitude of ~1 km. The figure also shows that air masses within the lower of the two upper layers overpassed Southern Spain and Marocco, descending from an elevation Figure 4: 48h back-trajectory ending in Candillargues at 15:30 UTC on 28 September 2012 as simulated by MESO-NH. Colors identify the altitude of the air masses during their transport to Candillargues, with the color legend being expressed in km. Numbers at the beginning of each trajectory identify the ending altitude in Candillargues. Red ellipse: back-trajectories ending in Candillargues within the surface humidity layer. 3.1 Model simulations and back-trajectory analysis Figure 5: 48h back-trajectory ending in Candillargues at 18:00 UTC on 28 September 2012 as simulated by MESO-NH. Red ellipses: back-trajectories ending in Candillargues at the altitude of elevated filamentary humidity layer and within the two upper layers. Figure 5: 48h back-trajectory ending in Candillargues at 18:00 UTC on 28 September 2012 as simulated by MESO-NH. Red ellipses: back-trajectories ending in Candillargues at the altitude of elevated filamentary humidity layer and within the two upper layers. Back-trajectory analysis ending in Candillargues at 18:00 UTC on 28 September 2012 and starting 240 hours (10 days) earlier at 18:00 UTC on 18 September 2012 have also been determined through the application of the NOAA HYSPLIT 3 EPJ Web Conferences 237, 03007 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023703007 Lagrangian back-trajectory model [13]. The back- trajectory analysis extending back in time by 10 days reveals that air masses in the uppermost humid layer are originated over the Saharan desert in Central Africa (Mali). Royal Meteorological Society 142 (Suppl 1): 259–274, doi: 10.1002/qj.2725 (2016). [4] Di Girolamo et al., Rotational Raman lidar measurements of atmospheric temperature in theUV, Geophys. Res. Lett. 31: L01106, doi: 10.1029/2003GL018342 (2004). Lagrangian back-trajectory model [13]. The back- trajectory analysis extending back in time by 10 days reveals that air masses in the uppermost humid layer are originated over the Saharan desert in Central Africa (Mali). Royal Meteorological Society 142 (Suppl 1): 259–274, doi: 10.1002/qj.2725 (2016). [4] Di Girolamo et al., Rotational Raman lidar measurements of atmospheric temperature in theUV, Geophys. Res. Lett. 31: L01106, doi: 10.1029/2003GL018342 (2004). Royal Meteorological Society 142 (Suppl 1): 259–274, doi: 10.1002/qj.2725 (2016). [4] Di Girolamo et al., Rotational Raman lidar measurements of atmospheric temperature in theUV, Geophys. Res. Lett. 31: L01106, doi: 10.1029/2003GL018342 (2004). ( ) Figure 6: 240 h back-trajectory ending in Candillargues at 18:00 UTC on 28 September 2012 as simulated by NOAA-HYSPLIT back-trajectory model. [5] Di Girolamo P, Behrendt A, Wulfmeyer V., Spaceborne profiling of atmospheric temperature and particle extinction with pure rotational Raman lidar and of relative humidity in combination with differential absorption lidar: performance simulations, Appl. Opt. 45: 2474–2494, doi: 10.1364/AO.45.002474 (2006). [6] P. Di Girolamo et al., Multiparameter Raman lidar measurements for the characterization of a dry stratospheric intrusion event, J. Atmos. Oceanic Technol. 26: 1742–1762, doi: 10.1175/2009JTECHA1253.1 (2009). 3.1 Model simulations and back-trajectory analysis [7] Bhawar et al., The water vapour intercomparison effort in the framework of the Convective and Orographically-induced Precipitation Study: Airborne- to-ground-based and airborne-to-airborne lidar systems, Q. J. R. Meteorol. Soc. 137: 325–348, doi: 10.1002/qj.697 (2011). [8] V. Griaznov et al., Spatial Distribution of Doubly Scattered Polarized Laser Radiation in the Focal Plane of a Lidar Receiver, Applied Optics 46, 6821-6830, doi: 10.1364/AO.46.006821 (2007). Figure 6: 240 h back-trajectory ending in Candillargues at 18:00 UTC on 28 September 2012 as simulated by NOAA-HYSPLIT back-trajectory model. As a continuation of this research effort, we intend to investigate more precisely the origin of the sounded air parcels and their possible modifications along their route, also referring to the time series of potential temperature and other relevant parameters, as the aerosol properties. [9] P. Di Girolamo et al., UV Raman Lidar measurements of relative humidity for the characterization of cirrus cloud microphysical properties, Atmospheric ChemistrY and Physics 9, 8799-8811, doi: 10.5194/acp-9-8799-2009 (2009). [10] P. Di Girolamo et al., Raman Lidar observations of a Saharan dust outbreak event: Characterization of the dust optical properties and determination of particle size and microphysical parameters, Atmospheric Environment 50, 66-78 (2012). ACKNOWLEDGEMENTS This work was supported by the Italian Ministry for Education, University and Research under the Grant OT4CLIMA. The authors gratefully acknowledge NOAA Air Resources Laboratory for the use of the HYSPLIT transport and dispersion model. This work was supported by the Italian Ministry for Education, University and Research under the Grant OT4CLIMA. The authors gratefully acknowledge NOAA Air Resources Laboratory for the use of the HYSPLIT transport and dispersion model. This work was supported by the Italian Ministry for Education, University and Research under the Grant OT4CLIMA. The authors gratefully acknowledge NOAA Air Resources Laboratory for the use of the HYSPLIT transport and dispersion model. [11] P. Di Girolamo et al., Lidar and radar measurements of the melting layer: observations of dark and bright band phenomena, Atmospheric Chemistry and Physics 12, 4143-4157, doi: 10.5194/acp-12-4143-2012 (2012). REFERENCES [12] P. Di Girolamo et al., Model simulations of melting hydrometeors: a new Lidar bright band from melting frozen drops, Geophysical Research Letters 30, doi: 10.1029/2002GL016825 (2003). [1] P. Drobinski et al., HyMeX, a 10-year multidisciplinary program on the Mediterranean water cycle, Bull. Am. Meteorol. Soc. 95: 1063–1082, doi: 10.1175/BAMS-D-12-00242.1 (2014). [2] V. Ducrocq et al., HyMeX-SOP1, the field campaign dedicated to heavy precipitation and flash- flooding in northwestern Mediterranean, Bull. Am. Meteorol. Soc. 95: 1083–1100, doi: 10.1175/BAMS-D- 12-00244.1 (2014). [3] F. Duffourg et al., Offshore deep convection initiation and maintenance during HyMeX IOP 16a heavy precipitation event, Quarterly Journal of the [1] P. Drobinski et al., HyMeX, a 10-year multidisciplinary program on the Mediterranean water cycle, Bull. Am. Meteorol. Soc. 95: 1063–1082, doi: 10.1175/BAMS-D-12-00242.1 (2014). [13] V. Griaznov et al., Numerical Simulation of Light Backscattering by Spheres With Off-Center Inclusion. Application for Lidar Case, Applied Optics 43, 5512- 5522, doi: 10.1364/AO.43.005512. [2] V. Ducrocq et al., HyMeX-SOP1, the field campaign dedicated to heavy precipitation and flash- flooding in northwestern Mediterranean, Bull. Am. Meteorol. Soc. 95: 1083–1100, doi: 10.1175/BAMS-D- 12-00244.1 (2014). [14] Draxler, R. R and Hess, G. D.: An overview of the HYSPLIT_4 modeling system for trajectories, dispersion and deposition, Australian Meteorological Magazine 47(4), 295–308 (1998). [3] F. Duffourg et al., Offshore deep convection initiation and maintenance during HyMeX IOP 16a heavy precipitation event, Quarterly Journal of the 4 4
https://openalex.org/W4234200102	https://www.qeios.com/read/F4DHG5/pdf	English	null	Intent to Compound 506E Drug	Definitions	2,020	cc-by	59	Qeios · Definition, February 8, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Intent to Compound 506E Drug National Cancer Institute Qeios ID: F4DHG5 · https://doi.org/10.32388/F4DHG5 Source National Cancer Institute. Intent to Compound 506E Drug. NCI Thesaurus. Code C112087. C112087. A compounder intends to produce a drug classified as 506E. Qeios ID: F4DHG5 · https://doi.org/10.32388/F4DHG5 1/1
https://openalex.org/W3179768959	https://europepmc.org/articles/pmc8253928?pdf=render	English	null	Decreasing glucocorticoid levels towards the expansion front suggest ongoing expansion in a terrestrial mammal	Conservation physiology	2,021	cc-by	10,524	Alexandre Azevedo1,2,, Liam Bailey3, Victor Bandeira4, Carlos Fonseca4,5, Jella Wauters1 and Katarina Jewgenow1 Alexandre Azevedo1,2,, Liam Bailey3, Victor Bandeira4, Carlos Fonseca4,5, Jella Wauters1 and Katarina Jewgenow1 , y , , , Katarina Jewgenow1 1Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany 2Instituto de Ciências Biomédicas Abel Salazar - University of Porto, R. Jorge de Viterbo Ferreira 228, 4050-313 Porto, Portugal 3Department of Evolutionary Genetics, Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany 4Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal 5ForestWISE - Collaborative Laboratory for Integrated Forest and Fire Management, Quinta de Prados, Campus da UTAD 5001-801 Vila Real, Portugal C di h D f R d i Bi l L ib i I i f Z d Wildlif R h Alf d K lk S ß 17 10315 1Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany 2Instituto de Ciências Biomédicas Abel Salazar - University of Porto, R. Jorge de Viterbo Ferreira 228, 4050-313 Porto, Portugal 3Department of Evolutionary Genetics, Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany 4Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal 5ForestWISE - Collaborative Laboratory for Integrated Forest and Fire Management, Quinta de Prados, Campus da UTAD 5001-801 Vila Real, Portugal Corresponding author: Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany. Email: ax.c.azevedo@gmail.com Understanding the causes of range expansions in abundant species can help predict future species distributions. During range expansions, animals are exposed to novel environments and are required to cope with new and unpredictable stressors. Glucocorticoids (GCs) are mediators of the hormonal and behavioural mechanisms allowing animals to cope with unpredictable changes in the environment and are therefore expected to differ between populations at expansion edge and the historic range. However, to date, very few studies have evaluated the relationship between GCs and range expansion. The Egyptian mongoose has been rapidly expanding its range in Portugal over the past 30 years. In this study, we applied an information theoretic approach to determine the most important spatial and environmental predictors of hair GCs (hGCs) in the population, after controlling for normal patterns of hGC variation in the species. We observed a decrease in hGC as distance from the historic range increased (i.e. closer to the expansion front). Volume 9 • 2021 10.1093/conphys/coab050 Research article Volume 9 • 2021 10.1093/conphys/coab050 Research article 10.1093/conphys/coab050 Volume 9 • 2021 Introduction Differences in HPA-axis physiology could influence the ability of animals to colonize new environments during range expansion, but studies evaluating this link are scarce and focus on birds, reptiles and amphibians (e.g. Atwell et al., 2012; Liebl and Martin, 2012; Brown et al., 2015; Martin et al., 2018). For example, in expanding populations of house sparrows (Passer domesticus), individuals at the range edge have been shown to exhibit increased stress-induced GC levels and differences in the expression of the receptors involved in GC pathways, which is thought to facilitate their ability to colonize novel environments (Liebl and Martin 2012, 2013; Martin et al., 2017). In dark-eyed juncos (Junco hyemalis) and cane toads (Rhinella marina), a difference in GC reac- tivity was also observed between individuals of the newly established and historic populations, but with the colonists or edge populations showing decreased stress-induced GC levels (Atwell et al., 2012; Brown et al., 2015). The abundance, richness and spatial distribution of natu- rally occurring populations of wild species are changing at an unprecedented rate as a consequence of anthropogenic environmental change (Pimm et al., 2014; Newbold et al., 2015). Ecologists and evolutionary biologists have sought to understand the factors that shape species’ spatial distributions for decades (Holt, 2003; Liebl and Martin, 2012). In the Anthropocene, a small number of species have expanded their ranges, contradicting the general trend of decline driven by anthropogenic environmental change (Liebl and Martin, 2012). While the study of these exceptions can offer impor- tant information to predict how populations respond to envi- ronmental change,opportunities for such studies are rare (Hui et al., 2012; Liebl and Martin, 2012, 2013). Range expansions take place through the dispersal of individuals from the core population into novel environments. This is influenced by the set of environmental factors that enable the species to persist (niche), their spatial distribution (habitat) and the ability of the species to move (disperse) and adapt to new areas (Holt, 2003). Range expansions can take place when species occupy niche opportunities that arise due to changes in biotic or abiotic factors, such as changes in climate, land use or extirpation of competing species, or alternatively, due to the emigration of individuals from core populations into habitats where their niche requirements are not met, creating sink populations (Holt, 2003). The neuroendocrine stress response allows organisms to cope with unpredictable stressors in the environment (Wingfield et al., 1998). Introduction Accordingly, the colonization of new and unfamiliar environments during range expansions is expected to be facil- itated by increased stress reactivity (Liebl and Martin, 2012; Martin et al., 2017). However, if the new and unknown envi- ronment leads to very frequent or chronic activation of the stress response, impacts of elevated glucocorticoids (GCs) on survival, reproduction and fitness may occur (Sapolsky et al., 2000; Bonier et al., 2009a). Baseline levels of GCs during range expansions have also shown inconsistent trends (Atwell et al., 2012; Liebl and Martin, 2012; Martin et al., 2017). A broad scale study including approximately one hundred species of birds and reptiles found little evidence of a relation between stress- induced or baseline GCs and edge/non-edge location within the population (Martin et al., 2018). The inconsistency of these findings could be due to methodological factors, such as the lability of point samples of blood GC measurements (Bonier et al., 2009a). Baseline GCs based on plasma samples have shown low intra-individual repeatability (Vitousek et al., 2018, 2019b) compared to GC measurements from matrices reflecting long-term GC levels (Taff et al., 2018) and are greatly influenced by environmental conditions. Alternatively, these inconsistencies could be explained by the context depen- dence of stress-induced and basline levels of GC (Vitousek et al., 2018) or the fact that they are simply separate traits shaped by different selective pressures (Vitousek et al.,2019a). Hair GC (hGC) measurements are thought to reflect both baseline and stress-induced GCs incorporated into hair over prolonged periods (D’Anna-Hernandez et al., 2011; Kapoor et al., 2018; Short et al., 2016) and suffer little influence from short-term variations such as those caused by capture or handling. This may allow the identification of long-term trends in overall GC exposure that would be difficult to detect using matrices reflecting short-term variations. For example, a wild population of red deer (Cervus elaphus) exhibited an increase in hGC levels in response to hunting activity that was not detectable in faeces, while baseline plasma GC levels actually tended to decrease (Vilela et al., 2020). Hence, information on long-term GC exposure obtained from hGC analysis could help understand range expansions and predict whether expanding populations are likely to become established and pose an invasion risk (Martin et al., 2017) or result in sink populations. Conservation Physiology • Volume 9 2021 Conservation Physiology • Volume 9 2021 Research article Research article Alexandre Azevedo1,2,*, Liam Bailey3, Victor Bandeira4, Carlos Fonseca4,5, Jella Wauters1 and Katarina Jewgenow1 This distance term was present in all of the top models and had a 95% confidence interval (95% CI) that did not overlap with zero, strongly supporting its influence on hGC. We estimated a 0.031 pg/mg (95% CI: −0.057, −0.004) decrease in hGCs for each kilometre distance to the Tagus River, which was once the limit of the species’ distribution. Our results indicate that the species’ expansion is unlikely to be limited by mechanisms related to or mediated by the physiological stress response. The decrease in hGC levels towards the expansion edge coupled with limited evidence of a negative effect of human population density suggests that the species’ northward expansion in Portugal could continue. © The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology. 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. .......................................................................................................................................................... 1 Key words: Egyptian mongoose, hair glucocorticoids, Herpestes ichneumon, range expansion, stress Editor: Steven Cooke Received 16 April 2021; Revised 2 June 2021; Editorial Decision 9 June 2021; Accepted 11 June 2021 Cite as: Azevedo A, Bailey L, Bandeira V, Fonseca C, Wauters J, Jewgenow K. (2021) Decreasing glucocorticoid levels towards the expansion front suggest ongoing expansion in a terrestrial mammal. Conserv Physiol 9(1): coab050; doi:10.1093/conphys/coab050. .......................................................................................................................................................... Key words: Egyptian mongoose, hair glucocorticoids, Herpestes ichneumon, range expansion, stress © The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology. 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. .......................................................................................................................................................... 1 .......................................................................................................................................................... hGC measurement hGCs were quantified by an EIA for which validation was previously performed (Azevedo et al., 2019). Briefly, 20 mg of guard hairs were separated from undercoat, washed twice with 90% methanol for 5–10 seconds and dried at 70◦C. Next, 10 mg of hair were weighed and ground to a fine pow- der in a Precellys24 tissue homogenizer (Bertin Technologies, France). Finally, GCs were extracted from the powdered hair with 90% methanol and centrifuged, and the supernatant was collected and frozen until the day of GC measurement. hGCs were measured with an EIA using a polyclonal antibody (rabbit) against cortisol-3-CMO-BSA and cortisol-3-CMO- peroxidase as label.The assay was validated by demonstrating parallelism of serially diluted hair extracts to the cortisol stan- dard curve, and the inter-assay coefficients of variation were 10.78% for extracts containing low and 15.95% for extracts containing high concentrations of cortisol. The intra-assay coefficients of variation were 6.72% for extracts containing low and 5.37% for extracts containing high concentrations of cortisol and the sensitivity of the assay was 0.40 pg/well. In order to determine if the EIA was targeting the intended steroids, it was validated by HPLC analysis that demonstrated that the cortisol-3CMO antibody was binding to cortisol and small amounts of cortisone, and finally by demonstrating a strong correlation between cortisol-3CMO-EIA measure- ments and HPLC-MS/MS measurements of cortisol and cor- tisone from the same extracts. The validation of the EIA is reported in detail in Azevedo et al. (2019). In the current study, we measured hGC in Egyptian mongoose over the species’ entire range within Portugal. We applied an information theoretic approach (Burnham and Anderson, 1998) to determine which spatial (historic vs. expansion region, distance to Tagus river within the expansion population) and environmental factors [area of favourable habitat, European rabbit (Oryctolagus cuniculus) harvest data, Egyptian mongoose harvest data, human population density] from our data set influence hGC levels in the population, while controlling for known effects of age, sex, season and sample storage time. To our knowledge, this is the first study assessing the relation of GC with a mammalian range expansion and the only study using integumentary long-term GC measurements. hGC measurement If higher long-term GC levels facilitate expansion just as stress-induced blood GC levels seem to (Liebl and Martin, 2012, 2013; Martin et al., 2017), we expect to see higher hGC levels in the expansion area, with tendency to increase as the distance to the Tagus river increases. Alternatively, a decrease in hGC levels towards the expansion front would be more likely in a scenario where animals in the expansion area were presented with less Introduction In vertebrates, GCs are released by activation of the hypothalamic–pituitary–adrenal axis (HPA-axis) in response to challenging environmental stimuli (Wingfield et al., 1998; Sapolsky et al., 2000). This neuroendocrine response allows animals to respond to environmental cues (Wingfield and Mukai, 2009) and adjust their physiology and behaviour to cope with and recover from unpredictable environmental change (Wingfield et al., 1998; Sapolsky et al., 2000; Zimmer et al., 2020). Due to the pervasive effects of GCs, chronic elevations are thought to result in deleterious effects on survival, reproduction (Sapolsky et al., 2000) and fitness (Breuner et al., 2008; Vitousek et al., 2018). However, relations between baseline and stress-induced blood and faecal GC levels and fitness have been inconsistent (Bonier et al., 2009a, 2009b; Dantzer et al., 2014) and context dependent (Creel et al., 2013; Vitousek et al., 2018). The Egyptian mongoose (Herpestes ichneumon) is a medium sized carnivore that is widely distributed across 2 Conservation Physiology • Volume 9 2021 Conservation Physiology • Volume 9 2021 Research article Africa and the Middle East. In Europe, it is only present in the Iberian Peninsula, most likely due to colonization in the Late Pleistocene (Gaubert et al., 2011). In the past three decades, the species has rapidly increased its range to the north of the Tagus River (Fig. 1), which was once considered a natural barrier to its expansion (Barros et al., 2016b). In the expansion area, the species experiences very different environmental conditions, such as higher human density and primary productivity, and lower availability of favourable habitat (Bandeira et al., 2016). Based on presence–absence data, changes in land use such as rural abandonment have been identified as drivers of the expansion of the Egyptian mongoose (Barros et al., 2015). Morphological differences have been identified between the populations inhabiting the historic and expansion areas, with lower size (Bandeira et al., 2016) and body condition (Bandeira et al., 2019) and higher testicular mass (Bandeira et al., 2021) in the expansion area. However, no information exists on GC levels across the species’ range. frequent or severe stressors or where lower baseline hGCs favoured colonization. Sample collection We obtained data and hair samples from 294 carcasses of wild Egyptian mongoose collected throughout the year, between January 2008 and December 2014 from hunting activities throughout mainland Portugal (Bandeira et al., 2016). After the exclusion of specimens for which data were missing, 236 samples remained and were used in this study. Carcasses were stored frozen at −20◦C and then thawed at the time of sample collection. Hair was clipped with scissors as close to the skin as possible from a standard location (between the ‘scapulae’) in order to account for variation in hGC with anatomical region (Azevedo et al., 2019). Hair samples were stored in paper envelopes in a dry and dark location until the date of GC extraction. In order to be informative, GC measures require species- specific validation (Touma and Palme, 2005; Azevedo et al., 2020; Jewgenow et al., 2020) as well as consideration of how ‘normal’ patterns of variation (such as age, sex and season) and interacting environmental factors influence the physiological response (Dantzer et al., 2014). Both cortisol and cortisone are stress hormones that have been identified in Egyptian mongoose hair (Jewgenow et al., 2020). In previ- ous work, we cross-validated an enzyme immunoassay (EIA) targeting cortisol and cortisone in guard hairs of this species with liquid chromatography coupled with mass spectrome- try (LC–MS/MS) and high-pressure liquid chromatography (HPLC) (Jewgenow et al., 2020) and characterized normative variations with age, sex and season within the free-ranging population inhabiting Portugal (Azevedo et al., 2019). Spatial variation For specimens captured in the ‘expansion’ region, the shortest distance from capture location to the Tagus River in kilometres was calculated, and the resulting variable ‘distance to river’ was included in model construction to assess whether being closer to the expansion edge (or further from the core population) influenced hGC levels. River (Fig. 1). For specimens captured in the ‘expansion’ region, the shortest distance from capture location to the Tagus River in kilometres was calculated, and the resulting variable ‘distance to river’ was included in model construction to assess whether being closer to the expansion edge (or further from the core population) influenced hGC levels. Spatial variation In order to assess how expansion is related to hGC levels, Egyptian mongoose specimens were assigned to the ‘historic’ region if they were captured south of the Tagus River, or to the ‘expansion’ region if they were captured north of the Tagus 3 Conservation Physiology • Volume 9 2021 Research article Figure 1: Geographic distribution of the Egyptian mongoose in Portugal. The species was confined to the South of the Tagus River (dark grey area). In the past three decades, it has been rapidly expanding northward (light grey area). Circles represent number of specimens sampled in each location. Figure 1: Geographic distribution of the Egyptian mongoose in Portugal. The species was confined to the South of the Tagus River (dark grey area) In the past three decades it has been rapidly expanding northward (light grey area) Circles represent number of specimens sampled in Figure 1: Geographic distribution of the Egyptian mongoose in Portugal. The species was confined to the South of the Tagus River (dark grey area). In the past three decades, it has been rapidly expanding northward (light grey area). Circles represent number of specimens sampled in each location. 9 and 12 months), type 2 juvenile (between 5.5 and 9 months) and type 1 juvenile (between 2.5 and 5.5 months of age) based on dental development (Bandeira et al., 2016). Specimens were designated as male or female based on the presence of testicles or ovaries. Storage time was defined as the total number of days between the date of capture of the mon- goose and the date of cortisol extraction from hair (1150 to 2266 days). Although not a significant factor in our prior analyses (Azevedo et al., 2019), seasonal variations in GCs have often been demonstrated in vertebrates (Romero, 2002). Season was included in our model to account for the species’ seasonal reproductive activity with a peak in spring, which is possibly delayed in the expansion region (Bandeira et al., 2021). Animals were assigned to winter (January to March), spring (April to June), summer (July to September) or autumn (October to December) according to date of capture. We included snout–tail length (STL) values obtained by standard River (Fig. 1). Statistical methods mammal measurement methods to account for the potential effect of metabolic rate on baseline GCs, and because smaller animals may have less energy reserves and hence require enhanced GC responsiveness to meet unpredictable energy demands (Haase et al., 2016; Francis et al., 2018; Vitousek et al., 2019a). Finally, we included an index of body condition score (BCS) to account for the amount of energy reserves present in each specimen at the time of capture. We expect body condition to influence GC levels differently from size because of the central role of GCs in the regulation of energy metabolism (Sapolsky et al., 2000). For calculation of the BCS, we used the ‘scaled mass index’ based on body mass scaled for STL (Peig and Green, 2009, 2010). We analysed the effect of spatial and environmental factors on Egyptian mongoose hGC using linear mixed effects models with a Gaussian error distribution. Input variables were standardized on two standard deviations to account for differences in scale and to enable comparison of effect sizes (Gelman, 2008; Schielzeth, 2010). Variance inflation factors (VIFs) were used to test for multi-collinearity between variables with a cut-off value of 4. Collinearity was detected between road network (VIF = 6.50) and human population density (VIF = 5.16), which were highly correlated (r(234) = 0.86, P < 0.001). We considered the latter a more robust measure of human presence, as it is likely to include the effect of road network and many other factors. Therefore, road network was excluded from further analyses. The global model included the effect of the fixed factors age, sex, season STL, BCS, storage time, region, distance to river, favourable habitat, human population density, relative prey availability and relative conspecific density on hGC concentration. We also included the interactions between STL and both sex and age to account for differing effects of body size according to age cohort or sex. Year of capture was included as a random factor to account for non-independence and differences in GC levels in different years. Residuals of the fitted model were visually inspected by plotting against fitted values and with a Q-Q plot, to check model assumptions. We identified and removed two outlier hGC values that were more than six standard deviations from the mean and were causing violations in assumptions of homoscedasticity and normality of residuals. Normative patterns of variation In our previous study age, sex and storage time were shown to influence hGC measurements in this species (Azevedo et al., 2019). hGC levels were higher in males compared to females and in juveniles younger than 5.5 months compared to other age cohorts and decreased with storage time (Azevedo et al., 2019). Therefore, the effect of these variables was accounted for by including them in the model. Each mongoose was classified as an adult (over 1 year of age), sub-adult (between 4 Conservation Physiology • Volume 9 2021 Research article Research article Statistical methods We cannot rule out the possibility that these values are the result of severe stressors, since 4-fold increases in hair cortisol have previously been documented (del Rosario et al., 2011; Mastromonaco et al., 2014). However, we did not consider the effect of these potentially severe and rare stressors useful to answer our current research questions. After outlier removal, residuals of the fitted model displayed an approximately normal distribution with no strong pattern of over-dispersion or heteroscedasticity. The candidate model set included 6656 models that were ranked based on AICc (AICc from the best model ≤2.0) (Burnham and Anderson, 1998). We determined the relative importance of each factor using the sum of Akaike weights (sw) in the entire candidate model set, with 1 being the most important and 0 the least important. Factors that appeared in a higher number of models from the top model set and had higher sum of weights were considered more likely to be contained in the model best approximating the truth. We performed model averaging (Burnham and Anderson, 2002; Lukacs et al., 2010) on the top model set (AICc ≤2.0; Grueber et al., 2011) to account for uncertainty in model selection and obtain more robust estimates. Statistical analyses were performed using the R statistical system v 4.0.3 (R Core Team, 2020); model selection for mixed models was conducted using ‘lme4’ package (Bates et al., 2015) and ‘MuMIn’ package for model selection (Barton, 2020). Results Data for a total of 234 specimens were included in the analysis (Table 1). Among these, 141 belonged to the adult cohort, 24 to the sub-adult, 39 to juvenile 2 and 30 to juvenile 1, with a balanced distribution of females (125) and males (109). A total of 173 specimens were captured in the historic region and 61 were captured in the expansion area. hGC levels in Egyptian mongoose hair had a mean of 18.98 ± 5.42 pg/mg and varied between 8.07 and 43.36 pg/mg. Model averaged effect sizes within the top models (AICc ≤2.0; Table 4) indicated that STL had the strongest effect on hGC concentration (−5.20), followed by age (−4.23, juvenile type 2), sample storage time (3.65), sex (2.08, male), distance to the Tagus River within the expansion area (−1.76) and BCS (−1.76). The effects of the number of harvested mon- goose (−1.35) and rabbits (1.19), as well as season (−1.43, summer) and human population density (1.22), were weak when compared to the other factors. Interaction of age and sex with STL, region and favourable habitat were not present in the top model set. Model selection resulted in 6656 candidate models (Table S1), with a set of 16 models with AICc ≤2.0 (Table 2). The distance to the Tagus River for specimens collected in the expansion area appeared in all of the top models and had a relative importance of 0.69 (Table 3). In terms of environmental variables, relative mongoose and rabbit availability were present in most of the top models and had moderate relative importance based on sum of Akaike weights. There was little evidence in support of an effect of human population density, relative area of favourable habitat and region on hGC levels. Age, sex, body condition, body size and sample storage time appeared in all or the majority of the top models and had sums of Akaike weights higher than 0.60, providing evidence for their influence on hCG levels. Environmental factors 5 Conservation Physiology • Volume 9 2021 Conservation Physiology • Volume 9 2021 Research article Table 1: hGC values (mean ± SD in pg/mg) and number of Egyptian mongoose specimens from each region, age cohort and sex included in statistical analyses Historic region 19.03 ± 5.68 (n = 173) Expansion region 18.83 ± 4.65 (n = 61) Female 18.44 ± 4.28 (n = 91) Male 19.68 ± 6.88 (n = 82) Female 18.01 ± 4.39 (n = 34) Male 19.84 ± 4.84 (n = 27) Adult 17.78 ± 4.74 (n = 141) 18.49 ± 4.26 (n = 56) 19.16 ± 5.47 (n = 46) 17.84 ± 4.75 (n = 24) 20.23 ± 3.96 (n = 15) Sub-adult 19.42 ± 6.35 (n = 24) 17.1 ± 2.4 (n = 10) 24.97 ± 9.47 (n = 7) 16.38 ± 2.11 (n = 4) 18.24 ± 2.45 (n = 3) Juvenile 2 16.99 ± 4.83 (n = 39) 16.77 ± 4.12 (n = 11) 16.62 ± 4.48 (n = 20) 18.5 ± 2.94 (n = 4) 17.96 ± 9.85 (n = 4) Juvenile 1 22.11 ± 7.02 (n = 30) 20.53 ± 4.9 (n = 14) 25.07 ± 10.65 (n = 9) 22.32 ± 5.35 (n = 2) 21.15 ± 3.7 (n = 5) R l Table 1: hGC values (mean ± SD in pg/mg) and number of Egyptian mongoose specimens from each region, age cohort and sex included in statistical analyses n ± SD in pg/mg) and number of Egyptian mongoose specimens from each region, age cohort and sex included in with each harvested rabbit, after accounting for storage time and normative patterns of variation in the species. with each harvested rabbit, after accounting for storage time and normative patterns of variation in the species. Environmental factors The environmental factors used for model construction were selected based on our predictions of their biological rele- vance for hGC measurement drawn from results of previ- ous studies with the species (Barros et al., 2015, 2016a; Bandeira et al., 2016, 2018, 2019). All environmental vari- ables were presented as mean values within the 2 × 2 km grid cell where the specimen was collected. The reported home range size for the Egyptian mongoose in the Iberian Peninsula is 3.10 ± 2.12 km2 (Palomares, 1994). Hence, the grid cell area of 4 km2 is likely to offer an approximation of the environmental conditions each specimen experiences in its territory. We used the area occupied by shrub and/or agro- forestry habitat in each grid cell to obtain a proxy of the availability of habitat types that have been shown to favour gene flow (Barros et al., 2016a) and expansion (Barros et al., 2015) of the species in Portugal. Our prediction was that habitat types that have been favourable to the species’ expan- sion would be associated with lower hGC levels. Human population density presented as the number of inhabitants per km2 in each grid cell (data from Eurostat) (European Commission, 2015) was included as a fixed factor because increased levels of GCs are generally observed with increasing human disturbance (Dantzer et al., 2014). The extent of road network represented by the total length of road in metres in each grid cell (IGP, Instituto Geográfico Português, 2015) was included as a candidate factor in the model but was excluded due to collinearity with human population density. We included the number of Egyptian mongoose reported from hunting bags for each grid cell in the year and month (ICNF, unpublished data) of each specimen’s capture as a proxy of relative abundance of conspecifics to account for the effect of mongoose density on GCs. Population density can influence circulating GC levels in vertebrates, especially in non-social territorial species like the Egyptian mongoose where the frequency of social interactions at higher densities leads to more frequent activation of the HPA-axis (Creel et al., 2013). Similarly, European rabbit (O. cuniculus) yields for each grid cell in the respective year and month (ICNF,unpublished data) were used as a proxy of relative prey availability because food scarcity or unpredictability may influence GC levels directly (Fokidis et al., 2012). Discussion In this study, we aimed to determine which spatial and envi- ronmental factors influence long-term adrenocortical activity in the Egyptian mongoose population in Portugal, in order to better understand its idiosyncratic expansion in the context of anthropogenic change. We found support for a relation of hGC levels with range expansion in the Egyptian mongoose. The distance between capture location and the Tagus River, in animals from the expansion area, appeared in all models with AICc ≤2.0 and had a sum of Akaike weights of 0.69, strongly supporting its inclusion in the model best approximating the truth. Based on the averaged estimates of the top 16 models using stan- dardized input variables, the effect size of the distance to the Tagus (−1.76) was of a magnitude comparable to the effect of sex (2.08) or body condition (−1.76) with a 95% confidence interval that did not include zero (−3.26, −0.26). hGC levels are estimated to decrease 0.031 pg/mg per kilometre as the Based on the estimates obtained by model averaging using untransformed data (Table 4), hGCs were estimated to decrease 0.031 pg/mg (95% CI: −0.057, −0.004) for each 1 kilometre increase in distance from the capture location to the Tagus River, in the expansion area (Fig. 2). In terms of environmental factors, hGCs were predicted to decrease 0.258 pg/mg (95% CI: −0.513, −0.002) for each additional mongoose harvested in the 2 × 2 km grid cell during that month and to increase 0.011 pg/mg (95% CI: 0.000, 0.023) 6 Conservation Physiology • Volume 9 2021 Research article Table 2: Model selection table; top ranked models with AICc ≤2.0 are shown here (for full model selection table see Table S1); R2 and adjusted R2 (Adj. R2) values for each model are included (Nakagawa and Schielzeth, 2013); w indicates model weights Age Region Sex Season Mongoose Rabbit BCS Habitat Population STL Storage Age:STL D. Discussion river Sex:STL R2 Adj.R2 df AICc AICc w + − 2.10 + −1.14 1.27 −1.86 − 1.09 −5.34 −3.78 − + − 0.33 0.33 17.00 1397.29 0.00 0.017 + − 2.11 + −1.44 1.28 −1.91 − − −5.62 −3.64 − + − 0.32 0.32 16.00 1397.34 0.05 0.016 + − 2.09 + −1.20 1.23 −1.77 − 1.09 −5.05 −3.84 − + −1.74 0.34 0.34 18.00 1397.50 0.22 0.015 + − 2.10 + −1.50 1.24 −1.81 − − −5.33 −3.70 − + −1.74 0.33 0.33 17.00 1397.56 0.27 0.015 + − 2.01 + − 1.18 −1.73 − 1.43 −5.17 −3.94 − + − 0.32 0.32 16.00 1397.84 0.55 0.013 + − 2.00 + − 1.14 −1.64 − 1.45 −4.89 −4.00 − + −1.61 0.33 0.33 17.00 1398.36 1.07 0.010 + − 2.17 − −1.47 1.08 −1.58 − 1.16 −5.42 − − + − 0.30 0.30 13.00 1398.82 1.53 0.008 + − 2.00 + −1.10 − −1.77 − 1.11 −4.67 −3.93 − + −1.84 0.33 0.33 17.00 1399.05 1.76 0.007 + − 2.01 + −1.03 − −1.87 − 1.11 −4.96 −3.87 − + − 0.32 0.32 16.00 1399.07 1.79 0.007 + − 1.94 + − − −1.75 − 1.42 −4.83 −4.01 − + − 0.31 0.31 15.00 1399.09 1.80 0.007 + − 2.16 − −1.52 1.06 −1.49 − 1.15 −5.15 − − + −1.67 0.30 0.31 14.00 1399.14 1.85 0.007 + − 2.01 + −1.41 − −1.82 − − −4.95 −3.79 − + −1.84 0.32 0.32 16.00 1399.20 1.91 0.006 + − 2.02 + −1.34 − −1.92 − − −5.25 −3.73 − + − 0.31 0.31 15.00 1399.21 1.93 0.006 + − 2.20 − −1.55 1.02 −1.57 − 1.23 −5.26 −1.96 − + − 0.30 0.30 14.00 1399.25 1.96 0.006 + − 2.18 − −1.61 1.00 −1.47 − 1.23 −4.95 −2.09 − + −1.81 0.31 0.31 15.00 1399.25 1.96 0.006 + −0.78 2.07 + −1.43 1.26 −1.88 − − −5.65 −3.67 − + − 0.32 0.33 17.00 1399.29 2.00 0.006 Legend: BCS, body condition score; STL, snout–tail length; Age:STL, interaction between age and snout–tail length; D. river, distance to the Tagus River for animals captured in the expansion region; sex:STL, interaction between sex and snout–tail length; df, degrees of freedom; AICc, Akaike information criteria; AICc, difference in AICc to the model with lowest AICc; w, Akaike weight. Discussion ‘+’ indicates a categorical variable that was included in the model, while ‘−’indicates a variable that was not present. 7 Conservation Physiology • Volume 9 2021 Research article .................................................................................................................................................. Figure 2: Model predictions of the effect of distance from the historic range (limited by the Tagus River) on hGC levels in the Egyptian mongoose. The plot shows model predictions and 95% CI (grey band) based on parametric bootstrapping with 5000 iterations. Figure 2: Model predictions of the effect of distance from the historic range (limited by the Tagus River) on hGC levels in the Egyptian mongoose. The plot shows model predictions and 95% CI (grey band) based on parametric bootstrapping with 5000 iterations. GCs towards the expansion edge could reflect lower energy requirements to maintain physiological balance (McEwen and Wingfield, 2003), individuals in better condition and facing less challenges, potentially resulting in increased fitness (Bonier et al., 2009a) or a reduced likelihood to exceed the normal and non-pathological response to environmental challenges (Romero et al., 2009). The decreasing hGC levels towards the expansion front suggest that the Egyptian mongooses’ expansion is not likely to be limited by mechanisms related to or mediated by the physiological stress response. The negative association between hGC and distance to the historic range is consistent with the colonization of a new area where stressors are less frequent or less severe (e.g. niche opportunity or forced dispersal), as well as a scenario where lower baseline GC levels favour dispersal or survival at the expansion front. distance from the Tagus River (and historic distribution) increased, which would equate to an estimated 1.0 pg/mg decrease in hGC every 32.26 km (Fig. 2). The estimated decrease in hGCs from the Tagus River to the expansion front (136.95 km) equates to 22% (4.24 pg/mg) of the mean hGC levels for the population (18.98 ± 5.42 pg/mg). These values were obtained while accounting for hGC variation with age, sex, season, sample storage time, body condition (BCS), size (STL) and environmental factors (conspecific and prey availability, human population density and relative area of favourable habitat). We found no evidence to support an effect of region (expansion vs. historic) on hGC levels. The effect of the distance to the Tagus River is influenced by three samples collected at distances above 125 km (Fig. 2), which, in case they were somehow related, would raise concern about possible confounding effects. Conservation Physiology • Volume 9 2021 Discussion Inspection of the data on the three samples revealed they were collected at different locations, in different years and seasons, by different people. Additionally, the effect of the distance to the historic region in an analysis performed without these three specimens resulted in qualitatively similar results. Our results apparently contradict the findings linking increased stress-induced GCs with expansion in house sparrows (Liebl and Martin, 2012). However, while stress- induced GC measurements in sparrows reflect the reactivity of the HPA-axis, long-term hGC levels are thought to reflect circulating GCs over several weeks. The latter method is inadequate to assess phenotypic differences in HPA- axis reactivity favouring dispersal and survival in novel environments. However, it is likely to provide a better indicator of chronic GC exposure, which is thought to It is not possible to discern whether our results reflect a phenotypic difference in long-term GC levels facilitating expansion or reduced GC exposure due to an environment with less frequent or severe stressors we could not account for (e.g. predators or competing carnivores). Decreasing 8 Conservation Physiology • Volume 9 2021 Research article Table 3: Relative importance of predictors based on the sum of Akaike weights in the complete set of 6656 candidate models Table 3: Relative importance of predictors based on the sum of Akaike weights in the complete set of 6656 candidate models partially driven by metabolic scaling or energy availability rather than exclusively by ontogenetic variation in endocrine mechanisms. Body size received the most support for inclusion in the model and had the strongest effect (−5.20, 95% CI: −7.56, −2.84) on hGCs that decreased with size. Energy reserves represented by BCS also received strong support for inclusion in the model and had a strong effect (−1.76, 95% CI: −3.09, −0.43), with hGC levels decreasing as body con- dition increased. Overall, the effects of variables accounting for normal patterns of hGC variation in this population were quite strong (with magnitudes ranging from 1.76 to 5.20) compared to the effect of spatial and environmental factors. Additionally, storage time was present in 14 of the 16 models with AICc ≤2.0, with a sum of Akaike weights of 0.63 and a standardized effect of −3.65 (95% CI: −5.40, −1.90), supporting its inclusion in the model. These results illustrate how failing to include known causes of GC variation could confound the results of studies aiming to investigate the effects of environmental or spatial factors. Discussion weights in the complete set of 6656 candidate models Sum of weights N containing models STL 1.00 4608 Sex 0.99 4096 Age 0.98 4096 BCS 0.84 3328 Mongoose 0.72 3328 D. river 0.69 3328 Storage 0.63 3328 Season 0.61 3328 Rabbit 0.60 3328 Population 0.52 3328 Sex:STL 0.48 1536 Region 0.34 3328 Habitat 0.29 3328 Age:STL 0.13 1536 Legend: BCS, body condition score; STL, snout–tail length; Age:STL, interaction between age and snout–tail length; D. river, distance to the Tagus River for animals captured in the expansion region. Human population density was expected to be associated with increased hGCs. However, we did not find strong sup- port for its inclusion in the model (present in 11 of 16 models with AICc ≤2.0, sw = 0.52 and 95% CI: −0.18, 2.62), questioning whether the species is severely stressed by human presence. Resilience to stress caused by human presence due to the species’ known behavioural plasticity (Monterroso et al., 2014; Streicher et al., 2020) could have facilitated expansion in spite of increasing human population density. Alternatively, an attenuation of the stress response due to habituation (Cyr and Romero, 2009; Dickens and Romero, 2013) could explain absence of an increase in hGC levels with human density. Nevertheless, the result is discordant with the general trend in vertebrates, where an increase in GC levels is usually observed with increasing human disturbance (Dantzer et al., 2014). In the specific case of the Egyptian mongoose, presence–absence data previously revealed a negative influ- ence of urban areas and human infrastructure on the species’ occurrence (Barros et al., 2015). However, the absolute values of human density in our data were quite low, with a mean (and inter-quartile range) of human population data of 1(1– 44) inhabitants per km2, compared to the country’s average of 112.5 (0–5244.6) inhabitants per km2 (from Eurostat, European Commission, 2015) Therefore, although our results suggest little influence of human presence on Egyptian mon- goose hGCs at these human population densities, an effect might be present at higher densities, warranting cautious interpretation of these results. Legend: BCS, body condition score; STL, snout–tail length; Age:STL, interaction between age and snout–tail length; D. river, distance to the Tagus River for animals captured in the expansion region. Legend: BCS, body condition score; STL, snout–tail length; Age:STL, interaction between age and snout–tail length; D. river, distance to the Tagus River for animals captured in the expansion region. Discussion reflect the extent of environmental challenge to homeostasis (McEwen and Wingfield, 2003; Romero et al., 2009) and to potentially influence fitness through the effects of chronically elevated GC levels on most peripheral tissues (Sapolsky et al., 2000). Different response patterns and trade-offs between the effects of baseline and stress-induced GCs on fitness could explain simultaneously low baseline and increased stress- induced GC in expanding populations (Vitousek et al., 2018, 2019a). Due to the existence of a (albeit dynamic) threshold above which GCs cause deleterious effects to the organism, it is expected that lower baseline levels are required to allow higher levels of stress-induced increases without reaching pathological levels of GC exposure. Body size (STL), sex, age and body condition (BCS) were included in all of the 16 models with AICc ≤2.0, providing strong support for their inclusion in the model explaining hGC variation. Based on the sum of Akaike weights, STL had the highest relative importance (1.00), followed by sex (0.99), age (0.98) and finally BCS (0.84). Regarding the effect of sex on hGCs, the results of this study are consistent with our prior research, with males presenting higher GC levels compared to females (Azevedo et al., 2019). However, the variation of hGC among age cohorts is strikingly different from our previous work, where only type 1 juveniles differed from other cohorts, exhibiting the highest hGC levels. Here, with the inclusion variables reflecting body size and BCS, adults presented the highest hGC levels, followed by sub-adults, type 1 juveniles and finally type 2 juveniles. These results suggest that increased GC levels observed in juveniles could be at least European rabbit and Egyptian mongoose harvest data were included in our model as proxies of relative prey avail- ability and relative conspecific abundance, respectively. We expected hGC to decrease with relative prey availability and to increase with conspecific density. However, our results showed the opposite relation in both cases, with hGC increas- ing with prey availability and decreasing with mongoose abundance. Discussion The European rabbit is the Egyptian mongoose’s main prey species, accounting for 28% of ingested biomass in 9 Conservation Physiology • Volume 9 2021 Research article Research article Table 4: Model averaging results presented as estimates and 95% confidence intervals Standardized variables Untransformed variables Factor Estimate 95% Confidence interval Estimate 95% Confidence interval Intercept 20.26 18.20 22.33 58.68 41.86 75.50 Age (juvenile 1) −2.39 −5.89 1.11 −2.39 −5.89 1.11 Age (juvenile 2) −4.23 −6.56 −1.90 −4.23 −6.56 −1.90 Age (Sub-adult) −1.91 −3.99 0.17 −1.91 −3.99 0.17 Sex (male) 2.08 0.88 3.28 5.14 −4.25 14.53 Season (spring) 1.24 −0.73 3.22 1.24 −0.73 3.22 Season (summer) −1.43 −3.31 0.45 −1.43 −3.31 0.45 Season (winter) 0.92 −1.39 3.22 0.92 −1.39 3.22 Mongoose −1.35 −2.69 −0.01 −0.26 −0.51 −2.3e−3 Rabbit 1.19 −0.05 2.42 0.01 −4.0e−4 0.02 Body condition (BCS) −1.76 −3.09 −0.43 −2.8e −3 −4.9e−3 −7.0e−4 Human population 1.22 −0.18 2.62 0.01 −9.0e−4 0.01 Body size (STL) −5.20 −7.56 −2.84 −0.21 −0.34 −0.09 Storage −3.65 −5.40 −1.90 −0.01 −0.01 −4.3e−3 Distance to Tagus River −1.76 −3.26 −0.26 −0.03 −0.06 −4.5e−3 Sex × body size (STL) −1.74 −4.10 0.62 −0.08 −0.18 0.03 Region (historic) −0.78 −3.28 1.72 −0.78 −3.28 1.72 Legend: estimates are presented for model averaging using input variables standardized on two standard deviations following Gelman (2008) and using untransformed input variables. Confidence intervals not including zero are highlighted in bold as variables are considered to significantly influence hGC concentration. Table 4: Model averaging results presented as estimates and 95% confidence intervals Legend: estimates are presented for model averaging using input variables standardized on two standard deviations following Gelman (2008) and using untransformed input variables. Confidence intervals not including zero are highlighted in bold as variables are considered to significantly influence hGC concentration. this specific population (Bandeira et al., 2018). Food scarcity has been associated with increases in GCs in some species (Bryan et al., 2013, 2014; Riechert et al., 2014; Barrett et al., 2015) but in others no effect was detected (Van Meter et al., 2009; Rector et al., 2012; Burstahler et al., 2019). mongoose removal through hunting, potentially alleviating territorial competition. Secondly, hunting activities can cause stress (Bryan et al., 2015; Vilela et al., 2020). The increasing number of rabbits removed by hunting activities could lead to an increased stress response in mongooses in those areas either by acting as competition for resources or by causing direct disturbance to mongoose. Discussion This 10 Conservation Physiology • Volume 9 2021 Research article result could be due to the mongooses’ behavioural and dietary plasticity or alternatively by the dependence on resource-rich favourable habitat in the core areas of its territory that is not necessarily proportional to the size of the home range of each individual (Streicher et al., 2020) or the area of favourable habitat available in each grid cell. Animal movement and high-resolution landscape data would be required to further analyse the relation between hGC and favourable habitat in the species. result could be due to the mongooses’ behavioural and dietary plasticity or alternatively by the dependence on resource-rich favourable habitat in the core areas of its territory that is not necessarily proportional to the size of the home range of each individual (Streicher et al., 2020) or the area of favourable habitat available in each grid cell. Animal movement and high-resolution landscape data would be required to further analyse the relation between hGC and favourable habitat in the species. Funding Barros T, Carvalho J, Pereira MJR, Ferreira JP, Fonseca C (2015) Following the trail: factors underlying the sudden expansion of the Egyptian mongoose (Herpestes ichneumon) in Portugal. PLoS One 10: 1–18, e0133768. This research was supported by the Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany. V.B. and C.F. were supported by national funds through Fun- dação para a Ciência e a Tecnologia and European funds through the Programa Operacional Temático Factores de Competitividade and European Regional Development Fund by co-funding through the project ‘Genetic assess- ment of a successful invasion: Population genetics of the Egyptian mongoose (Herpestes ichneumon) in Portugal’ (PTDC/BIA-BEC/104401/2008) and by Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior for the financial support to CESAM (UIDP/50017/2020 + UIDB/50017/2020), through national funds. Barros T, Cushman SA, Carvalho J, Fonseca C (2016a) Mediterranean scrubland and elevation drive gene flow of a Mediterranean carni- vore, the Egyptian mongoose Herpestes ichneumon (Herpestidae). Biol J Linn Soc 120. 10.1111/bij.12867. Barros T, Ferreira E, Rocha RG, Gaubert P, Bandeira V, Souto L, Mira A, Fonseca C (2016b) Genetic signature of the northward expansion of the Egyptian mongoose Herpestes ichneumon (Herpestidae) in the Iberian Peninsula. Biol J Linn Soc 118: 686–697. Barton K (2020) MuMIn: Multi-Model Inference. Bates D, Mächler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67: 1–48. References Atwell JW, Cardoso GC, Whittaker DJ, Campbell-Nelson S, Robertson KW, Ketterson ED (2012) Boldness behavior and stress physiology in a novel urban environment suggest rapid correlated evolutionary adaptation. Behav Ecol 23: 960–969. Azevedo A, Bailey L, Bandeira V, Dehnhard M, Fonseca C, de Sousa L, Jewgenow K (2019) Age, sex and storage time influence hair cortisol levels in a wild mammal population. PLoS One 14: e0221124. AzevedoA,WautersJ,KirschbaumC,SerraR,RivasA,JewgenowK (2020) Sex steroids and glucocorticoid ratios in Iberian lynx hair. Conserv Physiol 8. 10.1093/conphys/coaa075. Conclusion In the Anthropocene, species distributions are changing at an unprecedented rate. A small number of wild species have expanded their ranges, contradicting the general trend of decline. The study of these exceptions can help predict future species distributions. This study is the first to examine the relation between GCs and range expansion in mammals and uses a long-term measure of GC levels that is less subject to the short-term influence of environmental variables. The results show a decrease in hGC levels towards the expansion front, suggesting that the species’ expansion is unlikely to be limited by mechanisms related to- or mediated by the physiological stress response. Bandeira V, Virgós E, Azevedo A, Carvalho J, Cunha MV, Fonseca C (2019) Sex and season explain spleen weight variation in the Egyptian mongoose. Curr Zool 65: 11–20. BandeiraV,VirgósE,AzevedoA,CunhaMV,FonsecaC(2021)Association between reproduction and immunity in Herpestes ichneumon is sex- biased and unaffected by body condition. J Zool 313: 124–134. Bandeira V, Virgós E, Barros T, Cunha MV, Fonseca C (2016) Geographic variation and sexual dimorphism in body size of the Egyptian mon- goose, Herpestes ichneumon in the western limit of its European distribution. Zool Anz J Comp Zool 264: 1–10. Bandeira V, Virgós E, Carvalho J, Barros T, Cunha MV, Fonseca C (2018) Diet footprint of Egyptian mongoose along ecological gradients: effects of primary productivity and life history traits. Mamm Biol 88: 16–25. Discussion Rabbit hunting often involves the use of firearms and hunting dogs, while mongoose captures are usually undertaken by trapping (Ministério da Agricul- tura, Desenvolvimento Rural e Pescas, 2000). This difference in hunting methods could explain an increase in hCG with increasing rabbit harvest numbers. Finally, hunting data have no correction for sampling effort, which can bias abundance estimations. Even when a catch-per-unit-effort metric is used, catch data may overestimate abundance (Harley et al., 2001). In the specific case of the rabbit, discrepancies have been reported between abundance estimates using hunting data and field data (Ferreira et al., 2010). Therefore, while harvest data might provide a good metric for the assessment of the physiological impact of hunting activities on the Egyptian mongoose, caution is necessary when interpreting them as indicators of abundance. An equally surprising result was the negative effect of relative mongoose abundance on hGC. It is not clear whether high population density always leads to an increased stress response. Studies linking the GCs to conspecific density and intraspecific competition in aquatic (Leatherland, 1993; Glen- nemeier and Denver, 2002; Bolasina et al., 2006; Ramsay et al., 2006; Teixeira et al., 2012) and social (Hawley et al., 2006; Eggermann et al., 2013) species have shown inconsis- tent trends. In a non-social and aggressively territorial species like the Egyptian mongoose (Palomares and Delibes, 1993), the increased frequency of antagonistic social interactions is expected to result in increased HPA-axis activity at high population densities (Creel et al., 2013), especially during the breeding season. While a possible explanation is that aggressive interactions driven by territorial behaviour cause only transient stress responses that might not be reflected in hGC, the limitations of our harvest data on these results can- not be ignored. Firstly, hunting yields might be more reflec- tive of removal of individuals than indicators of abundance. Lower hGC levels could be a consequence of the continuous We found no support for our prediction that larger areas of Mediterranean shrub and agro-forestry habitats within each grid cell would be associated with lower levels of hGCs. Supplementary material Supplementary material is available at Conservation Physiol- ogy online. Barrett RT, Erikstad KE, Sandvik H, Myksvoll M, Jenni-Eiermann S, Kris- tensenDL,MoumT,ReiertsenTK,VikebøF(2015)Thestresshormone corticosterone in a marine top predator reflects short-term changes in food availability. Ecol Evol 5: 1306–1317. Acknowledgements Bolasina S, Tagawa M, Yamashita Y, Tanaka M (2006) Effect of stock- ing density on growth, digestive enzyme activity and cortisol level in larvae and juveniles of Japanese flounder, Paralichthys olivaceus. Aquaculture 259: 432–443. 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https://openalex.org/W3175638899	https://journals.plos.org/plosmedicine/article/file?id=10.1371/journal.pmed.1003682&type=printable	English	null	Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic: An interrupted time series analysis	PLoS medicine	2,021	cc-by	10,509	Methods and findings We performed interrupted time series (ITS) analyses of sales volumes reported in standard units (i.e., doses), collected at regular monthly intervals from January 2018 to December 2020 and obtained from IQVIA, India. As children are less prone to develop symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a predominant increase in non-child-appropriate formulation (non-CAF) sales. COVID-19- attributable changes in the level and trend of monthly sales of total antibiotics, azithromycin, and HCQ were estimated, accounting for seasonality and lockdown period where appropri- ate. A total of 16,290 million doses of antibiotics were sold in India in 2020, which is slightly less than the amount in 2018 and 2019. However, the proportion of non-CAF antibiotics increased from 72.5% (95% CI: 71.8% to 73.1%) in 2019 to 76.8% (95% CI: 76.2% to 77.5%) in 2020. Our ITS analyses estimated that COVID-19 likely contributed to 216.4 mil- lion (95% CI: 68.0 to 364.8 million; P = 0.008) excess doses of non-CAF antibiotics and 38.0 million (95% CI: 26.4 to 49.2 million; P < 0.001) excess doses of non-CAF azithromycin (equivalent to a minimum of 6.2 million azithromycin treatment courses) between June and September 2020, i.e., until the peak of the first epidemic wave, after which a negative change in trend was identified. In March 2020, we estimated a COVID-19-attributable change in level of +11.1 million doses (95% CI: 9.2 to 13.0 million; P < 0.001) for HCQ sales, whereas a weak negative change in monthly trend was found for this drug. Study limitations include the lack of coverage of the public healthcare sector, the inability to distinguish antibi- otic and HCQ sales in inpatient versus outpatient care, and the suboptimal number of pre- Academic Editor: Gwenan M. Knight, London School of Hygiene and Tropical Medicine, UNITED KINGDOM Received: February 3, 2021 Accepted: May 30, 2021 Published: July 1, 2021 Received: February 3, 2021 Accepted: May 30, 2021 Published: July 1, 2021 Received: February 3, 2021 Accepted: May 30, 2021 Published: July 1, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pmed.1003682 Copyright: © 2021 Sulis et al. OPEN ACCESS We assessed the impact of the coronavirus disease 2019 (COVID-19) epidemic in India on the consumption of antibiotics and hydroxychloroquine (HCQ) in the private sector in 2020 compared to the expected level of use had the epidemic not occurred. Citation: Sulis G, Batomen B, Kotwani A, Pai M, Gandra S (2021) Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic: An interrupted time series analysis. PLoS Med 18(7): e1003682. https://doi.org/10.1371/ journal.pmed.1003682 Giorgia SulisID1,2, Brice BatomenID3, Anita Kotwani4, Madhukar PaiID1,2, Sumanth Gandra5* 1 Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Quebec, Canada, 2 McGill International TB Centre, McGill University, Montreal, Quebec, Canada, 3 Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada, 4 Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India, 5 Division of Infectious Diseases, Department of Medicine, Washington University in St. Louis, Saint Louis, Missouri, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * gandras@wustl.edu Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic: An interrupted time series analysis Giorgia SulisID1,2, Brice BatomenID3, Anita Kotwani4, Madhukar PaiID1,2, Sumanth Gandra5* Giorgia SulisID1,2, Brice BatomenID3, Anita Kotwani4, Madhukar PaiID1,2, Sumanth Gandra5* Why was this study done? • There are concerns that the widespread and often inappropriate use of antibiotics has been aggravated by the COVID-19 pandemic, but little is known regarding the true impact of the pandemic on antibiotic use, particularly in low- and middle-income coun- tries (LMICs). hydroxychloroquine; ITS, interrupted time series; S. Typhi, Salmonella enterica serotype Typhi; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. hydroxychloroquine; ITS, interrupted time series; S. Typhi, Salmonella enterica serotype Typhi; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. • India is the largest antibiotic user in the world and is among the countries that are most severely affected by the pandemic. • About 75% of healthcare in India is private, and this unregulated and fragmented pri- vate sector accounts for 90% of antibiotic consumption, raising major concerns about the potential effects of COVID-19 on prescribing and dispensing practices. • About 75% of healthcare in India is private, and this unregulated and fragmented pri- vate sector accounts for 90% of antibiotic consumption, raising major concerns about the potential effects of COVID-19 on prescribing and dispensing practices. Author summary Abbreviations: ATC, Anatomical Therapeutic Chemical; AWaRe, Access, Watch, Reserve; CAF, child-appropriate formulation COVID-19, coronavirus disease 2019; ESBL, extended spectrum beta-lactamase; HCQ, hydroxychloroquine; ITS, interrupted time series; S. Typhi, Salmonella enterica serotype Typhi; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. Abbreviations: ATC, Anatomical Therapeutic Chemical; AWaRe, Access, Watch, Reserve; CAF, child-appropriate formulation COVID-19, coronavirus disease 2019; ESBL, extended spectrum beta-lactamase; HCQ, What did the researchers do and find? • Using an interrupted time series (ITS) design, we examined sales volumes of total anti- biotics, azithromycin alone, and hydroxychloroquine (HCQ) in India’s private sector from January 2018 to December 2020. • Focusing on non-pediatric formulations and adjusting for underlying seasonal and non-seasonal trends and accounting for the effect of lockdown, we estimated the impact of the first epidemic wave on monthly sales. • Based on our models, COVID-19 likely contributed to about 216 million excess doses (95% CI: 68.0 to 364.8 million; P = 0.008) of total antibiotics and 38.0 million excess doses (95% CI: 26.4 to 49.2 million; P < 0.001) of azithromycin between June and Sep- tember 2020 (i.e., after the lockdown and until the epidemic peak). • HCQ sales peaked in March 2020, reflecting the widespread use of this drug for both prophylaxis and treatment of COVID-19 (+11.1 million doses [95% CI: 9.2 to 13.0 mil- lion]; P < 0.001), followed by a slow decline afterwards. Conclusions A significant increase in non-CAF antibiotic sales, and particularly azithromycin, occurred during the peak phase of the first COVID-19 epidemic wave in India, indicating the need for urgent antibiotic stewardship measures. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MP is a member of the Editorial Board of PLOS Medicine, he coedits the PLOS Tuberculosis Channel and is the Editor- in-Chief of PLoS Global Public Health. All the other authors have no conflicts of interest to declare. Methods and findings This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data cannot shared publicly because of license agreement with the IQVIA Inc. The data underlying the results PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 1 / 18 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic and post-epidemic data points, which could have prevented an accurate adjustment for sea- sonal trends despite the robustness of our statistical approaches. and post-epidemic data points, which could have prevented an accurate adjustment for sea- sonal trends despite the robustness of our statistical approaches. and post-epidemic data points, which could have prevented an accurate adjustment for sea- sonal trends despite the robustness of our statistical approaches. presented in the study are available from IQVIA Consulting and Information Services India Pvt. Ltd. https://www.iqvia.com/locations/india. Funding: The authors received no specific funding for this work. presented in the study are available from IQVIA Consulting and Information Services India Pvt. Ltd. https://www.iqvia.com/locations/india. Funding: The authors received no specific funding for this work. Introduction India is the largest consumer of antibiotics in the world [1,2]. Broad spectrum antibiotics such as second- and third-generation cephalosporins, macrolides, and quinolones are overused for acute respiratory tract infections in India [3]. There is a concern that symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), could lead to a substantial increase in antibiotic consumption (often inappropriately), thus promoting antibiotic resistance [4]. In many countries, azithromycin and hydroxychloroquine (HCQ) are reportedly being used off label in prophylactic and therapeutic regimens either alone or in combination. In India, azithromycin is typically utilized to treat a range of conditions, including acute respira- tory tract infections, bacterial dysentery, and enteric fever [5]. This macrolide antibiotic was repurposed for the treatment of COVID-19 based on in its hypothetical anti-inflammatory and immunomodulatory properties [6–8]. On the other hand, HCQ in India is mainly utilized for treatment of autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, and post-viral infectious arthritis, such as chikungunya arthritis, and is not part of national malaria treatment guidelines [9–12]. It has been suggested that HCQ could have antiviral activity as well as indirect anti-inflammatory properties through the acti- vation of CD8+ T cells and the reduction of pro-inflammatory cytokine response, thus leading to its widespread use in the management of COVID-19 as well as in pre- and post-exposure prophylaxis [13,14]. However, an increasing number of studies have observed no beneficial effects from the use of azithromycin and/or HCQ, and a number of safety concerns have also been raised [15–19]. A growing body of evidence from observational studies across multiple countries consistently indicates that only a small proportion of hospitalized COVID-19 patients develop secondary bacterial infections, with higher rates observed in intensive care units [20,21]. The risk of developing bacterial co-infections remains presumably very low in non- hospitalized patients with mild disease, who represent the majority of individuals with COVID-19. These observations suggest against the routine empirical use of antibiotics in the treatment of COVID-19 cases unless there is evidence of bacterial infection, as recommended by WHO and Indian Ministry of Health guidelines [22,23]. A few before-and-after studies have been conducted to determine the impact of COVID-19 on antibiotic use, but these were all done in high-income countries (see S1 Text and S1 and S2 Tables) [24–31]. • Similar trends are likely to have occurred in other LMICs, where antibiotics are often overused. • Similar trends are likely to have occurred in other LMICs, where antibiotics are often overused. • The medium- and long-term consequences for bacterial resistance patterns are highly concerning, highlighting the need for urgent antibiotic stewardship measures. What do these findings mean? • Our findings indicate a significant increase in antibiotic sales, particularly of azithromy- cin, during the peak phase of the first COVID-19 epidemic wave in India. • Our findings indicate a significant increase in antibiotic sales, particularly of azithromy- cin, during the peak phase of the first COVID-19 epidemic wave in India. 2 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic • Similar trends are likely to have occurred in other LMICs, where antibiotics are often overused. PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Temporal data on COVID-19 in India The first imported case of SARS-CoV-2 infection in India was identified on 30 January 2020. Until late March 2020 the number of cases detected across the country remained very low (about 0.1 per 100,000 population), although this might be partly explained by the limited number of tests being performed. In order to examine associations between drug sales volumes and COVID-19 cases, national and state-wise data regarding the monthly number of new cases detected in India were obtained from the publicly accessible online repository compiled by the Indian non-profit organization PRS Legislative Research, based on data from the Minis- try of Health and Family Welfare, Government of India [32]. The monthly number of tests performed in the country was obtained from Our World in Data [36]; however, this informa- tion is not available for individual states. Projected population estimates as determined by the National Commission on Population, Ministry of Health and Family Welfare, were utilized to calculate cumulative monthly rates of new cases and tests per 100,000 population [37]. For the purpose of our regression analyses, the exposure was treated as a binary variable (pre-epidemic phase coded 0 versus epidemic phase coded 1) as detailed below. Introduction With about 27.2 million COVID-19 cases reported as of 25 May 2021, India is among the hardest hit countries in the world [32]. In this study, we assessed the impact of the first COVID-19 epidemic wave on the consumption of antibiotics and HCQ in 2020 in India’s pri- vate sector, which accounts for three-quarters of healthcare delivery and 90% of antibiotic sales in the country [33,34]. 3 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Methods Study design We conducted interrupted time series (ITS) analyses using total antibiotics, azithromycin, and HCQ sales volumes as our continuous outcome, and COVID-19 epidemic as the exposure of interest [35]. Our counterfactual (i.e., sales volumes had the pandemic not occurred) was thus the extrapolation of the pre-epidemic period. Although a formal detailed protocol was not developed, our analytic plan was designed a priori, before performing the analyses. However, our original study included sales data only up to September 2020 and was updated during the review process as more recent data became available to the study team. In the absence of a vali- dated checklist for impact evaluation studies such as this, we followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines (S1 STROBE Checklist). PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Data analysis We performed descriptive analyses of antibiotics and HCQ sales data throughout the observa- tion period, reporting the absolute number of doses sold along with crude percentages of each drug class relative to the total. Medians and interquartile ranges (IQRs) were also used to describe overall and stratum-specific monthly sales volumes. Descriptive analyses were also performed to explore trends up to September 2020 (peak of the epidemic wave) in selected states/territories reporting either a very high number of cases (Andhra Pradesh, Delhi, Karna- taka, Maharashtra, Tamil Nadu) or a very low number of cases (Bihar, Gujarat, Madhya Pra- desh, Rajasthan, West Bengal). Next we conducted segmented regression analyses of time series data to assess how much the epidemic onset affected monthly sales volumes of (1) all antibiotics (including azithromy- cin), (2) azithromycin only (categorized as Schedule H), and (3) HCQ (categorized as Schedule H1 since March 2020) [35,42]. We decided a priori to exclude CAFs from these assessments as we anticipated an increase in antibiotic sales mainly among adult patients. Children constitute a small proportion of reported COVID-19 cases and are much less likely to develop symptom- atic SARS-CoV-2 infection [43,44]. Furthermore, social distancing measures and school clo- sure remained in place in most Indian states throughout the study period. As also documented in the United States [31,45], such a scenario likely plays a role in reducing the transmission of many respiratory infections that typically spread among children, leading to lower antibiotic use. Our models for total non-CAF antibiotics and azithromycin estimated the following mea- sures: (1) pre-epidemic trend (January 2018 to March 2020), (2) average level of change in mean monthly sales during the preventive lockdown, (3) the slope (trend) change in the out- come after the lockdown phase (i.e., from June 2020 onwards), and (4) the slope (trend) change in the outcome during the declining phase of the first epidemic wave (i.e., after Sep- tember 2020) relative to the rising phase. We introduced the term “declining phase” when the analyses were updated to incorporate more recent data from October to December 2020, to better reflect the change in the epidemic trend during the study period. The model described above allowed us to account for the effect of the nationwide lockdown enforced by the Govern- ment of India between 24 March and 31 May 2020. Antibiotic and HCQ sales data The main outcomes of interest for this study were the sales volumes of antibiotics and HCQ in India, using data obtained from IQVIA, which is a reliable source of drug sales data [1,38]. IQVIA is a company that collects over-the-counter (OTC) and prescription-based sales data by auditing sales from a representative panel of drug stockists. The data are then extrapolated to all stockists in the country using a proprietary projection algorithm. This accounts for an estimated 95% of the total pharmaceutical market in terms of value sales combining the retail sector, hospitals, and dispensing doctors. In India, all antibiotics are included in Schedule H or H1. Schedule H is a class of prescription drugs that cannot be purchased without the prescrip- tion of a qualified doctor. For Schedule H1 drugs, in addition to having a prescription, the dis- penser should record the prescriber and patient details, the drug, and the quantity dispensed and maintain the record for 3 years, and the record should be open for inspection by regula- tory officials. However, OTC dispensing of antibiotics is common in India [39]. Regular monthly data points from January 2018 to December 2020 were available for the purpose of our analyses. Sales volumes were reported in standard units (SU), and 1 SU (i.e., 1 dose) was defined as a single tablet, capsule, ampoule, vial, or a 5-mL liquid preparation for oral con- sumption, as reported previously [38]. Information on formulation type with regard to the route of administration (oral, parenteral, topical) was also available. We further classified oral PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 4 / 18 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic drugs as child-appropriate formulations (CAFs) or non-CAFs based on the description of the package content (the list of formulation types considered for this purpose is provided in S3 Table), as reported previously [38]. Antibiotics were categorized according to the Anatomical Therapeutic Chemical (ATC) Index 2020 and the WHO Access, Watch, Reserve (AWaRe) framework 2019 [40,41]. The full list of drugs (intended as active molecule) included in our dataset is available in S4 Table. Pattern of antibiotic and HCQ sales throughout the study period The absolute cumulative volume of antibiotics sold in 2020 was 16,290 million doses, which is slightly lower than the 18,167 million doses and 18,002 million doses sold in the same period of 2019 and 2018, respectively (Table 1). The CAF sales volume amounted to 3,779 million doses in 2020 as opposed to 4,998 million doses in 2019 and 4,934 million doses in 2018. The proportion of non-CAF sales among total antibiotics, likely prescribed and dispensed to ado- lescents and adults (although pediatric and non-pediatric use are indistinguishable for inject- ables), increased from 72.5% (95% CI: 71.8% to 73.1%) in 2019 to 76.8% (95% CI: 76.2% to 77.5%) in 2020 (Fig 1). The distribution of AWaRe categories remained almost stable over time except for a slight decline in the use of “discouraged” fixed-dose antibiotic combinations, which could be ascribed to a policy change introduced in September 2018 and was accompanied by an increase in the use of “access” antibiotics (Table 1; S4 and S5 Figs). The median (IQR) percent- ages of the different AWaRe categories relative to the total non-CAF antibiotics sold monthly throughout the entire study period (2018–2020) were as follows: “access”, 43.0% (42.2%– 44.4%); “watch”, 36.8% (35.4%–37.6%); “reserve”, 0.8% (0.7%–0.9%); and “discouraged”, 18.8% (17.4%–19.7%). The distribution of antibiotics by ATC class remained stable except for a noteworthy increase in non-CAF macrolide (J01F) sales, jumping from 947 million doses in 2018 to 1,124 million doses in 2020 (Table 1; S6 Fig). After the end of lockdown, between June and Septem- ber 2020, azithromycin (J01FA10) sales were 34.4% higher than observed in the corresponding months of the previous year, followed by a decline after the peak of the first epidemic wave (Fig 1). Besides azithromycin, 2 other antibiotics, doxycycline and faropenem, that are com- monly used for respiratory tract infections showed increased consumption. Monthly doxycy- cline (J01AA02) sales did not change much until September 2020, when a considerable peak was observed (+25.9% compared to September 2019). Faropenem (J01DI03) use has been ris- ing constantly over the years, but a 23.4% increase was registered in September 2020 versus the year before (Fig 1). No major changes were observed in the sales volumes of other broad spec- trum antibiotic classes, such as second- and third-generation cephalosporins and quinolones. Data analysis During this time, several restrictions were imposed on the entire population, including—but not limited to—closure of schools and all nonessential services, ban on stepping out from home, and curfew. For this reason and because of the still limited circulation of the SARS-CoV-2 infection within the community, we hypoth- esized that antibiotic sales could have been negatively affected. A fixed effect term for the rainy season (July to October) was included in the model for antibiotics to adjust for seasonality. As this approach did not perform equally well for azithromycin sales, for this outcome we used a harmonic seasonal model to better account for seasonal changes [46], along with further adjustments for non-seasonal autocorrelation. Given the initial recommendation for HCQ-based prophylaxis, particularly among health- care workers [47], we expected a weaker effect of lockdown on HCQ sales and did not account for it in the model. We thus estimated the average change in level and the slope change in the 5 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic outcome assuming the start of the COVID-19 epidemic in March 2020. Autocorrelated errors were also included to correct for the remaining serial correlation in the data, whereas no adjustments for seasonality were deemed necessary. HCQ is not recommended for malaria treatment according to Indian guidelines, so no major seasonal changes are expected to occur in its use. A detailed description of model specification and diagnostics is provided in S2 Text and S1–S3 Figs. Descriptive analyses were performed in STATA version 16.1 (StataCorp, College Station, TX, US), and regression analyses were conducted in R (version 4.0.3). Ethics considerations The institutional review boards of Washington University in St. Louis and McGill University exempted this study from ethics review as no identifiable information about living individuals was obtained (i.e., secondary use of anonymous information). PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Pattern of antibiotic and HCQ sales throughout the study period Similarly, monthly sales of selected parenteral antibiotics that are typically used in inpatient care, such as carbapenems, glycopeptides, third-generation cephalosporins, and polymyxins, remained almost stable (S7 Fig). 6 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic PLOS MEDICINE Table 1. Cumulative antibiotic sales volume per year (2018–2020), and distribution by AWaRe category and ATC class for formulations other than child-appropri- ate ones (non-CAF). Category Cumulative sales volume, in million standard units 2018 2019 2020 Sales volume Percent Sales volume Percent Sales volume Percent All antibiotics 18,002 100 18,167 100 16,290 100 Non-CAFs 13,068 72.6 13,169 72.5 12,512 76.8 CAFs 4,934 27.4 4,998 27.5 3,779 23.2 AWaRe category (non-CAF) Access 5,479 41.9 5,656 42.9 5,621 44.9 Watch 4,821 36.9 4,820 36.6 4,569 36.5 Reserve 87 0.7 113 0.9 115 0.9 Discouraged 2,578 19.7 2,484 18.9 2,142 17.1 Not included in AWaRe 103 0.8 96 0.7 65 0.5 ATC class (non-CAF) Aminoglycosides 240 1.8 237 1.8 184 1.5 BL-BLI 1,228 9.4 1,380 10.5 1,141 9.1 Carbapenems 39 0.3 46 0.3 48 0.4 Cephalosporin-BLI 425 3.3 477 3.6 402 3.2 Cephalosporins (first generation) 397 3.0 395 3.0 374 3.0 Cephalosporins (second generation) 232 1.8 248 1.9 216 1.7 Cephalosporins (third generation) 1,451 11.1 1,657 12.6 1,489 11.9 Cephalosporins (fourth generation+) 2 <0.1 2 <0.1 1 <0.1 Combinations 2,206 16.9 2,053 15.6 1,768 14.1 Glycopeptides 3 <0.1 4 <0.1 3 <0.1 Imidazoles 1,419 10.9 1,485 11.3 1,445 11.5 Macrolides 947 7.2 1,009 7.7 1,124 9.0 Penicillins 1,114 8.5 1,154 8.8 1,207 9.6 Polymyxins 2 <0.1 2 <0.1 1 <0.1 Quinolones 1,718 13.1 1,664 12.6 1,546 12.4 Sulfonamides 329 2.5 203 1.5 321 2.6 Tetracyclines 869 6.6 669 5.1 611 4.9 Other antibiotics 446 3.4 484 3.7 467 3.7 018–2020), and distribution by AWaRe category and ATC class for formulations other than child-appropri- Furthermore, cumulative HCQ sales (only available as non-CAF) increased by approxi- mately 35.4% between 2019 and 2020 (from 274 million doses in 2019 to 371 million doses in 2020) (Fig 1). ATC, Anatomical Therapeutic Chemical; AWaRe, Access, Watch, Reserve; BL, beta-lactam; BLI, beta-lactamase inhibitor; CAF, child-appropriate formulation. Percentages are calculated relative to all antibacterial drugs. Percentages are calculated relative to non-CAFs of antibacterial drugs. Including combinations of carbapenems and BLI. ATC, Anatomical Therapeutic Chemical; AWaRe, Access, Watch, Reserve; BL, beta-lactam; BLI, beta-lactamase inhibitor; CAF, child-appropriate formulation. Percentages are calculated relative to all antibacterial drugs. Impact of COVID-19 on antibiotic and HCQ sales Crude monthly sales of non-CAF antibiotics increased with the number of new COVID-19 cases per 100,000 population, a pattern that is clearly observable both nationally (Fig 2) and in selected Indian states with different epidemic curves (Figs 3, S8, and S9). Rising trends are evi- dent both in states with a high number of reported cases and in those with lower incidence. The reported number of COVID-19 cases in India remained quite low until June, reflecting PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 7 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Fig 1. Trend in sales volumes of total antibiotics, azithromycin, doxycycline, faropenem, and HCQ in India from January 2018 to December 2020. CAF, non-CAF, and total sales are presented in the graphs, as relevant. Data on antibiotics are inclusive of azithromycin. HCQ and faropenem are only shown as non-CAFs because CAFs are not available for these drugs. As only a very small proportion of doxycycline is sold as CAF, this is omitted in the graph. CAF, child appropriate formulations; HCQ, hydroxychloroquine, SU, standard unit. https://doi.org/10.1371/journal.pmed.1003682.g001 Fig 1. Trend in sales volumes of total antibiotics, azithromycin, doxycycline, faropenem, and HCQ in India from January 2018 to December 2020. CAF, non-CAF, and total sales are presented in the graphs, as relevant. Data on antibiotics are inclusive of azithromycin. HCQ and faropenem are only shown as non-CAFs because CAFs are not available for these drugs. As only a very small proportion of doxycycline is sold as CAF, this is omitted in the graph. CAF, child appropriate formulations; HCQ, hydroxychloroquine, SU, standard unit. https://doi.org/10.1371/journal.pmed.1003682.g001 https://doi.org/10.1371/journal.pmed.1003682.g001 the difficulties in testing scale-up across the country, particularly during the first half of 2020 (S10 Fig). Antibiotic sales volumes declined during the lockdown phase (April to May 2020). As esti- mated through segmented regression analyses (Table 2; Fig 4), non-CAF antibiotic and azi- thromycin sales in April 2020 decreased on average by 197.3 million doses (95% CI: −294.7 to −99.9 million; P < 0.001) and 11.3 million doses (95% CI: −17.6 to −5.0 million; P < 0.001), respectively. PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Impact of COVID-19 on antibiotic and HCQ sales Moreover, we observed a monthly increase in trend after the lockdown period for both non-CAF antibiotics (+54.1 million doses [95% CI: 17.0 to 91.2 million]; P = 0.008) and non-CAF azithromycin (+9.5 million doses [95% CI: 6.6 to 12.3 million]; P < 0.001) from June to September 2020. Cumulative excess antibiotic sales from June to September 2020 amounted to 216.4 million doses (95% CI: 68.0 to 364.8 million; P = 0.008) for non-CAF anti- biotics and 38.0 million doses (95% CI: 26.4 to 49.2 million; P < 0.001) for non-CAF azithro- mycin. The latter is equivalent to about 6.2 million azithromycin treatment courses for respiratory tract infection, considering a course to be 500 mg daily for 5 days (S2 Text). After the epidemic peak in September 2020, a declining trend in sales was observed from October to December 2020, but this was significant only for azithromycin (−20.8 million doses [95% CI: −26.93 to −14.73 million]; P < 0.001) (Table 2). We also estimated a change of +11.1 million doses (95% CI: 9.2 to 13.0 million; P < 0.001) for HCQ sales in March 2020 (Table 3; Fig 4). After this peak, sales began declining slowly, as confirmed by the weak negative change in trend suggested by our model (−0.6 million doses 8 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Fig 2. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and national sales volumes of antibiotics, azithromycin, doxycycline, faropenem, and HCQ from January to December 2020. CAF, non-CAF, and total sales are reported. Data on antibiotics are inclusive of azithromycin. HCQ and faropenem are only shown as non-CAFs because CAFs are not available for these drugs. As only a very small proportion of doxycycline is sold as CAF, this is omitted in the graph. CAF, child-appropriate formulation; HCQ, hydroxychloroquine, SU, standard unit. Fig 2. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and national sales volumes of antibiotics, azithromycin, doxycycline, faropenem, and HCQ from January to December 2020. CAF, non-CAF, and total sales are reported. Data on antibiotics are inclusive of azithromycin. HCQ and faropenem are only shown as non-CAFs because CAFs are not available for these drugs. As only a very small proportion of doxycycline is sold as CAF, this is omitted in the graph. Impact of COVID-19 on antibiotic and HCQ sales CAF, child-appropriate formulation; HCQ, hydroxychloroquine, SU, standard unit. Fig 2. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and national sales volumes of antibiotics, azithromycin, doxycycline, faropenem, and HCQ from January to December 2020. CAF, non-CAF, and total sales are reported. Data on antibiotics are inclusive of azithromycin. HCQ and faropenem are only shown as non-CAFs because CAFs are not available for these drugs. As only a very small proportion of doxycycline is sold as CAF, this is omitted in the graph. CAF, child-appropriate formulation; HCQ, hydroxychloroquine, SU, standard unit. https://doi.org/10.1371/journal.pmed.1003682.g002 https://doi.org/10.1371/journal.pmed.1003682.g002 [95% CI: −1.0 to −0.1 million]; P = 0.010), which became more pronounced after September 2020 (−3.1 million doses [95% CI: −4.3 to −1.9 million]; P < 0.001). [95% CI: −1.0 to −0.1 million]; P = 0.010), which became more pronounced after September 2020 (−3.1 million doses [95% CI: −4.3 to −1.9 million]; P < 0.001). PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Fig 3. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and azithromycin sales volumes per 100,000 population (only non-child-appropriate formulations) in 10 states of India from January to September 2020. States with the highest rates of detected COVID-19 cases are shown on the left side of the graph, whereas states with the lowest rates of detected COVID-19 cases are on the right. https://doi.org/10.1371/journal.pmed.1003682.g003 Fig 3. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and azithromycin sales volumes per 100,000 population (only non-child-appropriate formulations) in 10 states of India from January to September 2020. States with the highest rates of detected COVID-19 cases are shown on the left side of the graph, whereas states with the lowest rates of detected COVID-19 cases are on the right. https://doi.org/10.1371/journal.pmed.1003682.g003 Fig 3. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and azithromycin sales volumes per 100,000 population (only non-child-appropriate formulations) in 10 states of India from January to September 2020. States with the highest rates of detected COVID-19 cases are shown on the left side of the graph, whereas states with the lowest rates of detected COVID-19 cases are on the right. https://doi.org/10.1371/journal.pmed.1003682.g003 https://doi.org/10.1371/journal.pmed.1003682.g003 healthcare facilities for conditions other than acute respiratory infections (i.e., COVID-19 sus- picion). Therefore, we expect antibiotics to be less commonly prescribed for other types of ill- ness as compared to the previous years, suggesting that the COVID-19-attributable excess sales indicated by our models might be an underestimation. On the other hand, we observed a notable reduction in CAF sales, suggesting that antibiotic use among children has declined since the start of the pandemic, which is in line with our hypotheses [31,43,45]. Notably, the massive increase in azithromycin use raises several serious concerns. First, a recent randomized controlled study investigating the effects of mass distribution of azithromy- cin in Nigerian children demonstrated an increase not only in macrolide resistance determi- nants but also non-macrolide resistance in the gut flora such as resistance to beta-lactams [49]. Multi-drug-resistant Enterobacterales, including extended spectrum beta-lactamase (ESBL)– producing strains, are highly prevalent among healthy adults in the community and could be further aggravated with this unexpected increase in azithromycin use [50]. Discussion We estimate that between June and September 2020, with peak epidemic activity, COVID-19 likely contributed to excess sales of 216 million doses of non-CAF antibiotics and 38 million doses of non-CAF azithromycin. The excess antibiotic sales likely resulted from the sudden surge in the number of patients seeking medical care for presumptive or confirmed COVID- 19 both in the community and in the hospitals, as suggested by the abrupt increase in use of azithromycin, often prescribed for this condition. Assuming perfect adherence to the recom- mended dosage and duration of azithromycin treatment for respiratory tract infections not related to COVID-19 per Indian national guidelines (i.e., 500 mg daily for 5 days), 38 million excess doses from June to September 2020 corresponds to about 6.2 million treatment courses. During this period, 6 million new COVID-19 cases were reported in India across both public and private sectors, suggesting empirical use of azithromycin in the private sector in the absence of confirmed SARS-CoV-2 infection [32]. Moreover, azithromycin is often prescribed for shorter duration (e.g., 500 mg per day for 3 days) [48], potentially indicating that more peo- ple could have been treated empirically without diagnostic confirmation of SARS-CoV-2 infection. To support this, in states like Bihar, Gujarat, and West Bengal, where the number of cases is reportedly low and tests are not widely available nor accessible, azithromycin con- sumption has risen considerably. It should also be noted that healthcare-seeking behaviors have changed substantially during the pandemic period, with fewer people presenting to PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 9 / 18 PLOS MEDICINE PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Second, the sudden ongoing mass consumption of azithromycin has the potential to further exacerbate the selec- tion of azithromycin-resistant typhoidal and non-typhoidal Salmonella strains [51]. This is of PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 10 / 18 Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic PLOS MEDICINE PLOS MEDICINE Table 2. Estimated change in monthly sales volume (expressed in million SU) according to adjusted segmented regression models for total antibiotics and azithromycin. Measure Sales volume in million SU All antibiotics Azithromycin Estimate (95% CI) P value Estimate (95% CI) P value Baseline level (January 2018) 1,014.2 (963.4 to 1,065.1) <0.001 39.4 (36.5 to 42.4) <0.001 Pre-epidemic trend (monthly change from January 2018 to March 2020) 0.6 (−2.6 to 3.8) 0.725 0.6 (0.5 to 0.8) <0.001 Average change in level during lockdown (April to May 2020) versus the pre-epidemic period −197.3 (−294.7 to −99.9) <0.001 −11.3 (−17.6 to −5.0) <0.001 Change in trend after lockdown (after May 2020) 54.1 (17.0 to 91.2) 0.008 9.5 (6.6 to 12.3) <0.001 Change in trend after the epidemic peak (i.e., after September 2020) relative to the rising phase of the epidemic −64.3 (−150.6 to 21.9) 0.154 −20.8 (−26.9 to −14.7) <0.001 SU, standard unit. Only non-child-appropriate formulations were considered for these analyses. Model adjusted for seasonality using a fixed effect term indicating the rainy season. Harmonic seasonal model to adjust for seasonality, and autocorrelated errors to account for the remaining serial correlation in the data. https://doi org/10 1371/journal pmed 1003682 t002 particular concern for India, where enteric fever is highly endemic, with an estimated annual incidence of 377 cases per 100,000 population, and azithromycin has been increasingly chosen for empirical treatment [52]. The recent emergence of azithromycin-resistant Salmonella enterica serotype Typhi (S. Typhi) strains in India sounds a further alarm bell [53]. Another threat coming from this unexpected increase in azithromycin use is the possible selection of Fig 4. Results of segmented regression analysis for monthly sales volumes of non-CAF antibiotics, azithromycin, and HCQ between January 2018 and December 2020. https://doi org/10 1371/journal pmed 1003682 g004 Fig 4. Results of segmented regression analysis for monthly sales volumes of non-CAF antibiotics, azithromycin, and HCQ between January 2018 and December 2020. https://doi.org/10.1371/journal.pmed.1003682.g004 Fig 4. Results of segmented regression analysis for monthly sales volumes of non-CAF antibiotics, azithromycin, and HCQ between January 2018 and December 2020. https://doi.org/10.1371/journal.pmed.1003682.g004 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 11 / 18 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Table 3. Estimated change in monthly sales volume (expressed in million SU) according to adjusted segmented regression models for HCQ. PLOS MEDICINE Measure HCQ sales volume in million SU Estimate (95% CI) P value Baseline level (January 2018) 20.2 (19.3 to 21.2) <0.001 Pre-epidemic trend (monthly change from January 2018 to March 2020) 0.2 (0.1 to 0.2) <0.001 Change in level in March 2020 versus the pre-epidemic period 11.1 (9.2 to 13.0) <0.001 Change in trend after March 2020 −0.6 (−1.01 to −0.14) 0.010 Change in trend after the epidemic peak (i.e., after September 2020) relative to the rising phase of the epidemic −3.08 (−4.3 to −1.9) <0.001 HCQ, hydroxychloroquine; SU, standard unit. Only non-child-appropriate formulations were considered for these analyses. Model adjusted for non-seasonal autocorrelation. In March 2020 (i.e., when the number of cases in India was still very low), India’s Ministry of Health and Family Welfare issued a recommendation for use of HCQ in prophylaxis. The effect of the lockdown phase was not considered in this model based on the assumption that HCQ was predominantly used for prophylaxis among healthcare workers and thus unlikely to be negatively impacted by the lockdown as observed for antibiotics, and azithromycin in particular. https://doi.org/10.1371/journal.pmed.1003682.t003 Table 3. Estimated change in monthly sales volume (expressed in million SU) according to adjusted segmented regression models for HCQ. PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Measure Among the biggest threats associated with the widespread use of HCQ is the occurrence of adverse events and toxic effects, particularly when given in combination with other drugs with similar adverse effects. There are some limitations in our study. First, IQVIA data only cover the private healthcare sector. Although this does not allow us to draw conclusions regarding the impact of the pan- demic on antibiotic usage in the public sector, it should be highlighted that the private sector accounts for 75% of healthcare in India and 90% of national antibiotic sales [33,34]. Second, our data could not distinguish between inpatient and outpatient use of antibiotics, but the lat- ter is known to be largely predominant. Third, while we applied the most appropriate tech- niques to adjust for seasonal variations in the outcome, the suboptimal number of pre- and post-pandemic data points available for our analyses remains a limitation in that sense. None- theless, our models were quite robust and fitted the data reasonably well, as the residuals of each model were behaving as white noise; yet we recommend caution in interpreting the esti- mated impact on HCQ sales owing to the non-stationary nature of the time series. Fourth, we did not have data in defined daily doses (DDDs); however, there is a very good correlation between DDDs and standard units when estimating antibiotic consumption per person [58]. Fifth, while data from IQVIA are widely utilized to evaluate pharmaceutical sales, it should be highlighted that the company adopts a propriety method for estimating national sales. Finally, there is a lag time between sales from stockists and purchase by the end customer at the retail pharmacy, which could not be determined through the dataset. This could be a source of mea- surement error in the outcome data, although the lag time was likely lower during the epi- demic due to the increase in demand for antibiotics. Our findings have important implications for antimicrobial resistance globally and even more so for low- and middle-income countries (LMICs). Like in India, the overuse of antibiot- ics is common in other LMICs [2,59], where similar prescribing patterns among presumptive or confirmed COVID-19 cases likely exist. The situation could be even worse in other coun- tries like Pakistan where azithromycin is the only treatment option for S. Typhi infections, and an outbreak of extremely drug-resistant strains recently occurred [60]. Measure pan-oral-drug-resistant S. Typhi, requiring hospitalization for parenteral treatment adminis- tration [51]. Furthermore, azithromycin is currently recommended by the US Centers for Dis- ease Control and Prevention for travelers’ diarrhea in South Asia and Southeast Asia due to increasing fluoroquinolone-resistant strains among common bacterial diarrheal pathogens such as Salmonella, Shigella, and Campylobacter spp. [54]. The growing use of azithromycin could further jeopardize the available therapeutic choices for travelers’ diarrhea. Finally, the empirical use of azithromycin for presumptive COVID-19 could lead to a progressive substitu- tion of beta-lactam antibiotics (J01C) for any acute respiratory tract illness, aggravating the concerns regarding resistance selection. Among other oral agents commonly used for respiratory tract infections, doxycycline and faropenem sales peaked in September 2020. Faropenem is an oral “penem” drug that has been approved in India for several clinical indications including community-acquired respiratory tract infections [55]. A recent in vitro study demonstrated cross-resistance to carbapenems among ESBL-producing Escherichia coli isolates [55]. The unnecessary use of faropenem could promote intestinal colonization of carbapenem-resistant Enterobacterales in a context like India where there is a high community burden of ESBL positivity. However, the decline in far- openem sales after September 2020 was not as apparent as the declines in azithromycin and doxycycline sales, indicating that faropenem may have been used for non-respiratory-tract- infection indications. Regarding HCQ, the sudden increase in consumption registered in March 2020 could be attributed to prophylaxis for healthcare workers, as initially recom- mended by the Ministry of Health and Family Welfare [47]. The national guidelines were sub- sequently revised on 27 June 2020, limiting the prescription of HCQ to moderate to severe COVID-19 cases and to patients with mild disease if immunocompromised or under 5 years old [56]. This change in recommendations is reflected in the slowly declining trend observed after the initial peak. Moreover, HCQ is unlikely to be prescribed for mild cases evaluated by primary care physicians or informal healthcare providers, who often recommend/dispense antibiotics like azithromycin but have less experience in handling HCQ-based treatment. PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 12 / 18 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic Additionally, in March 2020, the Indian government issued an emergency order imposing stronger restrictions on HCQ sales by including it in Schedule H1 [57]. Measure Policy makers in India and other LMICs should recognize this substantial overuse of antibiotics induced by COVID- 19. A second and substantially more devastating epidemic wave hit India from April 2021 onwards. This could result in severe antimicrobial resistance consequences if the amount of antibiotic use follows a similar pattern as in the first wave, and thus warrants reexamining the impact of the second wave on antibiotic use. Considering the ongoing trends, the very low vac- cination coverage level, and the amount of time necessary to eventually vaccinate the entire population, immediate action is needed to reduce the overuse of antibiotics for COVID-19. India will need to greatly increase COVID-19 testing access to reduce empirical treatments. Issuing further restrictions on azithromycin prescription by moving it from Schedule H to H1, as done with HCQ, could potentially help limit the widespread use of this important antibiotic. Similar restrictions on azithromycin use should also be considered in other LMICs. Antimi- crobial stewardship interventions have never been so critical, and mass media awareness cam- paigns targeting prescribers and the general public to discourage the routine use of antibiotics for COVID-19 need to be rapidly implemented in India and other LMICs. Supporting information S1 STROBE Checklist. (PDF) Supporting information S1 STROBE Checklist. (PDF) 13 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic S1 Fig. Autocorrelation, partial autocorrelation, and distribution of residuals from model 1 for total antibiotic sales. (TIF) S2 Fig. Autocorrelation, partial autocorrelation, and distribution of residuals from model 3 for azithromycin sales. (TIF) S3 Fig. Autocorrelation, partial autocorrelation, and distribution of residuals from model 5 for hydroxychloroquine sales. (TIF) S4 Fig. Monthly sales volume of each AWaRe category in India between January 2018 and December 2020, separated for child-appropriate formulations (CAFs) and non-CAFs. (TIF) S5 Fig. Cumulative volume of antibiotics sold per year (2018–2020), stratified by AWaRe category, presented separately for child-appropriate formulations (CAFs) and non-CAFs. (TIF) S6 Fig. Cumulative volume of antibiotics sold per year (2018–2020), stratified by ATC class and presented separately for child-appropriate formulations (CAFs) and non-CAFs. (TIF) S7 Fig. Monthly sales volumes between January 2018 and December 2020 for selected anti- biotic ATC classes: Parenteral carbapenems, glycopeptides, polymyxins, and parenteral third-generation cephalosporins (including those associated with a beta-lactamase inhibi- tor [BLI]). (TIF) S8 Fig. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and antibiotic sales volumes per 100,000 (only non-child appropriate formulations [non-CAFs]) in 10 states of India from January to September 2020. States with the highest rates of detected COVID-19 cases are shown on the left side of the graph, whereas states with the lowest rates of detected COVID-19 cases are on the right. (TIF) S9 Fig. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and hydroxychloroquine (HCQ) sales volumes per 100,000 (only non-child appro- priate formulations [non-CAFs]) in 10 states of India from January to September 2020. States with the highest rates of detected COVID-19 cases are shown on the left side of the graph, whereas states with the lowest rates of detected COVID-19 cases are on the right. (TIF) S10 Fig. Number of SARS-CoV-2 tests performed and number of new COVID-19 cases detected each month in India per 100,000 inhabitants between January and December 2020. (TIF) S1 Table. Search strategy used in the rapid systematic review regarding the impact of the COVID-19 pandemic on antibiotic use. (PDF) PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 Project administration: Sumanth Gandra. Resources: Madhukar Pai, Sumanth Gandra. Software: Giorgia Sulis. Software: Giorgia Sulis. Supervision: Sumanth Gandra. Supervision: Sumanth Gandra. Validation: Giorgia Sulis, Brice Batomen, Sumanth Gandra. Visualization: Giorgia Sulis. Visualization: Giorgia Sulis. Writing – original draft: Giorgia Sulis. Writing – original draft: Giorgia Sulis. Writing – review & editing: Giorgia Sulis, Brice Batomen, Anita Kotwani, Madhukar Pai, Sumanth Gandra. S2 Table. Features and findings of studies that evaluated the impact of COVID-19 on anti- biotic use. (PDF) S4 Table. List of all antibiotics included in our dataset, along with ATC class, AWaRe cate- gory (2019), and Schedule H/H1. (PDF) S4 Table. List of all antibiotics included in our dataset, along with ATC class, AWaRe cate- gory (2019), and Schedule H/H1. (PDF) S4 Table. List of all antibiotics included in our dataset, along with ATC class, AWaRe cate- gory (2019), and Schedule H/H1. (PDF) S1 Text. Summary of the evidence regarding the impact of the COVID-19 pandemic on antibiotic use. (PDF) S2 Text. Detailed methods. (PDF) S1 Text. Summary of the evidence regarding the impact of the COVID-19 pandemic on antibiotic use. (PDF) Acknowledgments The authors gratefully acknowledge Dr. Nimalan Arinaminpathy (School of Public Health, Imperial College London, UK), Dr. Puneet Dewan (Global Health Labs, Seattle, WA, US), and Dr. Sophie Huddart (School of Medicine, University of California, San Francisco, CA, US) for providing their valuable feedback on this work. The authors gratefully acknowledge Dr. Nimalan Arinaminpathy (School of Public Health, Imperial College London, UK), Dr. Puneet Dewan (Global Health Labs, Seattle, WA, US), and Dr. Sophie Huddart (School of Medicine, University of California, San Francisco, CA, US) for providing their valuable feedback on this work. S4 Fig. Monthly sales volume of each AWaRe category in India between January 2018 and December 2020, separated for child-appropriate formulations (CAFs) and non-CAFs. (TIF) S5 Fig. Cumulative volume of antibiotics sold per year (2018–2020), stratified by AWaRe category, presented separately for child-appropriate formulations (CAFs) and non-CAFs. (TIF) (TIF) S9 Fig. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and hydroxychloroquine (HCQ) sales volumes per 100,000 (only non-child appro- priate formulations [non-CAFs]) in 10 states of India from January to September 2020. States with the highest rates of detected COVID-19 cases are shown on the left side of the graph, whereas states with the lowest rates of detected COVID-19 cases are on the right. (TIF) S10 Fig. Number of SARS-CoV-2 tests performed and number of new COVID-19 cases detected each month in India per 100,000 inhabitants between January and December 2020. (TIF) S1 Table. Search strategy used in the rapid systematic review regarding the impact of the COVID-19 pandemic on antibiotic use. (PDF) S9 Fig. Relationship between new COVID-19 cases per 100,000 (cumulative rate per month) and hydroxychloroquine (HCQ) sales volumes per 100,000 (only non-child appro- priate formulations [non-CAFs]) in 10 states of India from January to September 2020. States with the highest rates of detected COVID-19 cases are shown on the left side of the graph, whereas states with the lowest rates of detected COVID-19 cases are on the right. (TIF) S10 Fig. Number of SARS-CoV-2 tests performed and number of new COVID-19 cases detected each month in India per 100,000 inhabitants between January and December 2020. (TIF) S1 Table. Search strategy used in the rapid systematic review regarding the impact of the COVID-19 pandemic on antibiotic use. (PDF) 14 / 18 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic S2 Table. Features and findings of studies that evaluated the impact of COVID-19 on anti- biotic use. (PDF) S3 Table. List of oral formulations considered child-appropriate. (PDF) S4 Table. List of all antibiotics included in our dataset, along with ATC class, AWaRe cate- gory (2019), and Schedule H/H1. (PDF) S1 Text. Summary of the evidence regarding the impact of the COVID-19 pandemic on antibiotic use. (PDF) S2 Text. Detailed methods. (PDF) Author Contributions Conceptualization: Giorgia Sulis, Anita Kotwani, Madhukar Pai, Sumanth Gandra. Conceptualization: Giorgia Sulis, Anita Kotwani, Madhukar Pai, Sumanth Gandra. Data curation: Giorgia Sulis. Data curation: Giorgia Sulis. Formal analysis: Giorgia Sulis. Formal analysis: Giorgia Sulis. Funding acquisition: Madhukar Pai, Sumanth Gandra. Investigation: Giorgia Sulis, Sumanth Gandra. Methodology: Giorgia Sulis, Brice Batomen, Sumanth Gandra. Project administration: Sumanth Gandra. References 1. Klein EY, Van Boeckel TP, Martinez EM, Pant S, Gandra S, Levin SA, et al. Global increase and geo- graphic convergence in antibiotic consumption between 2000 and 2015. Proc Natl Acad Sci U S A. 2018; 115(15):E3463–70. https://doi.org/10.1073/pnas.1717295115 PMID: 29581252 PLOS Medicine \| https://doi.org/10.1371/journal.pmed.1003682 July 1, 2021 15 / 18 PLOS MEDICINE Sales of antibiotics and hydroxychloroquine in India during the COVID-19 epidemic 2. 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https://openalex.org/W2806250458	https://www.nature.com/articles/s41598-018-23351-0.pdf	English	null	The effects of promoter variations of the N-Methylcanadine 1-Hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy	Scientific reports	2,018	cc-by	9,546	The effects of promoter variations of the N-Methylcanadine 1-Hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy Davar Abedini1, Sajad Rashidi Monfared1 & Alireza Abbasi2 Received: 12 June 2017 Accepted: 9 March 2018 Published: xx xx xxxx Received: 12 June 2017 Accepted: 9 March 2018 Published: xx xx xxxx Noscapine is an antitumor alkaloid produced in opium poppy (Papaver somniferum) and some members of the Papaveraceae family. It has been primarily used for its antitussive effects; more recently, its anticancer properties were shown. Herein, we detected an SSR embedded in the promoter region of the CYP82Y1 gene, which was found to be the first committed-step enzyme in the noscapine biosynthesis pathway, using the MISA program. Some collected ecotypes of P. somniferum were investigated for understanding of SSRs role in the regulation of gene expression and metabolite content. Quantitative PCR showed that a variation in the motif repeat number (either a decrease or increase) down-regulated the expression of the CYP82Y1 gene. Furthermore, the analysis of noscapine content suggested that a variation in the promoter region influence noscapine amount. Moreover, P. bracteatum was analyzed in both transcript and metabolite levels, and illustrated much less expression and metabolite level in comparison to P. somniferum. By exploiting the transcriptome data from the eight genera of the Papaveraceae family, we found that noscapine biosynthesis genes are present in P. bracteatum and are not shared in other genera of the Papaveraceae family. This results may explain production of a confined metabolite within a genus. Opium poppy, Papaver somniferum L., a member of Papaveraceae family, is one of the most agronomically and economically important plant and is one of the ancient herbal plants with powerful pharmacological features1,2. Even though more than 40 alkaloids have been identified in poppy, six represent almost all of the total alkaloid including: morphine (4–21%), thebaine (0.5–2%), codeine (0.8–2.5%), noscapine (4–8%), papaverine (0.5–2.5%), and reticuline (0.1–2%)1,3. Noscapine belongs to benzylisoquinoline alkaloid produced in opium poppy and other members of the Papaveraceae family. Noscapine has been used as a human cough suppressant and, recently was shown to possess anticancer activity. Regarding to its long its long history of safe usage as an antitussive (cough-suppressing), rapid absorption after oral administration, and apoptosis inducing effect on a variety of cancer cell lines, noscapine is more advantageous in comparison to other natural tubulin-binding anti-cancer compounds, such as the well-established taxanes4–7. Additionally, unlike codeine and other opiates, noscapine is neither addictive nor painkilling. This alkaloid accumulates in the cytoplasm or latex of a specialized cells known as laticifers. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. 2Department of Agronomy and Plant Breeding, University of Tehran, Karaj, Iran. Correspondence and requests for materials should be addressed to S.R.M. (email: rashidims@modares.ac.ir) The effects of promoter variations of the N-Methylcanadine 1-Hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy Davar Abedini1, Sajad Rashidi Monfared1 & Alireza Abbasi2 According to a comprehensive study, the accumulation of noscapine has further occurred in the latex. It has also been discovered that SDR1, which catalyzes the final step of noscapine biosynthesis, is localized into latex rather than adjust cells9,10. The SDR1 genes have been reported to possess both functions of either dehydrogenase or reductase activity in some species; however, however, in the noscapine biosynthesis pathway, it has only a dehydrogenation function8,9.h co-expressed genes for noscapine synthesis. Moreover, co-regulation analysis using in silico methods, based on promoter analysis, has been elucidated, and the putative cis/trans regulatory elements represented in the prompter region of these genes have been identified7,11. It has been shown that the noscapine pathway starts with the 9-O-methylation of scoulerine catalyzed by scoulerine 9-O-methyltransferase (MT1) and ends with a short-chain dehydrogenase/reductase (NOS or SDR1)7,9. In noscapine biosynthesis, in addition, CYP82Y1 has been reported as a first committed step enzyme. This enzyme catalyzes the 1-hydroxylation of N-methylcanadine to 1-hydroxy-N-methylcanadine12. Manipulation of each individual gene (MT1, CYP82Y1, and SDR1) has had a significant influence on noscapine accumulation8,12. According to a comprehensive study, the accumulation of noscapine has further occurred in the latex. It has also been discovered that SDR1, which catalyzes the final step of noscapine biosynthesis, is localized into latex rather than adjust cells9,10. The SDR1 genes have been reported to possess both functions of either dehydrogenase or reductase activity in some species; however, however, in the noscapine biosynthesis pathway, it has only a dehydrogenation function8,9.h y y y y g The exclusive levels and spatiotemporal patterns of expression of the majority of plant genes are assigned to the sophisticated regulation in their cognate promoters. Recent evidences suggest that a variation in the pro- moter region, particularly close to Translation Start Site (TSS), might affect the gene expression and, subsequently, would have impact on phenotype diversity in human13 and plant14,15 and secondary metabolite production in plants16,17. Promoter divides into two parts; the proximal part where is a central processor of transcription and the distal part where may contain additional regulatory elements such as enhancers and silencers. p y g y Our aim in the present research was to study the gene expression of three determinative genes—MT1, CYP82Y1, and SDR1—involved in the noscapine biosynthesis pathway, and their role within the noscapine con- tent among the collected ecotypes of P. somniferum and P. bracteatum. The effects of promoter variations of the N-Methylcanadine 1-Hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy Davar Abedini1, Sajad Rashidi Monfared1 & Alireza Abbasi2 After morphine, this compound is second abundant alkaloid in the latex8,9. According to the literature, stem contains high amount of noscapine in comparison to other tissues. Due to the occurrence of two chiral centers in this molecule, chemical synthesis is prevented and P. somniferum is the only commercial source for noscapine8,10. p Recently, Winzer and co-workers discovered a co-expressed cluster of 10 genes involved in the noscapine biosynthesis pathway. These genes putatively encode most of the noscapine biosynthetic enzymes7. They used a genetic mapping strategy in order to identify a 10-gene cluster representing a 401-kb region of the opium poppy genome in a high-noscapine variety. The BAC sequencing revealed a cluster of 10 physically linked, 1Department of Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. 2Department of Agronomy and Plant Breeding, University of Tehran, Karaj, Iran. Correspondence and requests for materials should be addressed to S.R.M. (email: rashidims@modares.ac.ir) SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 1 www.nature.com/scientificreports/ Primer no. Primer name Use Primer sequence 1 Spop1forward SSR 5′-TACCACTCCACTGGATTCCTC-3′ 2 Spop1reverse SSR 5′-CCTCAAACCCCTCTTCTTCTG-3′ 3 Spop2forward SSR 5′-AATGTGTTGGTGAGCACTAGC-3′ 4 Spop2reverse SSR 5′-TACACCCCTTGTGGAAAGTGT-3′ 5 CYP82Y1-1forward Promoter isolation 5′-CTGGTTGTATCACGGATTTCAC-3′ 6 CYP82Y1reverse Promoter isolation 5′-GTGTCAGCAGAAAAGCAAGTATA-3′ 7 CYP82Y1-2forward Promoter isolation 5′- AGAACACTTTTGCTTGCTCTTG-3′ 8 CYP82Y1-3forward Promoter isolation 5′- CTCCAATCATGTAGGTGAACAAC-3′ 9 CYP82Y1-4forward Promoter isolation 5′- GCGTATTTTGTGTTCACTGACA-3′ 10 CYP82Y1-5forward Promoter isolation 5′-GTAACGGAAATGAAACAACACTAC-3′ 11 PsMT1forward Promoter isolation 5′-GCATCTGGTTGTTCTGTAGTAC-3′ 12 PsMT1reverse Promoter isolation 5′-CTGTGGCTGATTGATGATTATGAC-3′ 13 SDR1forward Promoter isolation 5′-CCACCTGTTACACATACTATCTTC-3′ 14 SDR1reverse Promoter isolation 5′-GAAGGTAGGAAGAAGCTAGTTTG-3′ 15 qCYP82Y1forward qRT-PCR 5′-CTATTGCCTTGGACATGTTATC-3′ 16 qCYP82Y1reverse qRT-PCR 5′-ATCTACTTCTTCTTTAGCCTTGTC-3′ 17 qPsMT1forward qRT-PCR 5′-GTTGGTGGTGGTATTGGTACTT-3′ 18 qPsMT1reverse qRT-PCR 5′-ATGCTCTACACCTGGGTATTG-3′ 19 qSDR1forward qRT-PCR 5′-GACAGAAAGAGCTTGCCTAAAG-3′ 20 qSDR1reverse qRT-PCR 5′-CGACACATCACCTGAAATTATCG-3′ 21 Actinforward qRT-PCR 5′-CCAGGCTGTTCAGTCTCTGTAT-3′ 22 Actinreverse qRT-PCR 5′-CGCTCGGTAAGGATCTTCATCA-3′ Table 1. Primers used in qPCR, SSR detection and promoter isolation. Table 1. Primers used in qPCR, SSR detection and promoter isolation. co-expressed genes for noscapine synthesis. Moreover, co-regulation analysis using in silico methods, based on promoter analysis, has been elucidated, and the putative cis/trans regulatory elements represented in the prompter region of these genes have been identified7,11. It has been shown that the noscapine pathway starts with the 9-O-methylation of scoulerine catalyzed by scoulerine 9-O-methyltransferase (MT1) and ends with a short-chain dehydrogenase/reductase (NOS or SDR1)7,9. In noscapine biosynthesis, in addition, CYP82Y1 has been reported as a first committed step enzyme. This enzyme catalyzes the 1-hydroxylation of N-methylcanadine to 1-hydroxy-N-methylcanadine12. Manipulation of each individual gene (MT1, CYP82Y1, and SDR1) has had a significant influence on noscapine accumulation8,12. The effects of promoter variations of the N-Methylcanadine 1-Hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy Davar Abedini1, Sajad Rashidi Monfared1 & Alireza Abbasi2 Furthermore, the SSR embedded into the promoter region of CYP82Y1, as the first committed step involved in the noscapine biosynthesis pathway, was detected and its impact on the gene expression was assessed. Eventually, the evolution of genes involved in the noscapine pathway—particularly, CYP82Y1 in the Papaveraceae family—was also discussed. Figure 2. Agarose gel electrophoresis of PCR amplified of detected SSR embedded in upstream region of MT1 gene from different ecotypes of opium poppy; (‘Ps#1’-‘Ps#16’) P. somniferum L. ecotypes. M; 250 bp DNA ladder. biosynthesis pathway, therefore, variation in the promoter region of this gene, particularly close to the TSS might influence gene expression. Cloning and sequencing of SSR and upstream regions. In order to further understanding of the sequence characteristics of the promoter regions, we isolated the MT1 (using primers 11 and 12) and the SDR1 (using primers 13 and 14) from ‘Ps#7’ ecotype. After conducting PCR, the length of amplified fragments were confirmed using gel agarose (Supplementary Fig. S2A), then, the final products were cloned into pJET vector and subjected to sequencing. Pair-wise alignment between sequenced fragments of MT1 and SDR1 from ‘Ps#7’ (sub- mitted to GenBank as accession number; KY348335 and KY348336, respectively) and reported region from high noscapine variety (HN1), was performed (for alignment details, see Supplementary Fig. S3). Alignment results demonstrate that there were few in/del and point mutations in both promoter regions. d f h l f ‘ ’ d ‘ ’ h h d l d l In order to attempt a sequencing of CYP82Y1, we chose samples of ‘Ps#3’ and ‘Ps#7’, which displayed poly- morphism within the SSR region in CYP82Y1 promoter, and ‘Ps#12’ as a candidate of the other samples. To this end, we designed some primers (primers 5–10) from different regions of this promoter (Supplementary Fig. S4). After PCR amplification, using the mentioned primers, we amplified the ‘Ps#12’ ecotype (submitted to GenBank as accession number no.: KY348333) using the primers 5 and 6. We also designed primer 5 in close proximity to TSS; this decision was taken because the TSS’s proximity to the ATG seemed to be more conserved than the sequences of the upstream region. The PCR was conducted using primers 5 and 2, and the results showed that amplified fragments differ in size from the presumed promoter region. Regarding the sequencing results, there were several insertions just upstream from the TSS (Supplementary Fig. S7) in the isolated promoter region of CYP82Y1 from ‘Ps#7’ (accession no.: KY348334); this insertion appear neither in the promoter of CYP82Y1 in HN1 nor ‘Ps#12’. The promoter region is frequently divided into two—i.e. the proximal and the distal part. We had assumed that the distal region of CYP82Y1 in this ecotype is highly variable. Results CYP82Y1 and MT1 embrace SSR in their promoter region. By the analysis of 401 Kb obtained from opium poppy7, 14 SSRs were earned, scattering in different regions of the 401 Kb of sequenced region (Table 2). Two types of detected SSRs were located in the promoter region of CYP82Y1 [(AT)15] and MT1 [(A)22], taking place in the 59 and the 208 base pairs, just upstream from the ATG codon, respectively. PCR amplification (using genomic DNA as a template) on the specific primer pairs from the 12 P. somniferum ecotypes was performed. The difference between amplified products was shown on gel agarose 1.5%. According to the gel electrophoresis, two samples of studied ecotypes named ‘Ps#3’ and ‘Ps#7’ displayed polymorphism in the SSR embedded in pro- moter region of CYP82Y1 (Fig. 1a,b), whereas no polymorphism was detected in (A)22, which was located in the promoter region of MT1 (Fig. 2). The promoter regions of ‘Ps#3’, ‘Ps#7’, ‘Ps#6’ and ‘Ps#12’ were sequenced and the results demonstrated that a significant variation existed in the number of (AT)n microsatellite motifs in the promoter CYP82Y1 of these ecotypes, wherein, ‘Ps#3’ ecotype had [(AT)16], ‘Ps#7’ had [(AT)8], ‘Ps#6’ [(AT)10] and ‘Ps#12’ [(AT)11] motifs (Fig. 1a,b). CYP82Y1 was reported as a first committed step enzyme in the noscapine SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Agarose gel electrophoresis of PCR amplified of detected SSR located in CYP82Y1 upstream region from different collected ecotypes of opium poppy from Iran; (Ps#1-Ps#16) P. somniferum L. ecotypes. M; 100 bp DNA ladder (a). Sequence alignment of CYP82Y1 promoter region (approximately 200 bp) of the four P. somniferum L ecotypes, ‘Ps#7’, ‘Ps#3’, ‘Ps#6’ and ‘Ps#12’. Variation in the number of AT motifs is distinctly observed. AT motifs are colored in red in three ecotypes, which is also considered as a putative TATA-box. The translation start codon (ATG) was also highlighted in yellow. The blue sequences indicates a part of CDS, first exon (b). Figure 2. Agarose gel electrophoresis of PCR amplified of detected SSR embedded in upstream region of MT1 gene from different ecotypes of opium poppy; (‘Ps#1’-‘Ps#16’) P. somniferum L. ecotypes. M; 250 bp DNA ladder. SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 3 3 www.nature.com/scientificreports/ Figure 3. Relative abundance of CYP82Y1, MT1 and SDR1 transcripts in some geographical collected ecotypes of P.somniferum L. (‘Ps#3’, ‘Ps#6’, ‘Ps#7’, ‘Ps#9’ and ‘Ps#12’) and P. bracteatum from Iran in stem (a) and leaf (b) tissues. Figure 3. Relative abundance of CYP82Y1, MT1 and SDR1 transcripts in some geographical collected ecotypes of P.somniferum L. (‘Ps#3’, ‘Ps#6’, ‘Ps#7’, ‘Ps#9’ and ‘Ps#12’) and P. bracteatum from Iran in stem (a) and leaf (b) tissues. In silico analysis of CYP82Y1 promoter region and TATA-box prediction. Computational analysis using PlantCARE database was performed for insertion part of ‘Ps#7’, and several cis elements were identified in this part, that could play a role, whether positive or negative, in activity of the CYP82Y1 promoter in this ecotype (Supplementary Table S1). Kinds of TFs have been reported as a regular in BIA production. Silencing and over- expression of CjWRKY, as an example, was determinant in berberine accumulation18. Moreover, several putative WRKY and MYB elements placed within or near the promoter regions of reported noscapine gene cluster7,11, sug- gesting that noscapine biosynthesis may be regulated by wRKY and MYB factors. Previously, two TFs (PsWRKY and PsMYB) were isolated from different organ of P. somniferum and P.bracteatum in our laboratory11. Moreover, by utilizing the TRANSFAC and Softberry databases, several TATA-boxes were predicted, indicating that identi- fied SSR is a TATA-box region, with high probability based on its location, which is close to the TSS. A number of studies suggest that the basal level of gene expression is influenced by variations in the TATA-box sequence19–21. Estimating the effect of (AT)n and (A)n motif number variations on the expression level of the CYP82Y1 and the MT1 genes. To understand the impact of detected SSR, which was detected in the upstream region of CYP82Y1 in some ecotypes, on transcripts level, we checked gene expression level by qRT-PCR. The transcript level of stem and leaf from P. somniferum ecotypes i.e. -‘Ps#3’, ‘Ps#6’, ‘Ps#7’, ‘Ps#9’ and ‘Ps#12’- and P. bracteatum just before anthesis stage were measured. In the selected ecotypes to perform qRT-PCR, ‘Ps#3’, ‘Ps#7’ were picked up as an ecotypes having variation in the ATn motifs number and ‘Ps#6’, ‘Ps#9’ and ‘Ps#12’, which showed no variation. With respect to results shown in Fig. 3, presence of SSR has affected expression of CYP82Y1 gene. 4). It is apparent from the figure that the amount of noscapine in P. bracteatum is strikingly lower than all the ecotypes of P. somniferum. This species also showed a low rate of expression for both determinative genes, CYP82Y1 and SDR1, in this pathway. g p y Prior studies have noted the importance of the CYP82Y1 gene in the accumulation of noscapine, and as it is the first committed enzyme in this route, the suppression of CYP82Y1 transcript levels are dramatically reduced in the production of this agent12. Analysis of noscapine content revealed that the expression of this gene when it is extreme, could influence noscapine production. In other words, according to Figs 3a and 4, when the transcript level of this gene increase, subsequently, the amount of noscapine also peak and vice versa. For instance, ‘Ps#3’ and ‘Ps#12’ illustrated lowest and highest level in both transcript and metabolite content, respectively. However, there is no such a relation between ecotypes showing intermediate expression of CYP82Y1 gene, suggesting that in order to produce high amount of noscapine, high level expression of this gene is required. Evolution of the noscapine biosynthesis pathway in the Papaver genus as the only source of noscapine in the Papaveraceae family. Noscapine is a precious compound with important pharma- cological properties; therefore, this agent continues to draw considerable attention. Due to the occurrence of two chiral centers in this molecule, the de novo chemical synthesis is hindered; hence, opium poppy is the only commercial resource of noscapine8. Although this agent is substantially produced in P. somniferum, biosynthesis in other Papaver genus like P. bracteatum occurs. However, the genes that govern this pathway in P. bracteatum (which is known as Iranian poppy or Persian poppy) have not been reported. In this study, we analyzed sequence reads obtained from transcriptome projects (accession Nos.; SRX096061, SRX039638). In silico assembly of genes involved in this pathway such as; acetyltransferase (AT1), three cytochrome p450s (CYP82Y1, CYP719A21 and CYP82X1), carboxylesterase (CXE1), tetrahydroxyprotoberberine Nmethyltransferase (TNMT), short-chain dehydrogenase/reductase (SDR1) and O-methyltransferases (MT1), suggest that there were a close relationship of noscapine biosynthesis genes in both species (Supplementary Table and Fig. S6(A and B)). We recognized eight genes involved in noscapine biosynthesis pathway in Persian poppy transcriptome data (see Supplementary data S8 to access full length of these identified genes). According to the quantitative real-time PCR results, remarkably, the expression rate of the CYP82Y1 gene in ecotypes of ‘Ps#6’, ‘Ps#9’ and ‘Ps#12’, which did not show the variation in their promoter region, was more than both ‘Ps#3’ and ‘Ps#7’. For instance, the expression of this gene in Ps#6 is about 2fold com- pared to Ps# and 4fold compared to Ps#3 ecotype. y In this study, we have also checked the expressions of MT1 and SDR1 as important genes in the noscapine biosynthesis pathway. The transcript level of MT1 in P. bracteatum was higher than all of the ecotypes of P. som- niferum (Fig. 3). This gene converts scoulerine to tetrahydrocolumbamine at high efficiency, and silencing of this SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 4 www.nature.com/scientificreports/ Figure 4. Amount of noscapine in stem tissue from collected ecotypes of P.somniferum (‘Ps#3’, ‘Ps#6’, ‘Ps#7’, ‘Ps#9’ and ‘Ps#12’) and P. bracteatum. Figure 4. Amount of noscapine in stem tissue from collected ecotypes of P.somniferum (‘Ps#3’, ‘Ps#6’, ‘Ps#7’, ‘Ps#9’ and ‘Ps#12’) and P. bracteatum. gene was associated with accumulation of scoulerine in the latex and capsule7. However, the high expression rate of this gene is not related to noscapine content as after this step (conversion of scoulerine to tetrahydrocolum- bamine) there are two branch points that are able to change the route of noscapine production to berberine or tetrahydropalmatine. Moreover, the expression of this gene was approximately equal among the P. somniferum ecotypes, where there was no variation in their promoter regions of this gene. Another important gene in this pathway is short-chain reductase (SDR), which catalyses the final step of noscapine biosynthesis. The main func- tion of this enzyme takes place in the laticifer and its trace in the noscapine pathway has been recently mani- fested9. It should be noted that the expressions of all the studied genes except MT1 (which has been clarified above) in the P. bracteatum was much lesser than the lowest ecotypes of P. somniferum. Measurement of nocapine content using HPLC. Noscapine content in the extracted latex from the studied ecotypes was determined using the HPLC method. According to the HPLC results, the amount of noscap- ine in the studied ecotypes were different. As we had assumed, the content of noscapine in the P. bracteatum was less than the P. somniferum ecotypes. Noscapine content in the highest ecotype ‘Ps#12’ was 18-fold in comparison to the lowest ecotype ‘Ps#3’ (Fig. In addition, for AT1, SDR1, CYP719A21, and CXE1, whose functional motifs and residues had been analyzed7,12 were discussed. The results showed that these func- tional features are present in identified amino acid sequences, which have been obtained from P. bracteatum. All of the constructed phylogenic trees and alignments are shown and discussed in Supplementary Fig. S5(A–Z). Prior studies suggested that biosynthesis of this compound is confined to the members of the genus Papaver (Papaveraceae)8; nevertheless, using phylogenetic analysis by the exploitation of the transcriptome data set, we found that the enzymes involved in this pathway are likewise limited to this genus. It is to be noted that this anal- ysis takes account of only P. bracteatum as a member of the Papaver genus whose Transcriptome data is available. Regarding the investigations, there is a functional motif, YPA(G/S)XXX(E/D)R, which is distinctly present in the CYP82 family12,22; however, as far as alignment results are concerned, the motif of YPASXXXER is unique in PsCYP82Y1 and PbCYP82Y1. A notable example is PsCYP82Y1, which displays a remarkable sequence identity Regarding the investigations, there is a functional motif, YPA(G/S)XXX(E/D)R, which is distinctly present in the CYP82 family12,22; however, as far as alignment results are concerned, the motif of YPASXXXER is unique in PsCYP82Y1 and PbCYP82Y1. A notable example is PsCYP82Y1, which displays a remarkable sequence identity SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 5 www.nature.com/scientificreports/ Figure 5. Unrooted neighbor-joining phylogenetic tree for selected CYP82s, constructed using MEGA 7 software. Bootstrap frequencies for each clade were based on 1,000 iterations (a). Comparison of substrate recognition site across some CYP82s from Papaveraceae family. The red rectangle represents YPASXXXER conserved motif that is present in PsCYP82Y1 and PbCYP82Y1 (b). Abbreviated species names are given before gene identifiers for each protein related to CYP82Y1 are as follows: PbCYP82Y1, P. bracteatum CYP82Y1; PsCYP82Y1, P. somniferum (AFB74617); NtCYP82E4v2, Nicotiana tabacum nicotine demethylase (ABN42695.1); NtCYP82E3, N. tomentosiformis nicotine demethylase (ABM46919.1); PsCYP82N4, P. somniferum (L7X3S1.1); GmCYP82C1p, Glycine max cytochrome P450 (AAB94590.1); AlCYP82G1, Arabidopsis lyrata DMNT/TMTT homoterpene synthase (XP_002885694.1); PsCYP82X1, P. somniferum CYP82X1 (AFB74614); PbCYP82X1, P. bracteatum CYP82X1; AmCYP82Y1, Argemone Mexicana; CmCYP82Y1, Chelidonium majus; CcCYP82Y1, Corydalis cheilanthifolia; EcCYP82Y1, Eschscholzia californica; GfCYP82Y1, Glaucium flavum; ScCYP82Y1, Sanguinaria canadensis; SdCYP82Y1, Stylophorum diphyllum. Figure 5. Unrooted neighbor-joining phylogenetic tree for selected CYP82s, constructed using MEGA 7 software. Bootstrap frequencies for each clade were based on 1,000 iterations (a). Comparison of substrate recognition site across some CYP82s from Papaveraceae family. The red rectangle represents YPASXXXER conserved motif that is present in PsCYP82Y1 and PbCYP82Y1 (b). Abbreviated species names are given before gene identifiers for each protein related to CYP82Y1 are as follows: PbCYP82Y1, P. bracteatum CYP82Y1; PsCYP82Y1, P. somniferum (AFB74617); NtCYP82E4v2, Nicotiana tabacum nicotine demethylase (ABN42695.1); NtCYP82E3, N. tomentosiformis nicotine demethylase (ABM46919.1); PsCYP82N4, P. somniferum (L7X3S1.1); GmCYP82C1p, Glycine max cytochrome P450 (AAB94590.1); AlCYP82G1, Arabidopsis lyrata DMNT/TMTT homoterpene synthase (XP_002885694.1); PsCYP82X1, P. somniferum CYP82X1 (AFB74614); PbCYP82X1, P. bracteatum CYP82X1; AmCYP82Y1, Argemone Mexicana; CmCYP82Y1, Chelidonium majus; CcCYP82Y1, Corydalis cheilanthifolia; EcCYP82Y1, Eschscholzia californica; GfCYP82Y1, Glaucium flavum; ScCYP82Y1, Sanguinaria canadensis; SdCYP82Y1, Stylophorum diphyllum. with PsCYP82N4 (53%), and accepts N-methylstylopine and N-methylcanadine as substrates. However, while CYP82N4 converted both N-methylcanadine and N-methylstylopine at similar rates, PsCYP82Y1 converted N-methylcanadine with higher efficiency than N-methylstylopine12,22. In the noscapine pathway, PsCYP82Y1 converts N-methylcanadine to 1-hydroxy-N-methylcanadine; this step is known as the first committed step in the noscapine biosynthesis pathway. Admittedly, a high rate affinity of PsCYP82Y1 to this substrate would increase the rate of noscapine production. The specific residues for alkaloid binding in PsCYP82N4 are Ile and Leu, whereas the corresponding residues in PsCYP82Y1 are Leu and Ser. The mentioned motif is recognized in Fig. 5 among some CYP82s from some member of the Papaveraceae family. In the frame of this work, we also probed to further identify the genes involved in this pathway from other genus of the Papaveraceae family; however, a low identity in sequence (less than 30 percent sequence identity) was detected. This result demonstrates that these genes are only in the Papaver genus and, therefore, as metabolome analysis proved, the production of noscapine is limited to this genus8. In other words, in the other genus of the Papaveraceae family, orthologous noscapine biosynthesis genes were not evolved. with PsCYP82N4 (53%), and accepts N-methylstylopine and N-methylcanadine as substrates. However, while CYP82N4 converted both N-methylcanadine and N-methylstylopine at similar rates, PsCYP82Y1 converted N-methylcanadine with higher efficiency than N-methylstylopine12,22. In the noscapine pathway, PsCYP82Y1 converts N-methylcanadine to 1-hydroxy-N-methylcanadine; this step is known as the first committed step in the noscapine biosynthesis pathway. Admittedly, a high rate affinity of PsCYP82Y1 to this substrate would increase the rate of noscapine production. The specific residues for alkaloid binding in PsCYP82N4 are Ile and Leu, whereas the corresponding residues in PsCYP82Y1 are Leu and Ser. The mentioned motif is recognized in Fig. 5 among some CYP82s from some member of the Papaveraceae family. In the frame of this work, we also probed to further identify the genes involved in this pathway from other genus of the Papaveraceae family; however, a low identity in sequence (less than 30 percent sequence identity) was detected. This result demonstrates that these genes are only in the Papaver genus and, therefore, as metabolome analysis proved, the production of noscapine is limited to this genus8. In other words, in the other genus of the Papaveraceae family, orthologous noscapine biosynthesis genes were not evolved. www.nature.com/scientificreports/ However, a high level of similarity was detected between the promoter region of CYP82Y1 from the ‘Ps#12’ and the HN1 variety. Analysis of experimental results suggested that there are differences in the number of detected SSRs in the collected ecotypes of P. somniferum. Moreover, only two ecotypes, ‘Ps#3’ and ‘Ps#7’, exhibit the variation in the SSR region; while others demonstrated no signif- icant differences with each other.h f The given results of qRT-PCR revealed that the transcript level of CYP82Y1 in ecotypes, in which their SSR region in the CYP82Y1 promoter region are similar to the HN1 variety, rather than the ecotypes which possess variations in the SSR motif number. We realized that the variation of motif number in the SSR region in two modes, either increases or decreases the motif number, seems to contribute to the decline in the rate of CYP82Y1 gene expression. Studies reported that the presence of variation in the SSR region, specially located in the pro- moter region, could influence the gene expression, and this impact can be positive or negative. The details of this process differ from organism to organism and from gene to gene13,14,16,23. In chickpea, for instance, the presence of SSRs in the 5′UTR region of myo-inositol monophosphatase is associated with expression and metabolite content. Shorter repeats of the SSR in this case had shown two-fold expression in comparison to those that have longer repeats14. On the other hand, Kumar and Bhatia have demonstrated that the increase in the motif number of the same SSR in the 5′UTR region of tryptophan decarboxylase in Catharanthus roseus L. significantly enhances gene expression16. In this work, the expression of the CYP82Y1 gene in ‘Ps#7’, which carried smaller repeats of SSR, was up-regulated to three-folds in comparison to ‘Ps#3’, which had a longer repeat. This experiment was performed in leaf and stem tissues, and we obtained the same results. It should be noted that this SSR is close to TSS (just 59 bp upstream of ATG codon) and its impact on gene expression is logical. However, detected insertion could be a reason for the variation of gene expression. We assumed that because the SSR is near to TSS, this change could influence gene expression rather than that of occurred in distal region. In other words, the (AT)n motif is located in the proximal region, but insertion has occurred in the distal part of this promoter. www.nature.com/scientificreports/ somniferum by assisting with some known and unknown genes. This alkaloid accumulate in the cytoplasm or latex and for this metabolite, P. somniferum is the only commercial source6,8.hf somniferum by assisting with some known and unknown genes. This alkaloid accumulate in the cytoplasm or latex and for this metabolite, P. somniferum is the only commercial source6,8.hf f y This study examines the effect of variation in the promoter region and its impact on gene expression and metabolite content as well as the evolution of the noscapine biosynthesis pathway. Some collected ecotypes of P. somniferum and P. bracteatum were chosen for this study. The effect of geographical regions on metabolite content and the SSR variation have been reported14,23. Several lines of evidence suggest that SSRs are distributed non-randomly across transcribed regions of genomes24,25; however, UTRs harbor more SSRs than the coding regions. In particular, 5′UTRs contain a great number of various SSRs that can regulate gene expression13,16. In our study, two types of SSRs that were embedded into the CYP82Y1 [(AT)15] and the MT1 [(A)22] promoter regions were detected. CYP82Y1 was known as a fine-tuning enzyme in the noscapine biosynthesis pathway and a variation in its promoter region could influence differentiation in noscapine content as a final product in this pathway12,16,17. Two other genes that could influence the accumulation of this compound are MT1, which is responsible for the initiation of this pathway7, and SDR1, the catalyzing enzyme of the final step in this pathway9. Concrete evidence proved that all of these genes were certainly recruited for the production of noscapine and by silencing each of them the production of this compound would be prevented7,8,12. The PCR assay, in order to probe variation in the promoter region, demonstrated variation in two ecotypes (‘Ps#3’ and ‘Ps#7’) for CYP82Y1 (Fig. 1); in contrast, no significant polymorphism was detected for the motif located in the promoter region of MT1 gene. Sequenced promoter region of these genes revealed that there are no significant differences between these regions in ‘Ps#7’ and the HN1 variety. After attempts to isolate the promoter region of CYP82Y1 from ‘Ps#3’ and ‘Ps#7’, we found that this promoter is highly variable when compared with the presumed promoter region of this gene. The isolated promoter region of CYP82Y1 from ‘Ps#7’ demonstrated that there are a fragment insertion in the promoter region of CYP82Y1 in this ecotype. www.nature.com/scientificreports/ In this study, we also measured the SDR1 transcript level. The SDR1, which catalyzes the final step of this pathway, forms an inte- gral part in the accumulation of noscapine8,9. The expressions of CYP82Y1 and SDR1 in ‘Ps#12’ were more than all the P. somniferum collected ecotypes; however, in ‘Ps#3’, these were the lowest. The measuring of noscapine content revealed that the production of this compound in ‘Ps#12’ and ‘Ps#3’ is highest and lowest, respectively, in comparison to other ecotypes. These results confirm that ‘Ps#12’ and ‘Ps#3’ not only are extremes in the tran- script level of the CYP82Y1 gene but also are extreme in the noscapine content. Interestingly, the expression level of the CYP82Y1 gene in two ecotypes, ‘Ps#3’ and ‘Ps#7’, was notably less than the rest of ecotypes. On the other hand, intermediate ecotypes in the CYP82Y1 transcript abundance, such as ‘Ps#7’, ‘Ps#6’, and ‘Ps#9’ demonstrated less relation with the noscapine content. A study conducted by Dang and Facchini showed that the Natasha and Marianne chemotypes, which demonstrated high and low amount of noscapine, respectively, had high and low expression of CYP82Y1 gene respectively. However, intermediate chemotypes, including Roxanne and Veronica, had not depicted high relation between CYP82Y1 gene expression and noscapine amount 12. Therefore, it seems that in order to determine an ecotype with high level of noscapine amount, the high expression of the CYP82Y1 gene is the rate limiting step in the production of noscapine compound. In other words, the high level expression of CYP82Y1 gene is vital for higher noscapine production, although, CYP82Y1 enzyme is not the only factor in determining the rate of noscapine production. g p p Noscapine has a complicated pathway and its production occurs in three cell types. All characterized genes and enzymes involved in the formation of (S)-reticuline, the central branch point intermediate in the biosynthesis of most structural benzylisoquinoline alkaloid subgroups26, are localized in companion and sieve cells8. It seems that the final conversion of the noscapine catalyst by CXE1 and SDR1 occurs in the latex-bearing laticifers of the adjacent sieve elements8–10. In addition, it seems that the acetylation mechanism in this pathway plays a role in the transportation of pathway intermediates10. The transportation mechanism of 3-O-acetylpapaveroxine, which is transported from sieve elements to the laticifers, is unknown. SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 Discussion Noscapine is one of the first isolated alkaloids from opium poppy. It has long been used as a cough suppres- sant, and more recently, it has been investigated as a potential anti-cancer drug. This agent is produced in P. SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 6 www.nature.com/scientificreports/ Material and Method Plant material, growth conditions and sample harvesting. In this experiment, 12 seeds of the P. somniferum L were used. Ecotypes of opium poppy were collected from different geographical regions of Iran (the region of collected seeds are presented in Supplementary Table S9). These seeds were cultivated in a greenhouse (Tehran University) at 20 °C/18 °C (light/ dark) with a photoperiod of (16 h days and 8 h night)34. For metabolite and transcript analysis, 5 cm long shoot fragments were harvested immediately under flower bud from each plant stem, 1 to 2 days before anthesis stage35. Microsatellite mining, DNA extraction and PCR. The Perl script MISA (http://pgrc.ipkgatersleben. de/misa/misa.html) was utilized to detection of SSRs scattered within the partial sequenced poppy genome, which had recently been published. The given sequences had been cloned in Bacterial Artificial Chromosomes by Winzer et al.; the promoter region of CYP82Y1 and MT1 genes in BAC179L19 (with accession no.: JQ659009.1) and SDR1 in BAC193L09 (with accession no.: JQ659010.1). The parameters for the SSR search were deter- mined as follows: the size of motif was 1–6 nucleotides and the minimum repeat unit was ten, six and three for mononucleotides, dinucleotides, and all the higher order motifs, respectively, including tri-, tetra-, penta-, and hexanucleotides. Primers were designed using Primer3 (v. 0.4.0) (http://frodo.wi.mit.edu) and analyzed by Oligo Analyzer (http://eu.idtdna.com/site). Genomic DNA was extracted by the cetyl trimethyl ammonium bromide (CTAB) method and used as the template in the PCR assays. The PCR cycle consisted of an initial denaturation at 95 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 25 s, followed by a final extension of 10 min at 72 °C. Differentiation of amplified samples checked by 1.5% agarose gel electrophoresis. Upstream isolation, cloning and sequencing. Ten genes involved in the noscapine biosynthesis path- way had been cloned in four BACs. These BACs, GenBank accession nos. JQ659009 to JQ659012, were used for primer designing and pair-wise alignments as a reference sequence. Subsequently, approximately 1,500 bp upstream of the translation start site, for CYP82Y1, MT1, and SDR1, was isolated as a promoter region. It is worthwhile that the designed primers cover a part of the first exon for the verification of the amplified region. www.nature.com/scientificreports/ Comparing both the transcript and the metabolite levels of studied ecotypes suggested that the accumulation of noscapine in this plant depends on some factors such as the expression of determinative genes, the trafficking of substrates, and the enzymes within the mentioned SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 7 www.nature.com/scientificreports/ cells, as well as other presently unknown factors. Further investigations on the transportation mechanism will shed light on trafficking noscapine within opium poppy and the accumulation of this valuable compound3,10. gfi g p p p ppy p Our study on P. bracteatum as a member of the Papaver genus, on which lesser experiments related to noscap- ine biosynthesis pathway have been performed, demonstrated that this member contains much less noscapine than P. somniferum. The expression level of CYP82Y1 and SDR1 confirm metabolite studying results. In the follow-up phase of this study, questions are raised about the production of noscapine in other members of the Papaveraceae family. Our study, which included experimental and in silico methods, suggested that although the known genes which participated in noscapine formation were extant in P. bracteatum, the expression of these genes, mainly SDR1 and CYP82Y1, was lower than P. somniferum. It seems that the expression of these genes is highly restricted by some factors such as their promoter architecture and the presence/absence of specific TFs. In the case of the other members, transcriptome and phylogenetic analyses for all noscapine biosynthesis genes in the seven genera of the Papaveraceae family, whose transcriptome data are accessible on the Sequence Read Archive (SRA) database, suggest that the genes involved in noscapine biosynthesis may not be expressed in these genera or they are absent in the genome of these members. We analyzed the amino acid sequences of some CYP82 families and found that there were some changes in the sequences of these genes, which made PsCYP82Y1 a specific enzyme for this pathway. A conserved motif (YPASXXXER) in the CYP82 families12,22 is present only in PsCYP82Y1 and PbCYP82Y1. It is worth mentioning that the Papaver genus is merely the producer of this valuable compound in the Papaveraceae family8. We assumed that this motif plays a critical role in this activity, and eventually, in the formation of 1-hydroxy-N-methylcanadine, which is known as a first committed step in the noscapine biosynthesis pathway. It also reported that the variation in this motif influences the affinity rate of this enzyme to the substrate12. www.nature.com/scientificreports/ y Growing the physically-linked clusters of genes for specialized metabolic pathways highlighted an intriguing facet of a plant’s secondary metabolism27–29. Although it is a little difficult to reach a consensus on the evolution- ary origin of these clusters, it seems that some factors contributed to the formation of gene clusters. Duplication from primary metabolism genes, neofunctionalization, and transposable elements are the likely causes in this process30–33. With regard to the transcriptome and metabolome data, noscapine and the 10 genes, which formed the cluster, had been observed in only the Papaver genus and in none of the metabolite and transcripts in the other genera and noscapine-free varieties7,9. We hypothesized that the trapping of an ancestral gene that other genes derived from was the reason behind the confinement of the production of this compound in the Papaver genus. This research has opened up many questions in need of future investigations. All these supposed phenom- ena make the evolutionary Papaver genus the only source of noscapine in the Papaveraceae family and situate P. somniferum as a robust source of this valuable compound. Material and Method Detected SSRs embedded into sequenced genome introduced by Winzer et al., by running the Perl script MISA (http://pgrc.ipkgatersleben.de/misa/misa.html). The first and second line, which are colored in red, demonstrate the located SSRs in the promoter region of CYP82Y1 and MT1, respectively. liquid nitrogen. Thereupon, total RNA was extracted using RNeasy Plant Mini Kit (QIAGEN., Cat No.: 74904). Assessment of the extracted RNA integrity was performed by a NanoDrop spectrophotometer (BioTek, EPOCH, serial 121004C, USA), and confirmed by agarose gel electrophoresis. The single strand cDNA synthesis was car- ried out using HyperscriptTM Reverse Transcriptase (GeneAll Inc, South Korea Cat No.: 601–100) using 1 μg of extracted RNA following the manufacturer’s instruction. To avoid the risk of amplifying possible contaminat- ing genomic DNA, at least one of the primers in each pair was designed to spanning an exon-exon junction. This strategy was used for all three studied genes (i.e. CYP82Y1, MT1 and SDR1) (Supplementary Fig. S2-C1-3). Then, exclusivity performance of designed primers were checked using primer BLAST36 and after conducting the PCR, specificity of the primer amplicons were further verified by gel-electrophoretic analysis (Supplementary Fig. S2-C4). The qRT-PCR was performed using BioRad system with the fluorescent dye SYBR®Green Master Mix 2× (Ampliqon, Denmark (Lot No.: A322701)). The qRT-PCR was run at 95 °C for 15 min, 35 cycles at 94 °C 30 s, 58 °C for 20, 72 °C for 15 s. After each run was completed, the dissociation curves were obtained by slowly ramping up the temperature from 65 °C to 95 °C (0.5 °C increase per second) and fluorescence data verified a good specificity of PCR products. Three technical replicates were performed for each sample. For quantifying transcription levels, the reference actin (accession no.: EU531837) was used as an internal control. Cycle thresh- olds (C(t)s) were analyzed using Livak method (2−ΔΔCt) and relative expression levels were calculated using the Microsoft Excel software (Microsoft Office 2016). Primers are listed in Table 1. Noscapine content analysis and chromatographic conditions. Total alkaloid of opium poppy latex were extracted from the latex and approximately 10 µL of exuded latex were re-suspended in 100% methanol for 2 h at room temperature to extract total alkaloid. Extracts were centrifuged for 10 min in 12000 rpm to pellet debris and the supernatants were concentrated by negative pressure. Subsequently, pellets were re-suspended in 50 µL of 100% (v/v) methanol. Material and Method Promoter isolation was performed by the PCR cycle for each promoter region on the collected ecotypes as fol- lowing program: initial denaturation at 95 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 57 °C for 35 s, and 72 °C for 100 s, and final extension of 10 min at 72 °C. After validation via gel electrophoresis, amplified fragment were purified by using the GenElute™ PCR clean-up Kit (Sigma-Aldrich., Cat No.: NA1020) according to the manufacture’s instruction, and also cloned into pJET1.2/blunt vector (CloneJET PCR Cloning Kit, Fermentas Cat No.: K1231) then subjected to sequencing. RNA extraction and quantitative real-time PCR. Stem and leaf (8–16 g) of P. somniferum ecotypes including ‘Ps#3’, ‘Ps#7’, ‘Ps#6’, ‘Ps#9’ and ‘Ps#12’ ecotypes) and P. bracteatum were ground to a fine powder under SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 8 www.nature.com/scientificreports/ No. Defined Sequence length Type start end 1 29 (AT)15 237540 237569 2 21 (A)22 355895 355916 3 43 (AT)22 360632 360675 4 19 (T)20 9780 9799 5 23 (T)24 37164 37187 6 33 (TA)17 51682 51715 7 63 (T)64 56838 56901 8 20 (CTTTTAG)3 60229 60249 9 19 (T)20 67811 67830 10 19 (T)20 82607 82626 11 19 (CTTTT)4 131094 131113 12 278 (TTC)93 168005 168283 13 25 (TA)13 213392 213417 14 20 (CAT)7 225030 225050 Table 2. Detected SSRs embedded into sequenced genome introduced by Winzer et al., by running the Perl script MISA (http://pgrc.ipkgatersleben.de/misa/misa.html). The first and second line, which are colored in red, demonstrate the located SSRs in the promoter region of CYP82Y1 and MT1, respectively. No. Defined Sequence length Type start end 1 29 (AT)15 237540 237569 2 21 (A)22 355895 355916 3 43 (AT)22 360632 360675 4 19 (T)20 9780 9799 5 23 (T)24 37164 37187 6 33 (TA)17 51682 51715 7 63 (T)64 56838 56901 8 20 (CTTTTAG)3 60229 60249 9 19 (T)20 67811 67830 10 19 (T)20 82607 82626 11 19 (CTTTT)4 131094 131113 12 278 (TTC)93 168005 168283 13 25 (TA)13 213392 213417 14 20 (CAT)7 225030 225050 Table 2. Detected SSRs embedded into sequenced genome introduced by Winzer et al., by running the Perl script MISA (http://pgrc.ipkgatersleben.de/misa/misa.html). The first and second line, which are colored in red, demonstrate the located SSRs in the promoter region of CYP82Y1 and MT1, respectively. Table 2. Material and Method Noscapine content was determined by using an AZURA high performance liquid chroma- tography (HPLC) System (KNAUER, Berlin, Germany), diode array detector (DAD 2.1 L), on a Perfectsil® Target ODS-3, 250 mm × 4.6 mm column (5 μm particle size) (MZ-Analysentechnik, Mainz, Germany) with flow rate of 1 mL/min. Chromatographic data were recorded and analyzed by using ClarityChrom® (V. 6.1.0) software. The program was conducted with minor modification based on established method37. Separation was achieved using a gradient of solvent A [98% (v/v) H2O: 2% (v/v) acetonitrile: 0.04% (v/v) H3PO4] and solvent B [98% (v/v) acetoni- trile: 2% (v/v) H2O: 0.04% (v/v) H3PO4]. Chromatography was initiated in 90% solvent A for 2 min. Subsequently, the gradient was increased to 35% solvent B after 10 min, then increased to 80% solvent B over 5 min.it t Noscapine (Temad, Tehran, Iran) quantification were performed using Excel Analysis Tool Pack (Microsoft Office 2016). The calibration curve was constructed based on five different concentrations of noscapine i.e. 5, 12.5, 25, 50 and 60 ug/ml with the following regression equation: y = 150.07 × −28.13. The correlation coefficient (R2) was 0.99 for the data sets. Three replications per each sample were injected into the HPLC system. Sequence relatedness and evolutionary study of noscapine biosynthesis pathway genes in the Papaveraceae family. Reads sequences from 127 transcriptome sequencing projects of some Papaveraceae members, including P. bractatum A. mexicana, C. majus, C. cheilanthifolia, E. californica, G. flavum, S. canadensis, S. diphyllum were downloaded (http://www.ncbi.nlm.nih.gov/sra/). Afterward, Reads that had high similarity with noscapine biosynthesis pathway genes, i.e. acetyltransferase (AT1), three cytochrome p450s (CYP82Y1, CYP719A21 and CYP82X1), carboxylesterase (CXE1), tetrahydroxyprotoberberine Nmethyltransferase (TNMT), short-chain dehydrogenase/reductase (SDR1) and O-methyltransferases (MT1), were selected using the offline BLASTN 2.6.0 software38, and were used to build up consensus sequences. Then, the Read sequences were assembled with the “align-then assemble” strategy employing the Codon Code Aligner v. 5. 0.1. Program SCIenTIFIC Reports \| (2018) 8:4973 \| DOI:10.1038/s41598-018-23351-0 9 www.nature.com/scientificreports/ (http://www.codoncode.com/aligner/). Then consensus sequences for each group of Reads were created. ORF of each selected sequence was discovered by using the BioEdit software version 7.0.4.139. q y gt Protein Blast was run in order to obtain the proteins that were similar in the amino acid sequence with the query proteins. Meanwhile, multiple-sequence alignment was performed using the web-based Clustal Omega program. Material and Method The Molecular Evolutionary Genetic Analysis (MEGA) software version 7.0.14 (MEGA, PA, USA) was used in order to implement the maximum likelihood method and, subsequently, the phylogeny tree was con- structed using the neighbour-joining (NJ) tree reconstruction method. Bootstrap analysis was performed for each clade based on 1,000 replicates to evaluate the statistical significance of phylogenetic tree nodes. The graphical manipulations of aligned sequences were visualized by using the Sequence Manipulation Suite–Multiple Align Show (http://bioinformatics.org/sms). References k Short‐chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy. The Plant Journal 77, 173–184, https://doi.org/10.1111/tpj.12379 (2014). p p ppyh p g pj 10. Dang, T.-T. T., Chen, X. & Facchini, P. J. Acetylation serves as a protective group in noscapine biosynthesis in opium poppy. Nature chemical biology 11, 104–106, https://doi.org/10.1038/nchembio.1717 (2015).i h 10. Dang, T.-T. T., Chen, X. & Facchini, P. J. Acetylation serves as a protective group in noscapine biosynthesis in opium poppy. Nature chemical biology 11, 104–106, https://doi.org/10.1038/nchembio.1717 (2015).i gy p g 11. Kakeshpour, T., Nayebi, S., Monfared, S. R., Moieni, A. & Karimzadeh, G. Identification and expression analyses of MYB and WRKY transcription factor genes in Papaver somniferum L. Physiology and Molecular Biology of Plants 21, 465–478, https://doi.org/10.1007/ s12298-015-0325-z (2015). ( ) 12. Dang, T.-T. T. & Facchini, P. J. CYP82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium po Journal of Biological Chemistry 289, 2013–2026, https://doi.org/10.1074/jbc.M113.505099 (2014). f g y p g j 3. Sawaya, S. et al. 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https://openalex.org/W3212657605	https://www.frontiersin.org/articles/10.3389/fonc.2021.781471/pdf	English	null	The Circ_0001367/miR-545-3p/LUZP1 Axis Regulates Cell Proliferation, Migration and Invasion in Glioma Cells	Frontiers in oncology	2,021	cc-by	7,362	Keywords: circRNA, hsa_circ_0001367, miR-545-3p, LUZP1, glioma Edited by: Glioma is the most common primary intracranial malignant tumour in adults. It has a high incidence and poses a serious threat to human health. Circular RNA is a hotspot of cancer research. In this study, we aimed to explore the role of circ_0001367 in gliomagenesis and the underlying mechanism. First, qRT-PCR was conducted, which showed that circ_0001367 level was downregulated in glioma tissues and cells. Next, gain-of- function and loss-of-function assays were performed, which indicated that circ_0001367 inhibited the proliferation, migration and invasion of glioma cells. Subsequent bioinformatics analysis, dual-luciferase reporter assays, RNA immunoprecipitation assays and cell function assays demonstrated that circ_0001367 inhibited the proliferation, migration and invasion of glioma cells by absorbing miR-545-3p and thereby regulating the expression of leucine zipper protein (LUZP1). Finally, an in vivo experiment was conducted, which demonstrated that circ_0001367 inhibited glioma growth in vivo by modulating miR-545-3p and LUZP1. Taken together, the results of this study demonstrate that the circ_0001367/miR-545-3p/LUZP1 axis may be a novel target for glioma therapy. Reviewed by: Hailin Zhao, Imperial College London, United Kingdom Bruce A. Shapiro, National Cancer Institute at Frederick, United States Correspondence: Li Zhang lizhang@suda.edu.cn Jun Dong dongjun@suda.edu.cn †These authors have contributed equally to this work Reviewed by: Hailin Zhao, Imperial College London, United Kingdom Bruce A. Shapiro, National Cancer Institute at Frederick, United States Reviewed by: Hailin Zhao, Imperial College London, United Kingdom Bruce A. Shapiro, National Cancer Institute at Frederick, United States Correspondence: Li Zhang lizhang@suda.edu.cn Jun Dong dongjun@suda.edu.cn Correspondence: Li Zhang lizhang@suda.edu.cn Jun Dong dongjun@suda.edu.cn †These authors have contributed equally to this work Specialty section: This article was submitted to Neuro-Oncology and Neurosurgical Oncology, a section of the journal Frontiers in Oncology Received: 22 September 2021 Accepted: 01 November 2021 Published: 18 November 2021 Specialty section: This article was submitted to Neuro-Oncology and Neurosurgical Oncology, a section of the journal Frontiers in Oncology The Circ_0001367/miR-545-3p/LUZP1 Axis Regulates Cell Proliferation, Migration and Invasion in Glioma Cells Xuchen Dong 1,2†, Peng Zhang 1,3†, Liang Liu 1†, Haoran Li 1, Shan Cheng 1, Suwen Li 1, Yuan Wang 4, Chaonan Zheng 4, Jun Dong 1 and Li Zhang 4* 1 Department of Neurosurgery, Second Afﬁliated Hospital of Soochow University, Suzhou, China, 2 Medical College of Soochow University, Suzhou, China, 3 Department of Neurosurgery, Rugao Hospital Afﬁliated to Nantong University, Nantong, China, 4 College of Pharmaceutical Sciences, Soochow University, Suzhou, China INTRODUCTION Glioma, which originates from the malignant transformation of glial cells, is one of the most common primary intracranial malignant tumors in adults (1, 2). It has a high incidence and poses a serious threat to human health. According to the pathological characteristics of gliomas, the World Health Organization (WHO) classiﬁes gliomas into four grades (I, II, III and IV) (3). Almost all glioma patients diagnosed with high-grade glioma (HGG, comprising grades III and IV) experience recurrence after surgical resection, and the median survival time of HGG patients is only 30~39 weeks (4). Although advances have been made in surgical resection, radiotherapy, chemotherapy and targeted therapy in recent decades, the therapeutic effects of glioma treatment, especially glioblastoma treatment, remain unsatisfactory (5). To improve the efﬁcacy of glioma treatment, a greater understanding of the molecular mechanisms of gliomagenesis is urgently needed. Edited by: Kamalakannan Palanichamy, The Ohio State University, United States ORIGINAL RESEARCH published: 18 November 2021 doi: 10.3389/fonc.2021.781471 Cell Culture and Transfection Human normal astrocytes were purchased from Jennio Biological Technology (Guangzhou, China). Human glioma cell lines U87, LN229, U251 and T98G were purchased from Procell (Wuhan, China). All the cell lines were cultured in Dulbecco’s modiﬁed Eagle’s medium containing 10% fetal bovine serum (FBS, Gibco, USA). The cell lines were maintained in a humidiﬁed incubator (37°C and 5% CO2). Small interfering RNA (siRNA), miR-545-3p mimic, miR-545- 3p inhibitor, overexpression plasmid (full-length sequence of circ_0001367 was cloned into pGL3-Basic Vector) and the corresponding negative controls used in this study were all synthesized by GenePharma (Shanghai, China). Once the cells reached approximately 80% conﬂuence, they were transfected with synthesized oligonucleotides using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol. Western Blot Total protein was extracted from cells with the Total Protein Extraction Kit (KeyGEN BioTECH, Shanghai, China). Protein concentration was determined with the BCA Protein Assay Kit (KeyGEN BioTECH, Shanghai, China). Then, the proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After being blocked with blocking buffer for 10 min, the membranes were incubated with primary antibodies against LUZP1 (Proteintech, USA) and GAPDH (Proteintech, USA) overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Image Quant LAS (GE, USA) was used for signal detection. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was isolated from tissues and cells by using TRIzol (Invitrogen, USA). RNA was reverse transcribed into complementary DNA with the First Strand cDNA Synthesis Kit (MBI, Canada). SYBR Green Master Mix II (Takara, Japan) and the ABI 7900 system (Applied Biosystems, USA) were used to conduct qRT-PCR according to the manufacturers’ protocols. GAPDH and U6 were used as controls. The expression level of targets was determined by 2−DDCt method. The following primers used in this study. Circ_0001367, 5′-TGG GTC TAT CGT GCC GTT GA-3′ (forward) and 5′-GGA CAT CAT TTC ATT CCC AAG TA-3′ (reverse); miR- 545-3p, 5′-TGG CTC AGT TCA GCA GGA AC-3′ (forward) and 5′- TGG TGT CGT GGA GTCG-3′ (reverse); LUZP1, 5′-ATG GCC GAA TTT ACA AGC TAC-3′ (forward) and 5′-TCA GTT CTC CTC AGC ACA GG-3′ (reverse); GAPDH, 5′-CAT CAC TGC CAC CC AG-3′ (forward) and 5′-ATG CCAG TGA GCT TC CC-3′ (reverse); U6, 5′-CTC GCT TCG GCA GCA CA-3′ (forward) and 5′-AAC GCT TCA CGA ATT TGC GT-3′ (reverse). MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that regulate gene expression by mediating post- transcriptional silencing (16, 17). MiRNAs are widely involved in cell proliferation, cell differentiation, immune responses and other processes, and increasing evidence shows that miRNAs are important regulatory factors in disease progression (18, 19). Recent studies suggest that miRNAs can serve as targets of circRNAs and thereby participate in regulating cancer progression (20). An analysis via an online database (starBase, http://starbase.sysu.edu.cn/index.php) indicated that miR-545- 3p may be a target of hsa_circ_0001367; however, the regulatory network containing miR-545-3p and hsa_circ_0001367 has not been reported. Leucine zipper protein (LUZP1), predominantly expressed in brain, is reportedly involved in many diseases (21–23); however, its role in glioma remains unknown. Data from starBase indicate that the 3’-untranslated regions (3’-UTRs) of LUZP1 contain binding sites of miR-545-3p. In this study, we detected and correlated the expression of hsa_circ_0001367, miR-545-3p and LUZP1 in glioma tissues and cell lines. Moreover, function assays were performed to explore the mechanisms underlying the roles of hsa_circ-0001367/miR-545-3p/LUZP1 in gliomagenesis. Citation: Dong X, Zhang P, Liu L, Li H, Cheng S, Li S, Wang Y, Zheng C, Dong J and Zhang L (2021) The Circ_0001367/ miR-545-3p/LUZP1 Axis Regulates Cell Proliferation, Migration and Invasion in Glioma Cells. Front. Oncol. 11:781471. doi: 10.3389/fonc.2021.781471 November 2021 \| Volume 11 \| Article 781471 Frontiers in Oncology \| www.frontiersin.org Dong et al. circ_0001367 Inhibit Glioma Progress excision. Written informed consent was obtained from all the glioma patients who participated in this study. Circular RNAs (circRNAs) are a kind of non-coding RNA that are widespread in mammals and exhibit a circular structure formed by covalent bonds (6, 7). Researchers formerly considered circRNAs to be junk RNAs that do not play roles in regulating biological activities (8, 9). However, support for the competing endogenous RNA (ceRNA) hypothesis proposed by Pandolﬁet al. overturned this conventional view, and an increasing number of biological functions of circRNAs are gradually being explored (10). The circRNAs involved in human disease progression mainly exert their regulatory roles through the following six mechanisms: 1) by acting as sponges or traps of microRNA (miRNA); 2) by acting as sponges or traps of protein; 3) by affecting the transcription of downstream genes; 4) by affecting the variable splicing of target genes; 5) by regulating the translation function of target proteins; and 6) by participating in the epigenetic modiﬁcation of target genes or proteins (11, 12). Numerous studies have reported that circRNAs play important roles in glioma. Liu et al. showed that circHIPK3 promotes glioma progression by targeting miR-124 (13), and Lou et al. reported that CDR1as inhibits gliomagenesis by disrupting the p53/MDM2 complex (14). Furthermore, Yang et al. reported that circ-FBXW7 encodes a functional protein to regulate glioma progression (15). However, the potential role of hsa_circ_0001367, a novel circRNA located on chromosome 3 (q27.1), in glioma has not been fully studied. Patient Enrollment Glioma tissues and samples of corresponding adjacent normal brain tissue (36 paired samples) were obtained from patients diagnosed with glioma and admitted to Soochow University. The tissues were stored in liquid nitrogen immediately after 5-Ethynyl-20-Deoxyuridine (EdU) Assay y y y ( ) y EdU assays were performed by using the EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, Shanghai, China). Cells were incubated with culture medium and EdU labelled for 4 h. After being ﬁxed with anhydrous ethanol and treated with Triton-X- 100, the cells were incubated with reaction buffer for 30 min and stained with DAPI for 5 min in darkness. The number of EdU- positive cells was measured with a ﬂuorescence microscope (Olympus, Japan). RNase R Treatment After being isolated from LN229 and T98G cells, total RNA was incubated with RNase R or the mock treatment for 30 min (37°C). Then, the expression of circ_0001367 and GAPDH was determined by qRT-PCR (24). After being isolated from LN229 and T98G cells, total RNA was incubated with RNase R or the mock treatment for 30 min (37°C). Then, the expression of circ_0001367 and GAPDH was determined by qRT-PCR (24). Actinomycin D Assay Actinomycin D (2 mg/ml, APExBIO, USA) was added to culture medium to culture LN229 and T98G cells for 24 h. Next, the cells were harvested, and their expression of circ_0001367 and GAPDH was determined by qRT-PCR (24). Dual-Luciferase Reporter Assay The wild-type (WT) and mutant (MUT) sequences of circ_0001367 and miR-545-3p were synthesized and inserted into pmirGLO November 2021 \| Volume 11 \| Article 781471 Frontiers in Oncology \| www.frontiersin.org 2 Dong et al. circ_0001367 Inhibit Glioma Progress vectors. Next, LN229 and T98G cells were transfected with miR- 545-3p mimic or NC mimic along with WT or MUT reporter plasmids by using Lipofectamine 3000 (Invitrogen, USA). After 48 h, the luciferase activity was determined by the Dual-Luciferase Reporter Assay (Promega, USA). assay was conducted following the same steps described above for the migration assay except that the chambers were precoated with Matrigel (BD Biosciences, USA). Hsa_circ_0001367 Inhibits the Proliferation, Migration and Invasion of Glioma Cells To explore the role of circ_0001367 in gliomagenesis, circ_0001367 was upregulated or knocked down in LN229 and T98G cells by transfecting the cells with overexpression plasmid or small interfering RNA (siRNA). The transfection efﬁciency was veriﬁed by qRT-PCR (Figures S1A, B). MTT assays and EdU assays demonstrated that circ_0001367 overexpression obviously suppressed the proliferation of glioma cells. In contrast, the proliferation capacity of glioma cells was markedly promoted in circ_0001367-knockdown groups (Figures 2A–F). Furthermore, Transwell assays were performed to assess the effects of circ_0001367 on the migration and invasion of glioma cells. We Tumor Xenograft Experiment g Nude mice aged 4-5 weeks were obtained from Shanghai SLAC Laboratory Animal Company (Shanghai, China). Stable cell lines LN229 overexpressing circ_0001367 or corresponding negative control were administered into the dorsal surface of the mice shoulder. Three weeks later, the tumors formed in the mice were excised. Tumor volume and weight were determined (volume = 0.5 ×length×width2). In addition, the expression of circ_0001367, miR-545-3p and LUZP1 was determined by qRT-PCR, western blot or immunohistochemistry staining. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Staining TUNEL staining was conducted by using the TUNEL Assay Kit (Beyotime, Shanghai, China). Cells were ﬁxed with anhydrous ethanol and washed three times with PBS. Next, the cells were successively treated with Triton-X-100, terminal deoxynucleotide transferase (TdT), streptavidin HRP solution and DAB substrate according to the manufacturer’s protocol. The results were evaluated via microscope (Nikon, Japan). Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Staining TUNEL staining was conducted by using the TUNEL Assay Kit (Beyotime, Shanghai, China). Cells were ﬁxed with anhydrous ethanol and washed three times with PBS. Next, the cells were successively treated with Triton-X-100, terminal deoxynucleotide transferase (TdT), streptavidin HRP solution and DAB substrate according to the manufacturer’s protocol. The results were evaluated via microscope (Nikon, Japan). Hsa_circ_0001367 Was Downregulated in Glioma qRT-PCR was used to measure circ_0001367 in the clinical samples. As shown in Figure 1A, circ_0001367 was signiﬁcantly downregulated in glioma tissues compared with adjacent normal brain tissues (ANTs). To determine whether circ_0001367 expression was associated with clinical features, Kaplan-Meier analysis was performed. The results indicated that patients in the low circ_0001367-expression group had a shorter survival time than those in the high-expression group (Figure 1B). In addition, we detected circ_0001367 expression in glioma cell lines and found that circ_0001367 was obviously downregulated in glioma cell lines compared with NAs (Figure 1C). RNase R digestion demonstrated that circ_0001367 was resistant to RNase R (Figure 1D). In addition, actinomycin D treatment showed that the circular structure of circ_0001367 was stable (Figure 1E). These ﬁndings suggested that circ_0001367 is involved in gliomagenesis. 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide (MTT) Assay MTT assays were performed with an MTT kit (Beyotime, Shanghai, China). Cells were incubated with MTT solution (20 µl, 5 mg/ml) for 0, 24, 48 or 72 h. Then, dimethyl sulfoxide (DMSO, 4% 200 µl, Beyotime, Shanghai, China) was added to the cells. The absorbance at 490 nm was then determined with a microplate reader (Bio-Tek, Germany). Statistical Analysis Statistical Analysis The results of this study are presented as the mean ± standard deviation. SPSS software 19.0 (SPSS, USA) was used for statistical analysis. Student’s t-test was used for comparisons between groups. P <0.05 was considered statistically signiﬁcant. Transwell Assay y Cells were suspended in FBS-free culture medium (200 ml) and added to the upper chamber of Transwell inserts (Millipore, Germany). Medium (500 ml) containing 10% FBS was added to the lower chamber. After incubation in a humidiﬁed incubator (37°C and 5% CO2) for 24 h, the non-migrated cells in the upper chamber were wiped with a wet cotton swab. The remaining cells were ﬁxed in anhydrous ethanol and stained with 0.5% crystal violet. After being washed with PBS, the stained cells were imaged and counted under a microscope (Nikon, Japan). The invasion November 2021 \| Volume 11 \| Article 781471 Frontiers in Oncology \| www.frontiersin.org 3 Dong et al. circ_0001367 Inhibit Glioma Progress A B D E C FIGURE 1 \| Hsa_circ_0001367 was downregulated in glioma. (A) Expression of hsa_circ_0001367 in glioma tissues and adjacent non-neoplastic tissues (ANTs) detected by qRT-PCR. (B) Survival was compared between patients with high and low levels of hsa_circ_0001367 by Kaplan-Meier analysis. (C) Expression of hsa_circ_0001367 in glioma cell lines and normal human astrocytes (NHAs). (D) Expression of hsa_circ_0001367 and GAPDH in T98G and LN229 cells treated with RNase R or the mock treatment. (E) Expression of hsa_circ_0001367 and GAPDH in T98G and LN229 treated with Actinomycin D (Act D) or Mock. P < 0.01. A B C B D E D E E FIGURE 1 \| Hsa_circ_0001367 was downregulated in glioma. (A) Expression of hsa_circ_0001367 in glioma tissues and adjacent non-neoplastic tissues (ANTs) detected by qRT-PCR. (B) Survival was compared between patients with high and low levels of hsa_circ_0001367 by Kaplan-Meier analysis. (C) Expression of hsa_circ_0001367 in glioma cell lines and normal human astrocytes (NHAs). (D) Expression of hsa_circ_0001367 and GAPDH in T98G and LN229 cells treated with RNase R or the mock treatment. (E) Expression of hsa_circ_0001367 and GAPDH in T98G and LN229 treated with Actinomycin D (Act D) or Mock. P < 0.01. A B D E F G H C FIGURE 2 \| Hsa_circ_0001367 inhibits the proliferation, migration and invasion of glioma cells. (A–D) The effect of circ_0001367 on cell viability in T98G and LN229 cells was assessed with MTT assays. (E, F) EdU assays were used to evaluate the effect of circ_0001367 on the proliferation of T98G and LN229 cells. (G, H) Transwell assays were performed to analyse the effects of circ_0001367 on the migration and invasion of T98G and LN229 cells. P < 0.05, P < 0.01. Hsa_circ_0001367 Act as a Sponge of miR-545-3p The data obtained from starBase showed the presence of binding sites between circ_0001367 and miR-545-3p (Figure 3A). The luciferase reporter assay indicated that miR-545-3p was able to obviously reduce the luciferase activity of circ_0001367-WT cells; however, it had no pronounced effect in the circ_0001367-MUT group (Figure 3B). Furthermore, RIP assay veriﬁed that circ_0001367 and miR-545-3p were enriched in the anti-Ago2 group (Figures 3C, D). Next, we detected the expression of miR-545-3p in glioma tissues and cell lines. The results showed that miR-545-3p was upregulated in glioma tissues and cell lines compared with ANTs and NAs, respectively (Figures 3E, F). We also detected the level of miR- 545-3p in circ_0001367-overexpression LN229 and T98G cells. The results demonstrated that miR-545-3p was downregulated when circ_0001367 was overexpressed (Figure 3G). These ﬁndings suggest that circ_0001367 acts as a sponge of miR- 545-3p. LUZP1 Is the Direct Target of miR-545-3p The data from starBase indicated that the 3’-UTR of LUZP1 contains sites that can bind to miR-545-3p (Figure 5A). The luciferase reporter assay indicated that miR-545-3p could reduce the luciferase activity of LUZP1-WT cells much more than that of LUZP1-MUT cells (Figure 5B). By searching the online database GEPIA, we found that LUZP1 was downregulated in glioma tissues (Figure 5C). Next, we detected LUZP1 expression in clinical samples by qRT-PCR. The results demonstrated that LUZP1 was downregulated in glioma tissues compared with ANTs (Figure 5D). Consistent with this ﬁnding, LUZP1 was downregulated in LN229 and T98G cells compared with NAs (Figures 5E, F). Furthermore, we detected LUZP1 expression in the cell models we constructed previously. qRT-PCR and western blot indicated that hsa_circ_0001367 overexpression could promote expression of LUZP1, meanwhile, miR-545-3p mimic could suppress expression of LUZP1 (Figures 5G–J). These ﬁndings indicate that LUZP1 is the direct target of miR-545-3p. Hsa_circ_0001367 Suppresses Glioma Progression by Absorbing miR-545-3p To investigate whether circ_0001367 exerts its function in LN229 and T98G cells by sponging miR-545-3p, function assays were A B D E F G C FIGURE 3 \| Hsa_circ_0001367 act as a sponge of miR-545-3p. (A) The binding sites between hsa_circ_0001367 and miR-545-3p. (B) Dual-luciferase reporter assay showed that miR-545-3p could more greatly decrease the luciferase activity of circ_0001367-WT than circ_0001367-MUT. (C, D) RIP assay was performed to verify the relationship between circ_0001367 and miR-545-3p. (E) qRT-PCR was used to detect the expression of miR-545-3p in clinical samples. Transwell Assay A B D E F C F H H G H H FIGURE 2 \| Hsa_circ_0001367 inhibits the proliferation, migration and invasion of glioma cells. (A–D) The effect of circ_0001367 on cell viability in T98G and LN229 cells was assessed with MTT assays. (E, F) EdU assays were used to evaluate the effect of circ_0001367 on the proliferation of T98G and LN229 cells. (G, H) Transwell assays were performed to analyse the effects of circ_0001367 on the migration and invasion of T98G and LN229 cells. P < 0.05, P < 0.01. November 2021 \| Volume 11 \| Article 781471 Frontiers in Oncology \| www.frontiersin.org circ_0001367 Inhibit Glioma Progress Dong et al. conducted. The transfection efﬁciency of circ_0001367- overexpression plasmid and miR-545-3p mimics was veriﬁed by qRT-PCR (Figure S1C). The MTT assays and EdU assays demonstrated that circ_0001367 overexpression obviously decreased the proliferation of glioma cells and that this inhibitory effect could be reversed by miR-545-3p mimic (Figures 4A–D). Similarly, the Transwell assays showed that miR-545-3p mimic could reverse the inhibition of migration and invasion caused by circ_0001367 overexpression (Figures 4E, F). These results suggest that circ_0001367 suppresses the proliferation, migration and invasion of glioma cells by absorbing miR-545-3p. found that circ_0001367 overexpression signiﬁcantly inhibited the migration and invasion ability of glioma cells, whereas circ_0001367 knockdown markedly enhanced the migration and invasion of glioma cells (Figures 2G, H). Taken together, these results suggest that circ_0001367 inhibits the proliferation, migration and invasion of glioma cells in vitro. Hsa_circ_0001367 Act as a Sponge of miR-545-3p (F) qRT-PCR was used to detect the expression of miR-545-3p in NHAs and glioma cell lines. (G) The expression of miR-545-3p in T98G and LN229 cells transfected with circ_0001367 plasmid was detected by qRT-PCR. P < 0.01. B C A D E F G D FIGURE 3 \| Hsa_circ_0001367 act as a sponge of miR-545-3p. (A) The binding sites between hsa_circ_0001367 and miR-545-3p. (B) Dual-luciferase reporter assay showed that miR-545-3p could more greatly decrease the luciferase activity of circ_0001367-WT than circ_0001367-MUT. (C, D) RIP assay was performed to verify the relationship between circ_0001367 and miR-545-3p. (E) qRT-PCR was used to detect the expression of miR-545-3p in clinical samples. (F) qRT-PCR was used to detect the expression of miR-545-3p in NHAs and glioma cell lines. (G) The expression of miR-545-3p in T98G and LN229 cells transfected with circ_0001367 plasmid was detected by qRT-PCR. *P < 0.01. November 2021 \| Volume 11 \| Article 781471 5 Frontiers in Oncology \| www.frontiersin.org Dong et al. circ_0001367 Inhibit Glioma Progress A B D E F C FIGURE 4 \| Hsa_circ_0001367 suppresses glioma progression by absorbing miR-545-3p. (A, B) Cell proliferation was investigated by MTT assay after transfection. (C, D) Cell proliferation was tested by EdU assay after transfection. (E, F) Transwell assay was conducted to measure the migration and invasion capacity of T98G and LN229 cells after transfection. P < 0.05, *P < 0.01. E F F F FIGURE 4 \| Hsa_circ_0001367 suppresses glioma progression by absorbing miR-545-3p. (A, B) Cell proliferation was investigated by MTT assay after transfection. (C, D) Cell proliferation was tested by EdU assay after transfection. (E, F) Transwell assay was conducted to measure the migration and invasion capacity of T98G and LN229 cells after transfection. P < 0.05, *P < 0.01. A B D E F G I H J C FIGURE 5 \| LUZP1 is the direct target of miR-545-3p. (A) The binding sites of miR-545-3p and LUZP1 predicted by starBase. (B) The relative luciferase activity in T98G and LN229 cells was detected by dual-luciferase reporter assay. (C) The expression of LUZP1 in the public database (GEPIA). (D) The expression of LUZP1 in clinical samples detected by qRT-PCR. (E, F) The expression of LUZP1 in glioma cell lines detected by qRT-PCR and western blot. (G, H) LUZP1 expression in T98G and LN229 which transfected with miR-NC or miR-545-3p detected by qRT-PCR and western blot. Hsa_circ_0001367 Act as a Sponge of miR-545-3p (I, J) LUZP1 expression in T98G and LN229 cells transfected with vector, circ_0001367, circ_0001367+miR-NC or circ_0001367+miR-545-3p was detected by qRT-PCR and western blot. P < 0.05, *P < 0.01. B G C A D E F F F J I H H FIGURE 5 \| LUZP1 is the direct target of miR-545-3p. (A) The binding sites of miR-545-3p and LUZP1 predicted by starBase. (B) The relative luciferase activity in T98G and LN229 cells was detected by dual-luciferase reporter assay. (C) The expression of LUZP1 in the public database (GEPIA). (D) The expression of LUZP1 in clinical samples detected by qRT-PCR. (E, F) The expression of LUZP1 in glioma cell lines detected by qRT-PCR and western blot. (G, H) LUZP1 expression in T98G and LN229 which transfected with miR-NC or miR-545-3p detected by qRT-PCR and western blot. (I, J) LUZP1 expression in T98G and LN229 cells transfected with vector, circ_0001367, circ_0001367+miR-NC or circ_0001367+miR-545-3p was detected by qRT-PCR and western blot. P < 0.05, *P < 0.01. November 2021 \| Volume 11 \| Article 781471 6 Frontiers in Oncology \| www.frontiersin.org circ_0001367 Inhibit Glioma Progress Dong et al. DISCUSSION Glioma is the most common malignant tumor of the central nervous system. In the past 30 years, despite numerous studies on glioma pathogenesis, the prognosis of glioma patients has not improved signiﬁcantly (25). Therefore, it is of great signiﬁcance for the clinical diagnosis and treatment of glioma to focus on the pathogenesis of glioma. LUZP1 Downregulation Restored the Effect of miR-545-3p Knockdown or circ_0001367 Overexpression on Glioma Cells plasmid or corresponding negative control were subcutaneously injected into mice. Three weeks later, tumors had formed. Compared with those in the negative control group, the tumors formed in the circ_0001367-overexpression group were lower in weight and smaller in volume (Figures 8A–D). qRT- PCR showed that circ_0001367 was upregulated whereas miR- 545-3p was downregulated in the tumors formed in the circ_0001367-overexpression group (Figure 8E). Furthermore, qRT-PCR and western blot revealed that LUZP1 was upregulated in the circ_0001367-overexpression group relative to the control group (Figures 8E, F). Furthermore, Ki-67 staining indicated that circ_0001367 overexpression decreased the proportion of Ki-67-positive cells (Figure 8G). TUNEL staining showed that more apoptosis occurred in the circ_0001367-overexpression group than in the control group (Figure 8H). To investigate whether LUZP1 is the functional target of miR- 545-3p in LN229 and T98G cells, we constructed four cell models. The efﬁciency of miR-545-3p inhibitor and si-LUZP1 transfection was veriﬁed by qRT-PCR (Figure S1D). The MTT and EdU assays suggested that LUZP1 deletion could block the suppressive effect of miR-545-3p inhibitor on glioma proliferation (Figures 6A–D). Consistent with this ﬁnding, the Transwell assays showed that the migration and invasion ability of LN229 and T98G cells was impaired by miR-545-3p inhibitor, whereas LUZP1 deletion reversed this inhibitory effect (Figures 6E, F). In addition, function assays indicated that LUZP1 deletion could block the suppressive effect of circ_0001367 overexpression on glioma proliferation, migration and invasion (Figures 7A–F). These results suggested that LUZP1 downregulation restored the effect of miR-545-3p knockdown and circ_0001367 overexpression on glioma cells. Hsa_circ_0001367 Inhibited Glioma Growth In Vivo To evaluate the effect of circ_0001367 on glioma growth in vivo, LN229 cells transfected with circ_0001367-overexpression A B D E F C FIGURE 6 \| LUZP1 downregulation restored the effect of miR-545-3p knockdown on glioma cells. (A, B) The proliferation of indicated cells was tested by MTT. (C, D) The proliferation of indicated cells was tested by EdU. (E, F) Transwell assay was conducted to measure the migration and invasion capacity of T98G and LN229 cells after transfection. P < 0.05, *P < 0.01. A B D C B F E F E F FIGURE 6 \| LUZP1 downregulation restored the effect of miR-545-3p knockdown on glioma cells. (A, B) The proliferation of indicated cells was tested by MTT. (C, D) The proliferation of indicated cells was tested by EdU. (E, F) Transwell assay was conducted to measure the migration and invasion capacity of T98G and LN229 cells after transfection. P < 0.05, *P < 0.01. November 2021 \| Volume 11 \| Article 781471 7 Frontiers in Oncology \| www.frontiersin.org Dong et al. circ_0001367 Inhibit Glioma Progress A B D E F C FIGURE 7 \| LUZP1 downregulation restored the effect of circ_0001367 overexpression on glioma cells. (A, B) The proliferation of cells transfected with si-LUZP1, circ_0001367 or si-LUZP1 plus circ_0001367 was tested by MTT assay. (C, D) The proliferation of cells transfected with si-LUZP1, circ_0001367 or si-LUZP1 plus circ_0001367 was tested by EdU assay. (E, F) Transwell assay was conducted to measure the migration and invasion capacity of cells which transfected with si- LUZP1, circ_0001367 or si-LUZP1 plus circ_0001367. P < 0.05, *P < 0.01. A B D C D D E F F F E FIGURE 7 \| LUZP1 downregulation restored the effect of circ_0001367 overexpression on glioma cells. (A, B) The proliferation of cells transfected with si-LUZP1, circ_0001367 or si-LUZP1 plus circ_0001367 was tested by MTT assay. (C, D) The proliferation of cells transfected with si-LUZP1, circ_0001367 or si-LUZP1 plus circ_0001367 was tested by EdU assay. (E, F) Transwell assay was conducted to measure the migration and invasion capacity of cells which transfected with si- LUZP1, circ_0001367 or si-LUZP1 plus circ_0001367. P < 0.05, P < 0.01. A B D E F G H C FIGURE 8 \| Hsa_circ_0001367 inhibited glioma growth in vivo. (A, B) Tumour formation was examined in nude mice following the implantation of LN229 xenografts with vector or circ_0001367 overexpression. Hsa_circ_0001367 Inhibited Glioma Growth In Vivo (C, D) The volume and weight of tumours formed in the nude mice. (E) The expression of circ_0001367, miR-545-3p and LUZP1 in the tumours formed in the nude mice was measured by qRT-PCR. (F) The expression of LUZP1 in the tumours formed in the nude mice was measured by western blotting. (G) Immunohistochemistry for Ki-67 in the tumours formed in the nude mice. (H) TUNEL staining of the tumours formed in the nude mice. P < 0.01. D C B A B A F E E G F G E H H G G F FIGURE 8 \| Hsa_circ_0001367 inhibited glioma growth in vivo. (A, B) Tumour formation was examined in nude mice following the implantation of LN229 xenografts with vector or circ_0001367 overexpression. (C, D) The volume and weight of tumours formed in the nude mice. (E) The expression of circ_0001367, miR-545-3p and LUZP1 in the tumours formed in the nude mice was measured by qRT-PCR. (F) The expression of LUZP1 in the tumours formed in the nude mice was measured by western blotting. (G) Immunohistochemistry for Ki-67 in the tumours formed in the nude mice. (H) TUNEL staining of the tumours formed in the nude mice. **P < 0.01. November 2021 \| Volume 11 \| Article 781471 8 Frontiers in Oncology \| www.frontiersin.org circ_0001367 Inhibit Glioma Progress Dong et al. CircRNAs have become a hot topic in cancer research since the recent replacement of the traditional view of circRNAs as non-functional molecules with the understanding that they inﬂuence various cellular activities (9, 26). CircRNAs are not sensitive to nucleases, so it is more stable than the corresponding linear RNAs, which makes them a signiﬁcant advantage as a new clinical diagnostic marker and target for therapy (27). Accumulating studies have shown that several circRNAs are closely associated with human cancers. For example, circ_0000039 has been found to be a biomarker of tissue differentiation in gastric cancer (28); circ_0000282 has been shown to be upregulated in osteosarcoma and associated with the proliferation of osteosarcoma cells (29); and circ_0026416 functions as an oncogene in colorectal cancer and may be a novel biomarker for diagnosis (30). In the present study, we found that circ_0001367 was signiﬁcantly downregulated in glioma tissues and cells and that its expression level was negatively associated with the survival of glioma patients. ETHICS STATEMENT The animal study was reviewed and approved by Ethics Committee of Soochow University. Hsa_circ_0001367 Inhibited Glioma Growth In Vivo The gain-of-function and loss-of-function assays revealed that circ_0001367 overexpression inhibits the proliferation, migration and invasion of glioma cells, whereas circ_0001367 silencing promotes these processes. These ﬁndings suggest that circ_0001367 acts as a suppressor in glioma. cytoskeleton and has been reported to be associated with colorectal cancer, neural tube closure and Townes-Brocks syndrome (21–23); however, its role in glioma has not been studied. Herein, we showed that LUZP1 was signiﬁcantly downregulated in glioma tissues and cells. Furthermore, function assays indicated that LUZP1 functions as a suppressor in glioma and is regulated by miR-545-3p. In summary, our study revealed that circ_0001367 was markedly downregulated in glioma tissues and cells. In addition, it demonstrated that circ_0001367 suppresses the proliferation, migration and invasion of glioma cells by sponging miR-545-3p to regulate LUZP1. The circ_0001367/ miR-545-3p/LUZP1 axis may be a novel target for glioma therapy. 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 authors. It is currently theorized that circRNAs are mainly distributed in the cytoplasm and play a role in regulating tumor progression through the adsorption of miRNAs. For example, circ_0001785 can sponge miR-942 or miR-1200 to regulate breast cancer or osteosarcoma progression, respectively (31, 32); and circ_0000285 can absorb miR-409-3p, miR-197-3p and miR- 599, thereby affecting the progression of cervical cancer and osteosarcoma (24, 33, 34). Analysis using an online database revealed 29 miRNAs that may serve as the target of circ_0001367. Among them, miR-545-3p aroused our great interest. MiR-545-3p dysregulation has been reported in diverse cancers. Shi et al. reported that miR-545-3p was downregulated in epithelial ovarian cancer (35); Hao et al. showed that miR-545-3p inhibits the proliferation and differentiation of osteoblasts (36); and Zhong et al. reported that miR-545-3p functions as a tumor suppressor in endometrial carcinoma (37). Furthermore, some studies indicate that miR- 545-3p may be the target of circRNAs. Chen et al. reported that miR-545-3p can be sponged by circ_0007580 and is thus involved in non-small lung cancer progression (38); and Li et al. showed that circ_0072083 promotes non-small lung cancer progression by absorbing miR-545-3p (39). To date, the role of miR-545-3p in glioma and the associated circRNA- miRNA network have not been studied. In our study, employing a dual-luciferase reporter assay and RIP assay, we veriﬁed that miR-545-3p can be sponged by circ_0001367. In addition, function assays indicated that miR-545-3p can reverse the effects of circ_0001367 overexpression on glioma proliferation, migration and invasion. All of these ﬁndings suggest that circ_0001367 sponges miR-545-3p to regulate gliomagenesis. AUTHOR CONTRIBUTIONS XD conducted the cell assays and wrote the manuscript. PZ, HL, and CZ collected clinical samples and conducted the in vivo experiments. LL, SC, and SL analyzed the data, search literatures and prepared the ﬁgures. 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Circular RNA Circ_0000039 Enhances Gastric Cancer Progression Through miR-1292-5p/DEK Axis. Cancer Biomark (2021) 30(2):167–77. doi: 10.3233/CBM-201754 11. Guarnerio J, Zhang Y, Cheloni G, Panella R, Mae Katon J, Simpson M, et al. Intragenic Antagonistic Roles of Protein and circRNA in Tumorigenesis. Cell Res (2019) 29(8):628–40. doi: 10.1038/s41422-019-0192-1 29. Li H, He L, Tuo Y, Huang Y, Qian B. Circular RNA hsa_circ_0000282 Contributes to Osteosarcoma Cell Proliferation by Regulating miR-192/XIAP Axis. BMC Cancer (2020) 20(1):1026. doi: 10.1186/s12885-020-07515-8 30. Liang Y, Shi J, He Q, Sun G, Gao L, Ye J, et al. Hsa_circ_0026416 Promotes Proliferation and Migration in Colorectal Cancer via miR-346/NFIB Axis. Cancer Cell Int (2020) 20:494. doi: 10.1186/s12935-020-01593-1 12. FUNDING This study was supported by Hui-Chun Chin and Tsung- Dao Lee Chinese Undergraduate Research Endowment (CURE); National Natural Sciences Foundation of China (82072798, 82073873, 81803616); Jiangsu province key research and development program: Social development project (BE2021653); Natural Science Foundation of Jiangsu Province, China (BK20201172), Key project of Jiangsu Health Commission (ZDB2020016); Research and Practice Innovation Program for Postgraduates in Jiangsu (SJCX19_0183); School-level scientiﬁc research project of Jiangsu Health Vocational College (JKC202022). Using an online database, we found that miR-545-3p directly targets LUZP1. LZUP1 is a regulator of primary cilia and actin November 2021 \| Volume 11 \| Article 781471 Frontiers in Oncology \| www.frontiersin.org 9 circ_0001367 Inhibit Glioma Progress Dong et al. SUPPLEMENTARY MATERIAL Supplementary Figure 1 \| (A, B) The expression of hsa_circ_0001367 in T98G and LN229 cells transfected with vector, circ_0001367, si-NC, si-circ_0001367-1 or circ_0001367-2. (C) The expression of hsa_circ_0001367 in T98G and LN229 cells transfected with vector, circ_0001367, circ_0001367+miR-NC or circ_0001367+ miR-545-3p. (D) LUZP1 expression in T98G and LN229 cells transfected with anti- NC, anti-mir-545-3p, anti-miR-545-3p+si-NC or anti-miR-545-3p+si-LUZP1. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021.781471/ full#supplementary-material 17. Pisarska J, Baldy-Chudzik K. MicroRNA-Based Fingerprinting of Cervical Lesions and Cancer. J Clin Med (2020) 9(11):3668. doi: 10.3390/jcm9113668 REFERENCES Guarnerio J, Bezzi M, Jeong JC, Paffenholz SV, Berry K, Naldini MM, et al. Oncogenic Role of Fusion-circRNAs Derived From Cancer-Associated Chromosomal Translocations. Cell (2016) 166(4):1055–6. doi: 10.1016/ j.cell.2016.07.035 31. Li Z, Zheng J, Lin W, Weng J, Hong W, Zou J, et al. Circular RNA hsa_Circ_0001785 Inhibits the Proliferation, Migration and Invasion of Breast Cancer Cells In Vitro and In Vivo by Sponging miR-942 to Upregulate SOCS3. Cell Cycle (2020) 19(21)2811–25. doi: 10.1080/15384101.2020.1824717 j 13. Liu Z, Guo S, Sun H, Bai Y, Song Z, Liu X. Circular RNA CircHIPK3 Elevates CCND2 Expression and Promotes Cell Proliferation and Invasion Through miR-124 in Glioma. Front Genet (2020) 11:1013. doi: 10.3389/ fgene.2020.01013 32. Li S, Pei Y, Wang W, Liu F, Zheng K, Zhang X. Circular RNA 0001785 Regulates the Pathogenesis of Osteosarcoma as a ceRNA by Sponging miR- 1200 to Upregulate HOXB2. Cell Cycle (2019) 18(11):1281–91. doi: 10.1080/ 15384101.2019.1618127 14. Lou J, Hao Y, Lin K, Lyu Y, Chen M, Wang H, et al. Circular RNA CDR1as Disrupts the P53/MDM2 Complex to Inhibit Gliomagenesis. Mol Cancer (2020) 19(1):138. doi: 10.1186/s12943-020-01253-y 33. Zhang W, Zhang S. Downregulation of circRNA_0000285 Suppresses Cervical Cancer Development by Regulating Mir197-3p-ELK1 Axis. Cancer Manag Res (2020) 12:8663–74. doi: 10.2147/CMAR.S253174 15. Yang Y, Gao X, Zhang M, Yan S, Sun C, Xiao F, et al. Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis. J Natl Cancer Inst (2018) 110(3):301–15. doi: 10.1093/jnci/djx166 16. Ghafouri-Fard S, Gholipour M, Taheri M, Shirvani Farsani Z. MicroRNA Proﬁle in the Squamous Cell Carcinoma: Prognostic and Diagnostic Roles. Heliyon (2020) 6(11):e05436. doi: 10.1016/j.heliyon.2020.e05436 34. Yang D, Jin Y, Cheng S, Yang Y. The Interaction Between Circular RNA hsa_circ_0000285 and miR-599 in Thyroid Cancer. Eur Rev Med Pharmacol Sci (2020) 24(13):7219. doi: 10.26355/eurrev_202007_21870 November 2021 \| Volume 11 \| Article 781471 Frontiers in Oncology \| www.frontiersin.org 10 Dong et al. circ_0001367 Inhibit Glioma Progress 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. 35. Shi J, Xu X, Zhang D, Zhang J, Yang H, Li C, et al. Long Non-Coding RNA PTPRG-AS1 Promotes Cell Tumorigenicity in Epithelial Ovarian Cancer by Decoying microRNA-545-3p and Consequently Enhancing HDAC4 Expression. J Ovarian Res (2020) 13(1):127. doi: 10.1186/s13048-020-00723-7 36. Hao R, Wang B, Wang H, Huo Y, Lu Y. 35. Shi J, Xu X, Zhang D, Zhang J, Yang H, Li C, et al. Long Non-Coding RNA PTPRG-AS1 Promotes Cell Tumorigenicity in Epithelial Ovarian Cancer by Decoying microRNA-545-3p and Consequently Enhancing HDAC4 Expression. J Ovarian Res (2020) 13(1):127. doi: 10.1186/s13048-020-00723-7 39. Li H, Liu F, Qin W. Circ_0072083 Interference Enhances Growth-Inhibiting Effects of Cisplatin in Non-Small-Cell Lung Cancer Cells via miR-545-3p/ CBLL1 Axis. Cancer Cell Int (2020) 20:78. doi: 10.1186/s12935-020-1162-x 38. Chen S, Lu S, Yao Y, Chen J, Yang G, Tu L, et al. Downregulation of hsa_circ_0007580 Inhibits Non-Small Cell Lung Cancer Tumorigenesis by Reducing miR-545-3p Sponging. Aging (Albany NY) (2020) 12(14):14329–40. doi: 10.18632/aging.103472 November 2021 \| Volume 11 \| Article 781471 REFERENCES lncRNA TUG1 Promotes Proliferation and Differentiation of Osteoblasts by Regulating the miR-545-3p/CNR2 Axis. Braz J Med Biol Res (2020) 53(11):e9798. doi: 10.1590/1414-431X20209798 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. 37. Zhong Y, Wang Y, Dang H, Wu X. LncRNA AFAP1-AS1 Contributes to the Progression of Endometrial Carcinoma by Regulating miR-545-3p/VEGFA Pathway. Mol Cell Probes (2020) 53:101606. doi: 10.1016/j.mcp.2020.101606 38. Chen S, Lu S, Yao Y, Chen J, Yang G, Tu L, et al. Downregulation of hsa_circ_0007580 Inhibits Non-Small Cell Lung Cancer Tumorigenesis by Reducing miR-545-3p Sponging. Aging (Albany NY) (2020) 12(14):14329–40. doi: 10.18632/aging.103472 Copyright © 2021 Dong, Zhang, Liu, Li, Cheng, Li, Wang, Zheng, Dong and Zhang. 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. 39. Li H, Liu F, Qin W. Circ_0072083 Interference Enhances Growth-Inhibiting Effects of Cisplatin in Non-Small-Cell Lung Cancer Cells via miR-545-3p/ CBLL1 Axis. Cancer Cell Int (2020) 20:78. doi: 10.1186/s12935-020-1162-x November 2021 \| Volume 11 \| Article 781471 Frontiers in Oncology \| www.frontiersin.org
https://openalex.org/W2101441559	https://eprints.gla.ac.uk/103782/7/1037482coversheet.pdf	English	null	Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy	Cell communication and signaling	2,015	cc-by	187	Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Sin, Y. Y., Martin, T. P., Wills, L., Currie, S., and Baillie, G. S. (2015) Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy. Cell Communication and Signaling, 13. p. 16. Copyright © 2015 The Authors. This work is made available under the Creative Commons Attribution – Creative Commons Attribution 4.0 International License. (CC BY 4.0) Version: Accepted http://eprints.gla.ac.uk/103782/ Deposited on: 06 March 2015 Sin, Y. Y., Martin, T. P., Wills, L., Currie, S., and Baillie, G. S. (2015) Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy. Cell Communication and Signaling, 13. p. 16. Sin, Y. Y., Martin, T. P., Wills, L., Currie, S., and Baillie, G. S. (2015) Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy. Cell Communication and Signaling, 13. p. 16. Copyright © 2015 The Authors. Copyright © 2015 The Authors. Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk
https://openalex.org/W3018863570	https://zenodo.org/record/3988057/files/Trofa_et_al_Rheology.pdf	English	null	Rheology of a Dilute Suspension of Aggregates in Shear-Thinning Fluids	Micromachines	2,020	cc-by	10,697	Article Rheology of a dilute suspension of aggregates in shear-thinning ﬂuids Marco Trofa ‡ and Gaetano D’Avino ‡ * Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università di Napoli Federico II, Piazza Giorgio Ascarelli 80, 80125 Napoli, Italy; marco.trofa@unina.it (M.T.) * Correspondence: gadavino@unina.it; Tel.: +39-081-7682241 ‡ These authors contributed equally to this work. Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università di Napoli Federico II, Piazza Giorgio Ascarelli 80, 80125 Napoli, Italy; marco.trofa@unina.it (M.T.) * Correspondence: gadavino@unina.it; Tel.: +39-081-7682241 ‡ These authors contributed equally to this work. Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università di Napoli Federico II, Piazza Giorgio Ascarelli 80, 80125 Napoli, Italy; marco.trofa@unina.it (M.T.) * Correspondence: gadavino@unina.it; Tel.: +39-081-7682241 ‡ These authors contributed equally to this work. Version August 17, 2020 submitted to Micromachines Abstract: The prediction of the viscosity of suspensions is of fundamental importance in several 1 ﬁelds. Most of the available studies have been focused on particles with simple shapes, e.g., spheres 2 or spheroids. In this work, we study the viscosity of a dilute suspension of fractal-shape aggregates 3 suspended in a shear-thinning ﬂuid by direct numerical simulations. The suspending ﬂuid is 4 modeled by the power-law constitutive equation. For each morphology, a map of particle angular 5 velocities is obtained by solving the governing equations for several particle orientations. The map is 6 used to integrate the kinematic equation for the orientation vectors and reconstruct the aggregate 7 orientational dynamics. The intrinsic viscosity is computed by a homogenization procedure along the 8 particle orbits. In agreement with previous results on Newtonian suspensions, the intrinsic viscosity, 9 averaged over different initial orientations and aggregate morphologies characterized by the same 10 fractal parameters, decreases by increasing the fractal dimension, i.e., from rod-like to spherical-like 11 aggregates. Shear-thinning further reduces the intrinsic viscosity showing a linear dependence with 12 the ﬂow index in the investigated range. The intrinsic viscosity can be properly scaled with respect 13 to the number of primary particles and the ﬂow index to obtain a single curve as a function of the 14 fractal dimension. 15 Keywords: Rheology; shear-thinning ﬂuids; intrinsic viscosity; fractal aggregates; numerical 16 simulations 17 Submitted to Micromachines, pages 1 – 17 www.mdpi.com/journal/micromachines 1. Introduction 18 The intrinsic viscosity was found to be similar to the spherical particle case for clusters 42 with high fractal dimension, indicating no preferential orientation in the ﬂow. Deviations from the 43 Einstein’s coefﬁcient was, instead, found for aggregates with low fractal dimension due to their more 44 anisotropic shape. 45 p p All the aforementioned works deal with Newtonian suspensions. In several applications, however, 46 the suspending ﬂuid shows non-Newtonian properties such as shear-thinning and viscoelasticity. 47 A typical example is in the tire industry where, during the processing stage, particles of carbon 48 black or silica are added to a polymer melt and can agglomerate and form complex structures. It is 49 well-known that non-Newtonian rheological properties alter the suspension rheology as compared to 50 the Newtonian case [11,12]. For instance, for a dilute suspension of spherical particles in a power-law 51 ﬂuid, it has been shown that shear-thinning reduces the intrinsic viscosity [13,14]. Concerning more 52 complex particle shapes, the rheology of a dilute suspension of spheroids in a generalized Newtonian 53 ﬂuid is recently investigated by numerical simulations [15]. Different ﬂows of a Carreau ﬂuid around 54 spheroidal particles are simulated and a homogenization procedure is adopted to obtain the intrinsic 55 viscosity of the suspension as function of the applied rate of deformation, thinning exponent and 56 particle aspect ratio. The results show that the intrinsic viscosity strongly depends on the particle 57 aspect ratio along with the rheological parameters of the constitutive equation [15]. Very recently, these 58 calculations have been extended to a dilute suspension of rigid rods in a power-law ﬂuid showing no 59 similarity of the rheological coefﬁcients between rods and spheroids with large aspect ratio [16]. Similar 60 studies for dilute suspensions of particles with irregular shape, such as fractal-like morphologies, are 61 not available. 62 In this paper, we investigate the rheology of a dilute suspension of aggregates with complex shape 63 suspended in a shear-thinning ﬂuid by direct numerical simulations. Aggregates made of primary 64 spherical particles are built through a fractal-like model. The ﬂuid is modeled by the power-law 65 constitutive equation. The dynamics of a single particle in an unbounded shear ﬂow ﬁeld is ﬁrst 66 computed. To this aim, ﬁnite element simulations are employed to calculate the angular velocity of the 67 particle for several orientations on the unity sphere. 1. Introduction 18 Suspensions of solid particles are encountered in a variety of industrial and biological systems. 19 The knowledge of the viscosity and, more in general, of the rheological properties of these materials is 20 fundamental for correctly designing the processing stage and predicting the hydrodynamics resistance. 21 It is well-known that the addition of solid particles in a ﬂuid increases the viscosity as compared with 22 the suspending liquid [? ]. Particle shape and size, as well as the solid concentration strongly alter the 23 suspension viscosity giving rise to non-Newtonian phenomena such as shear-thinning and normal 24 stresses [1? ,2]. 25 For the simplest case of a dilute suspension of spherical particles in a Newtonian ﬂuid, Einstein 26 calculated that the suspension viscosity is related to the ﬂuid viscosity ηs according to the formula 27 ηs(1 + Bφ) where φ is the solid volume fraction and the factor B, usually referred as ‘intrinsic viscosity’, 28 is equal to 2.5 [3,4]. Einstein’s calculation was extended by Jeffery to spheroidal particles [5], ﬁnding 29 that the intrinsic viscosity depends on the particle orientation and aspect ratio. Speciﬁcally, the 30 minimum and maximum average viscosities are obtained for particles aligned along the vorticity axis 31 or tumbling on the ﬂow-gradient plane, respectively. For randomly-oriented particles, integration of 32 www.mdpi.com/journal/micromachines www.mdpi.com/journal/micromachines 2 of 17 Version August 17, 2020 submitted to Micromachines the instantaneous intrinsic viscosity for several initial orientations over the corresponding orbits leads 33 to an average value of B higher than the Einstein’s coefﬁcient for both prolate and oblate spheroids [6]. 34 In many processes, however, the suspended particles have a complex and irregular shape, without 35 symmetry axes or planes. This is, for instance, the case when primary spherical particles undergo an 36 aggregation process and form clusters with fractal-like morphology [7–9]. As for the spheroidal particle 37 case, the prediction of the intrinsic viscosity requires the calculation of the orientational dynamics of 38 the particles subjected to an external shear ﬂow. This approach is carried out in the work by Harshe 39 and Lattuada [10] where the average rigid body resistance matrix of arbitrary shaped clusters made of 40 uniform sized spheres is computed through the Stokesian dynamics method and Brownian dynamic 41 simulations. 1. Introduction 18 The orientational dynamics is then reconstructed 68 by integrating the kinematic equations for the orientation vector interpolating the angular velocity ﬁeld. 69 The ﬁrst-order contribution to the viscosity is computed by means of a homogenization procedure and 70 time-averaged over the particle orbits. The effect of particle morphology and power-law index on the 71 ensemble-average intrinsic viscosity is investigated. 72 2.1. Governing equations 74 2.1. Governing equations 74 We consider a dilute suspension of rigid, non-Brownian aggregates in a continuous shear ﬂow. 75 The computational domain consists in a single aggregate placed at the center of a spherical domain 76 with radius much larger than the maximum size of the aggregate. A Cartesian reference frame is 77 selected with x, y, and z denoting the ﬂow, gradient, and vorticity directions, respectively. Shear ﬂow 78 boundary conditions are applied on the spherical surface whereas a rigid-body motion is imposed on 79 Version August 17, 2020 submitted to Micromachines 3 of 17 Figure 1. Examples of aggregate shapes obtained from the particle-cluster method for Np = 20, kf = 1.3, and Df = 1.5 (a) or Df = 2.5 (c). To avoid numerical issues in the region between tangent particles, the centers of the spheres in contact are connected with a set of cylinders with radius 0.732a. In panels (b) and (d) the ﬁnal geometry of the aggregates and the surface mesh are shown. Figure 1. Examples of aggregate shapes obtained from the particle-cluster method for Np = 20, kf = 1.3, and Df = 1.5 (a) or Df = 2.5 (c). To avoid numerical issues in the region between tangent particles, the centers of the spheres in contact are connected with a set of cylinders with radius 0.732a. In panels (b) and (d) the ﬁnal geometry of the aggregates and the surface mesh are shown. the particle boundary. For the investigated problem (unbounded shear ﬂow) the only relevant particle 80 kinematic quantity is the angular velocity denoted by ωp = dθp/dt with θp the rotation angle. 81 The aggregate is made by primary spherical particles with radius a. The morphology is described 82 by the following fractal equation [17]: 83 kinematic quantity is the angular velocity denoted by ωp = dθp/dt with θp the rotation angle. 81 The aggregate is made by primary spherical particles with radius a. The morphology is described 82 by the following fractal equation [17]: 83 e c q y s e g e oc y e o e y p p/ p e o o The aggregate is made by primary spherical particles with radius a. 2.1. Governing equations 74 The morphology is 82 by the following fractal equation [17]: 83 Np = kf Rg a Df (1) (1) where Np is the number of primary particles, Rg is the radius of gyration, Df is the fractal dimension 84 (between 1 and 3), and kf is the fractal pre-factor. Low or high values of the fractal dimension 85 correspond to more rod-like or spherical-like particles, respectively. Several algorithms have been 86 proposed to build morphologies obeying the fractal equation [18–21]. In this work, we adopt the 87 procedure proposed in References [22,23] based on a particle–cluster aggregation method, which 88 has been veriﬁed to generate structures that fulﬁll the fractal equation also for very few primary 89 particles [23]. We point out that real fractal aggregates can be approximated by coarsened structures 90 where each sphere already represents an agglomerate of smaller real primary particles. In Figure 1, 91 two examples of aggregate morphologies with Np = 20, kf = 1.3, and for Df = 1.5 (Figure 1a) or 92 Df = 2.5 (Figure 1c) are shown. Notice that the algorithm for aggregate generation is based on a 93 sequence of pseudo-random numbers. Hence, by changing the random seed, different morphologies 94 are obtained for the same set of parameters of the fractal equation. Due to the asymmetric shape 95 of the particle, two orthogonal vectors, p and q, are needed to track its orientation. The selection 96 of these orientation vectors will be discussed later. Finally, we denote by Ωand P(t) the ﬂuid and 97 particle domain, respectively. The particle boundary is denoted by ∂P(t) and the surface of the external 98 spherical domain by Σ. 99 where Np is the number of primary particles, Rg is the radius of gyration, Df is the fractal dimension 84 (between 1 and 3), and kf is the fractal pre-factor. Low or high values of the fractal dimension 85 correspond to more rod-like or spherical-like particles, respectively. Several algorithms have been 86 proposed to build morphologies obeying the fractal equation [18–21]. In this work, we adopt the 87 procedure proposed in References [22,23] based on a particle–cluster aggregation method, which 88 has been veriﬁed to generate structures that fulﬁll the fractal equation also for very few primary 89 particles [23]. 2.1. Governing equations 74 109 n = 1, a Newtonian ﬂuid with (constant) viscosity m is recovered. 109 Concerning the boundary conditions, we impose shear ﬂow at the external spherical surface of 110 the domain: 111 Concerning the boundary conditions, we impose shear ﬂow at the external spherical surface of 110 the domain: 111 u = ( ˙γexty, 0, 0) on Σ (6) (6) with ˙γext the imposed shear ﬂow. No-slip boundary conditions are set on the particle surface resulting 112 in the rigid-body motion equation: 113 with ˙γext the imposed shear ﬂow. No-slip boundary conditions are set on the particle surface resulting 112 in the rigid-body motion equation: 113 u = ωp × (x −xc) on ∂P(t) (7) (7) where x is a point of the surface ∂P(t) and xc is the particle center of volume. As remarked above, the 114 particle translational velocity is irrelevant for the problem under investigation. 115 To close the set of equations, the hydrodynamic torque acting on the particle needs to be speciﬁed. 116 Under the assumptions of inertialess particle and no ‘external’ torques, such balance equation is given 117 by: 118 T = Z ∂P(t)(x −xc) × (σ · n) dS = 0 (8) (8) where T is the total torque on the particle boundary ∂P(t) and n is the unit vector normal to the particle 119 surface pointing from the ﬂuid to the boundary. 120 where T is the total torque on the particle boundary ∂P(t) and n is the unit vector normal to the particle 119 surface pointing from the ﬂuid to the boundary. 120 where T is the total torque on the particle boundary ∂P(t) and n is the unit vector normal to the particle 119 surface pointing from the ﬂuid to the boundary. 120 The solution of the governing equations gives the ﬂuid velocity and pressure ﬁelds, and the 121 particle angular velocity. The orientational dynamics can be computed by integrating the following 122 equation: 123 dθp dt = ωp (9) \| dθp dt = ωp (9) (9) with initial condition θp\|t=0 = θp,0. with initial condition θp\|t=0 = θp,0. 124 p p The governing equations can be conveniently made dimensionless by choosing appropriate 125 characteristic quantities for time, length, and stress. 2.1. Governing equations 74 We point out that real fractal aggregates can be approximated by coarsened structures 90 where each sphere already represents an agglomerate of smaller real primary particles. In Figure 1, 91 two examples of aggregate morphologies with Np = 20, kf = 1.3, and for Df = 1.5 (Figure 1a) or 92 Df = 2.5 (Figure 1c) are shown. Notice that the algorithm for aggregate generation is based on a 93 sequence of pseudo-random numbers. Hence, by changing the random seed, different morphologies 94 are obtained for the same set of parameters of the fractal equation. Due to the asymmetric shape 95 of the particle, two orthogonal vectors, p and q, are needed to track its orientation. The selection 96 of these orientation vectors will be discussed later. Finally, we denote by Ωand P(t) the ﬂuid and 97 particle domain, respectively. The particle boundary is denoted by ∂P(t) and the surface of the external 98 spherical domain by Σ. 99 Assuming inertialess conditions and negligible gravity effects, the governing equations for the 100 ﬂuid domain, Ω−P(t), read as follows: 101 mitted to Micromachines 4 of 17 4 of 17 Version August 17, 2020 submitted to Micromachines ∇· u = 0 (2) ∇· σ = 0 (3) σ = −pI + 2η( ˙γ)D (4) (2) (3) (2) (2) (3) (4) (3) (4) σ = −pI + 2η( ˙γ)D (4) (4) where u, σ, p, I, η, and D are the velocity vector, the stress tensor, the pressure, the 3 × 3 unity tensor, 102 the viscosity, and the rate-of-deformation tensor D = (∇u + (∇u)T)/2, respectively. The viscosity is 103 assumed to be a function of the effective deformation rate deﬁned as ˙γ = √ 2D : D. Equations (2)–(4) 104 are the mass balance (continuity), the momentum balance and the expression for the total stress, 105 respectively. 106 p y In this work, we consider a power-law constitutive equation for the ﬂuid given by: 107 η( ˙γ) = m ˙γn−1 (5) (5) with m the consistency index and n the ﬂow index. This model predicts shear-thinning for n < 1. For 108 n = 1, a Newtonian ﬂuid with (constant) viscosity m is recovered. 109 with m the consistency index and n the ﬂow index. This model predicts shear-thinning for n < 1. For 108 n = 1, a Newtonian ﬂuid with (constant) viscosity m is recovered. Version August 17, 2020 submitted to Micromachines 2.1. Governing equations 74 Notice that the radius of the 135 primary particles a is related to Reff through Np. Also, the radius of gyration Rg is determined once 136 the aforementioned three parameters are speciﬁed. In what follows, all the symbols (without stars) 137 will refer to dimensionless quantities. 138 q Aim of this work is to predict the viscosity of a dilute suspension of fractal-like aggregates in a 139 power-law ﬂuid. In the dilute regime, the suspension viscosity can be expressed as: 140 η = ηs (1 + B φ) (16) (16) where ηs is the matrix viscosity deﬁned by Equation (5), φ is the (low) particle volume fraction, and B 141 is the intrinsic viscosity. Once the local stress ﬁeld is calculated by solving the governing equations, 142 the intrinsic viscosity is computed through the numerical homogenization procedure as described in 143 References [15,24]. In brief, this procedure consists in evaluating ﬁrst the average power density: 144 where ηs is the matrix viscosity deﬁned by Equation (5), φ is the (low) particle volume fraction, and B 141 is the intrinsic viscosity. Once the local stress ﬁeld is calculated by solving the governing equations, 142 the intrinsic viscosity is computed through the numerical homogenization procedure as described in 143 References [15,24]. In brief, this procedure consists in evaluating ﬁrst the average power density: 144 ⟨P⟩= ⟨σ : D⟩= 1 V0 Z Vf σ : D dV (17) (17) where V0 and Vf are the suspension and ﬂuid volumes. The suspension viscosity is, then, evalu η = ⟨P⟩ 2Dext : Dext = ⟨P⟩ ˙γ2 ext (18) (18) with Dext the rate-of-deformation at the external boundaries of the domain that, for a simple shear 146 ﬂow, reduces to the last term of Equation (18). From the suspension viscosity, we can readily evaluate 147 the intrinsic viscosity B as: 148 with Dext the rate-of-deformation at the external boundaries of the domain that, for a simple shear 146 ﬂow, reduces to the last term of Equation (18). From the suspension viscosity, we can readily evaluate 147 the intrinsic viscosity B as: 148 with Dext the rate-of-deformation at the external boundaries of the domain that, for a simple shear 146 ﬂow, reduces to the last term of Equation (18). 2.1. Governing equations 74 As characteristic length, we select the effective 126 radius of the particle deﬁned as the radius of a sphere with equivalent volume V of the aggregate, 127 Reff = ( 3V 4π )1/3. The inverse of the external shear rate 1/ ˙γext is chosen as characteristic time (giving 128 Reff ˙γext as characteristic velocity) and m ˙γn ext as characteristic stress. 129 The governing equations can be conveniently made dimensionless by choosing appropriate 125 characteristic quantities for time, length, and stress. As characteristic length, we select the effective 126 radius of the particle deﬁned as the radius of a sphere with equivalent volume V of the aggregate, 127 Reff = ( 3V 4π )1/3. The inverse of the external shear rate 1/ ˙γext is chosen as characteristic time (giving 128 Reff ˙γext as characteristic velocity) and m ˙γn ext as characteristic stress. 129 With these characteristic quantities, the dimensionless governing equations an boundary 130 conditions read as: 131 With these characteristic quantities, the dimensionless governing equations an boundary 130 conditions read as: 131 Version August 17, 2020 submitted to Micromachines 5 of 17 ∇∗· u∗= 0 (10) −∇∗p∗+ ∇∗· 2 ˙γ∗,n−1D∗ = 0 (11) u∗= (y∗, 0, 0) on Σ (12) u∗= ω∗ p × (x∗−x∗ c) on ∂P(t) (13) T∗= Z ∂P(t)(x∗−x∗ c) × (σ∗· n) dS∗= 0 (14) dθp dt∗= ω∗ p (15) (10) (14) dθp dt∗= ω∗ p (15) (15) where the starred symbols denote dimensionless quantities. Hence, the only dimensionless parameter 132 appearing in the governing equations is the ﬂow index n. Of course, we need also to consider the 133 parameters related to the aggregate morphology appearing in Equation (1), that are the number of 134 particles Np, the fractal dimension Df, and the fractal pre-factor kf. Notice that the radius of the 135 primary particles a is related to Reff through Np. Also, the radius of gyration Rg is determined once 136 the aforementioned three parameters are speciﬁed. In what follows, all the symbols (without stars) 137 will refer to dimensionless quantities. 138 where the starred symbols denote dimensionless quantities. Hence, the only dimensionless parameter 132 appearing in the governing equations is the ﬂow index n. Of course, we need also to consider the 133 parameters related to the aggregate morphology appearing in Equation (1), that are the number of 134 particles Np, the fractal dimension Df, and the fractal pre-factor kf. 2.2. Numerical method 164 Except for the simplest case of spherical particles, the intrinsic viscosity is a function of the particle 165 orientation. Hence, the calculation of B in Equation (20) requires the knowledge of the evolution of the 166 particle orientation dynamics. To this aim, starting from an initial orientation, the governing Equations 167 (10)-(14) should be solved at each time step followed by the integration of the kinematic Equation 168 (15). This procedure is time-consuming since, as we will see later, the ﬁnal integration time must be 169 3-4 orders of magnitude higher than the characteristic time in order to get an accurate estimate of the 170 intrinsic viscosity. Furthermore, for each aggregate morphology, the procedure should be repeated 171 for several initial orientations. However, since no time-derivatives appear in Equations (10)-(14), a 172 strategy to speed-up the simulations has been recently proposed [26]. We run single-step simulations 173 for several orientations of the particles (i.e., without performing any time-integration). From these 174 simulations, we build a look-up table containing the particle angular velocity and the intrinsic viscosity 175 for each orientation. Then, the orientation dynamics is reconstructed by solving the kinematic Equation 176 (15) where the angular velocity on the right-hand side is taken by interpolating the simulation data. 177 Once the look-up table has been constructed, it can be used for calculating the particle orbits for any 178 initial orientation, thus greatly reducing the computational cost. 179 Let us describe in more details the adopted procedure. Since the particle shape has no symmetry 180 axes, two (orthogonal) orientation vectors p and q are required to identify the orientation of the particle 181 in a ﬁxed frame. The choice of these two vectors to build the look-up table is done as follows. Let 182 us consider the unit vector ˆx = (1, 0, 0) in the reference frame of the aggregate generated by the 183 particle-cluster method [22,23]. Notice that this vector identiﬁes an arbitrary direction (depending 184 on the algorithm used to build the aggregate) and, as such, does not have any physical meaning. A 185 triangulated icosahedral mesh over the unit sphere is generated, i.e., the unit sphere is divided in 186 triangles with icosahedral symmetry. The aggregate is then rigidly rotated so that the orientation vector 187 p, originally aligned with the unit vector ˆx, is brought to coincide with the vertices of the triangulated 188 mesh. 2.1. Governing equations 74 From the suspension viscosity, we can readily evaluate 147 the intrinsic viscosity B as: 148 B = η −ηs ηs φ (19) (19) Due to the anisotropic particle shape, the intrinsic viscosity depends on the particle orientation. 149 As we will show later, the orientational dynamics is rather complex and neither a steady-state nor a 150 simple periodic regime is achieved. Hence, we integrate the instantaneous intrinsic viscosity over the 151 particle orbit for a sufﬁciently long time T so that the average value deﬁned as: 152 B = 1 T Z T 0 B(t)dt (20) (20) does not change within a tolerance (chosen as 1%). For each particle morphology, we run several 153 simulations for different initial orientations. The time-average intrinsic viscosity is, then, averaged 154 over M initial orientations to get: 155 does not change within a tolerance (chosen as 1%). For each particle morphology, we run several 153 simulations for different initial orientations. The time-average intrinsic viscosity is, then, averaged 154 over M initial orientations to get: 155 Version August 17, 2020 submitted to Micromachines 6 of 17 August 17, 2020 submitted to Micromachines 6 of 17 Version August 17, 2020 submitted to Micromachines 6 of 17 ⟨B⟩= 1 M∑ M B (21) (21) M Finally, to make the results independent of the seed used to generate the aggregates, we 156 repeat the simulations for different seeds for ﬁxed values of the fractal parameters, and deﬁne the 157 ensemble-average intrinsic viscosity as: 158 Finally, to make the results independent of the seed used to generate the aggregates, we 156 repeat the simulations for different seeds for ﬁxed values of the fractal parameters, and deﬁne the 157 ensemble-average intrinsic viscosity as: 158 ⟨B⟩m = 1 Nseed ∑ Nseed ⟨B⟩ (22) (22) with Nseed the number of seeds. 159 In this work, we ﬁx the fractal pre-factor kf = 1.3, which is a value commonly used in the literature 160 to describe realistic aggregate morphologies [25]. The suspension intrinsic viscosity is, then, studied by 161 varying the power-law index, the number of primary particles forming the aggregate, and the fractal 162 dimension. 163 2.2. Numerical method 164 208 Algorithm 1 Procedure used to update the particle orientation dynamics 1: p0, q0 ←initial orientation 2: for t ←1, num_time_steps do 3: Identify the spherical triangle containing p with vertices vi (i = 1, 2, 3) 4: for i ←1, 3 do 5: Compute the rotation matrix Rp,vi from p to vi 6: q′ i ←Rp,vi · q 7: Compute ωi and Bi by interpolating the look-up table with entries vi and q′ i 8: end for 9: Compute ω and B by interpolating ωi and Bi over the spherical triangle in p 10: Update p and q using quaternions 11: end for Algorithm 1 Procedure used to update the particle orientation dynamics 7: Compute ωi and Bi by interpolating the look-up table with entries vi and q′ i 8: end for 9: Compute ω and B by interpolating ωi and Bi over the spherical triangle in p The same triangular mesh chosen for building the look-up table is used for interpolation. First 209 of all, the triangle containing p needs to be identiﬁed (step 3 of Algorithm 1). We use the search 210 procedure described in Reference [28]. The interpolation of the angular velocity and intrinsic viscosity 211 inside a spherical triangle mesh requires the knowledge of these quantities at the triangle vertices. To 212 do this, we compute the rotation matrix needed to rotate p on the unit vectors corresponding to the 213 triangle vertices vi and apply this rotation matrix to q in order to get the values q′ i in these vertices 214 (steps 5 and 6). Then, we enter into the look-up table with vi and q′ i to perform the interpolation (step 215 7). Notice that the values vi are already present in the look-up table since, as mentioned above, the 216 same mesh is used for building the table and for performing the interpolation. This is not the case for 217 q′ i. Hence, a mono-dimensional interpolation is required over the block of Nq data in the table. This 218 is done by using a quadratic interpolation. At the end of the inner loop in Algorithm 1 we get the 219 angular velocity and the intrinsic viscosity in the three vertices of the triangle. 2.2. Numerical method 164 189 For each of these orientations p, we select the vectors q as the equally-distributed vectors over 190 the unit circle around p. Speciﬁcally, the calculation of the vectors q is carried out by ﬁrst deﬁning Nq 191 vectors qxy,i uniformly distributed over the unit circle in the xy−plane, i.e., qxy,i = (cos ψ, sin ψ, 0) with 192 ψ = −π + 2π(i −1)/Nq for i = 1, ..., Nq; then, the vectors q are computed as qi = Rˆz,p · qxy,i where 193 Rˆz,p is the rotation matrix transforming ˆz = (0, 0, 1) to p. In this way, the vectors q are orthogonal to p 194 (since qxy are orthogonal to ˆz) and the vector q1 corresponds to the transformation of −ˆx according 195 to the rotation matrix Rˆz,p. In summary, the aggregate generated by the particle-cluster method is 196 Version August 17, 2020 submitted to Micromachines 7 of 17 ﬁrst rotated so that p coincides with one vertex of the icosahedral mesh, and then it is further rotated 197 around p to get one of the Nq vectors q as discussed above. 198 We select an icosahedral subdivision with triangulation number Tn = 4 resulting in 42 vertices 199 over the unit sphere and Nq = 12 for a total of 504 entries (i.e., single-step simulations) in the look-up 200 table. In this way, the angular distance of p on the unit sphere and of q on the unit circle (around p) is 201 approximately the same (about π/6). We have veriﬁed that this subdivision is sufﬁcient to assure a 202 good accuracy of the interpolation. 203 g y p Once the look-up table has been computed, integration of Equation (15) is done by interpolating 204 the angular velocity. Notice that two kinds of interpolations need to be performed, i.e., one over the 205 unit sphere (for p) and one over a unit circle orthogonal to p (for q). After obtaining the angular 206 velocity, we update both orientation vectors instead of the rotation angle θp by using quaternions [27]. 207 The entire procedure (interpolation and orientation update) is summarized in Algorithm 1. 2.2. Numerical method 164 A linear interpolation 220 inside the spherical triangle over p is ﬁnally performed to obtain the values of the two quantities in 221 the desired point [28,29] (step 9). Once the angular velocities are known, the quaternions are updated 222 Table 1. Mesh and geometrical parameters used in the simulations. Np ∆x Rext ∆xext Nelem 10 0.20 40 10 ∼20000 20 0.25 40 10 ∼20000 50 0.30 50 20 ∼30000 Table 1. Mesh and geometrical parameters used in the simulations. Version August 17, 2020 submitted to Micromachines 8 of 17 Df = 1.5 P 0.05 0.1 0.15 n = 1.0 Df = 2.5 0.1 0.2 0.3 0.4 0.5 0.6 n = 1.0 P 0.05 0.1 0.15 n = 0.7 0.1 0.2 0.3 0.4 0.5 0.6 n = 0.7 P 0 5 10 15 20 25 0.05 0.1 0.15 n = 0.4 B 0 5 10 15 20 25 0.1 0.2 0.3 0.4 0.5 0.6 n = 0.4 B Figure 2. Probability distribution of the intrinsic viscosity corresponding to the initial aggregate orientations used to build the look-up table, for low (left) and high (right) fractal dimension and three ﬂow indexes. The dashed line represents the median of the distribution. Df = 2.5 0.1 0.2 0.3 0.4 0.5 0.6 n = 1.0 0.1 0.2 0.3 0.4 0.5 0.6 n = 0.7 0 5 10 15 20 25 0.1 0.2 0.3 0.4 0.5 0.6 n = 0.4 B Df = 1.5 P 0.05 0.1 0.15 n = 1.0 P 0.05 0.1 0.15 n = 0.7 P 0 5 10 15 20 25 0.05 0.1 0.15 n = 0.4 B Figure 2. Probability distribution of the intrinsic viscosity corresponding to the initial aggregate orientations used to build the look-up table, for low (left) and high (right) fractal dimension and three ﬂow indexes. The dashed line represents the median of the distribution. in time by using a third-order Adams-Bashforth scheme. The rotation matrix in terms of quaternions is 223 constructed and used to update p and q (step 10). 224 The one-step direct numerical simulations are carried out by solving the governing equations by 225 the ﬁnite element method. The particle angular velocity are treated as additional unknowns, and are 226 included in the weak form of momentum equation. The torque-free condition is imposed through 227 Lagrange multipliers in each node of the particle surface [30]. 2.2. Numerical method 164 228 The one-step direct numerical simulations are carried out by solving the governing equations by 225 the ﬁnite element method. The particle angular velocity are treated as additional unknowns, and are 226 included in the weak form of momentum equation. The torque-free condition is imposed through 227 Lagrange multipliers in each node of the particle surface [30]. 228 The spherical primary particles forming the aggregate generated by the particle-cluster method 229 are tangent. To avoid numerical problems in the region of the contact point, we perform a Boolean 230 union operation of the spheres with a set of cylinders connecting the centers of the spheres in contact 231 (that are readily identiﬁed since the generation algorithm provides the connectivity list). The radius of 232 the cylinders is chosen 0.732a. We have veriﬁed that, by reducing the cylinder radius, the rotational 233 dynamics and the resulting intrinsic viscosity is weakly affected. The aggregate surface is, then, 234 smoothed and meshed. These operations are performed through the library PyMesh [31]. Examples 235 of the surface meshes for the aggregates in Figures 1a and 1c are shown in Figures 1b and 1d. The 236 volume mesh, that is the mesh between the external spherical domain and the aggregate, is made by 237 tetrahedral elements and is generated by Gmsh [32]. 238 Mesh and time convergence, the dimension of the external spherical domain as well as the validity 239 of the procedure described above are carefully checked. The parameters used in the simulations are 240 reported in Table 1 where ∆x and ∆Rext (made dimensionless by the primary particle radius a) are 241 the size of the elements on the aggregate surface and the external domain, respectively, Rext is the 242 radius of the external sphere, and Nelem is the number of tetrahedral elements in the volume mesh. We 243 veriﬁed that, by reducing ∆x and ∆Rext and increasing Rext by about 20%, the particle angular velocity 244 and the intrinsic viscosity change by less than 1.5%. The time-step size is ﬁxed at ∆t = 0.05, giving 245 the same orientation dynamics by repeating the computation with ∆t = 0.025. 2.2. Numerical method 164 We have also veriﬁed 246 9 of 17 Version August 17, 2020 submitted to Micromachines B 5 10 15 Df = 1.5 B n = 1.0 n = 0.8 n = 0.6 5 10 15 Df = 2.0 B 1 2 3 4 5 6 7 8 9 10 5 10 15 Df = 2.5 seed Figure 3. Box plot of the intrinsic viscosity as a function of aggregate random seed, ﬂow index and fractal dimension. The black dash within each box represents the median of the distribution. B 5 10 15 Df = 1.5 B n = 1.0 n = 0.8 n = 0.6 5 10 15 Df = 2.0 B 1 2 3 4 5 6 7 8 9 10 5 10 15 Df = 2.5 seed Figure 3. Box plot of the intrinsic viscosity as a function of aggregate random seed, ﬂow index and f l di i Th bl k d h i hi h b h di f h di ib i Figure 3. Box plot of the intrinsic viscosity as a function of aggregate random seed, ﬂow index and fractal dimension. The black dash within each box represents the median of the distribution. the accuracy of the solution of the governing equations for a spherical particle in a power-law ﬂuid 247 and the validity of the homogenization procedure for the most critical case of n = 0.4 by comparing 248 the results with those reported in Reference [24] (deviations are less than 2%). Finally, the procedure 249 described in Algorithm 1 is applied to a spheroid. The particle dynamics and the time-dependent 250 intrinsic viscosity is in excellent agreement with the Jeffery results [6]. 251 the accuracy of the solution of the governing equations for a spherical particle in a power-law ﬂuid 247 and the validity of the homogenization procedure for the most critical case of n = 0.4 by comparing 248 the results with those reported in Reference [24] (deviations are less than 2%). Finally, the procedure 249 described in Algorithm 1 is applied to a spheroid. The particle dynamics and the time-dependent 250 intrinsic viscosity is in excellent agreement with the Jeffery results [6]. 251 3. Results 252 In particular, B is higher when the aggregate is aligned 263 10 of 17 Version August 17, 2020 submitted to Micromachines Px -1 -0.5 0 0.5 1 Py -1 -0.5 0 0.5 1 Pz -1 -0.5 0 0.5 1 ωP 0 200 400 600 800 1000 -0.5 0 0.5 t Figure 4. Time evolution of the components of the particle smallest principal axis of inertia P and of the angular velocity around it ωP, for an initial orientation close to the vorticity axis z. The aggregate is the one reported in Figure 1b, the other parameters are Np = 20, Df = 1.5, and n = 1.0. Px -1 -0.5 0 0.5 1 Py -1 -0.5 0 0.5 1 Pz -1 -0.5 0 0.5 1 ωP 0 200 400 600 800 1000 -0.5 0 0.5 t Figure 4 Time evolution of the components of the particle smallest principal axis of inertia P and of Figure 4. Time evolution of the components of the particle smallest principal axis of inertia P and of the angular velocity around it ωP, for an initial orientation close to the vorticity axis z. The aggregate is the one reported in Figure 1b, the other parameters are Np = 20, Df = 1.5, and n = 1.0. with the gradient direction (i.e., y), and lower when it is aligned with the ﬂow or vorticity direction, 264 similarly to prolate spheroids. On the other hand, the distribution variance reduces at higher Df 265 as the particles are more sphere-like. In agreement with the results for a sphere in power-law ﬂuid 266 [24], shear-thinning determines a reduction of the intrinsic viscosity, but the shape of the distribution 267 remains the same. 268 As stated above, to make the results independent of the speciﬁc random seed used to generate 269 the aggregates, for every combination of the other parameters, we repeat the simulations with ten 270 different seeds. The effect of the speciﬁc morphology on the intrinsic viscosity distribution is reported 271 in Figure 3 with a box plot showing the ﬁrst, second (i.e., the median), and third quartile of every 272 distribution. In all cases we ﬁnd again the same trend seen in Figure 2 with fractal dimension and ﬂow 273 index, whereas the effect of the random seed is of secondary importance. 3. Results 252 In this section we present results by varying the fractal dimension, the ﬂow index, and the number 253 of primary particles. Speciﬁcally, we consider three values for each parameter, i.e., Df = [1.5, 2.0, 2.5], 254 n = [1.0, 0.7, 0.4], and Np = [10, 20, 50]. Notice that, since the intrinsic viscosity is normalized with 255 respect to the aggregate volume, the number of primary particles deﬁnes the structure resolution (ﬁner 256 for many particles) and, for low fractal dimension, it is also connected to the aggregate aspect ratio. 257 Figure 2 shows the probability distribution of the intrinsic viscosity with respect to the same 258 initial orientations considered to build the look-up table, for the two aggregates reported in Figure 1 259 and three ﬂow indexes. The dashed lines represent the medians of the distributions, which are all 260 approximately unimodal. Comparing the results for different fractal dimensions, it can be seen that 261 at lower Df the distribution of the intrinsic viscosity is wider, as the particles are more anisotropic 262 and their orientation becomes more relevant. In particular, B is higher when the aggregate is aligned 263 Figure 2 shows the probability distribution of the intrinsic viscosity with respect to the same 258 initial orientations considered to build the look-up table, for the two aggregates reported in Figure 1 259 and three ﬂow indexes. The dashed lines represent the medians of the distributions, which are all 260 approximately unimodal. Comparing the results for different fractal dimensions, it can be seen that 261 at lower Df the distribution of the intrinsic viscosity is wider, as the particles are more anisotropic 262 and their orientation becomes more relevant. In particular, B is higher when the aggregate is aligned 263 Figure 2 shows the probability distribution of the intrinsic viscosity with respect to the same 258 initial orientations considered to build the look-up table, for the two aggregates reported in Figure 1 259 and three ﬂow indexes. The dashed lines represent the medians of the distributions, which are all 260 approximately unimodal. Comparing the results for different fractal dimensions, it can be seen that 261 at lower Df the distribution of the intrinsic viscosity is wider, as the particles are more anisotropic 262 and their orientation becomes more relevant. 3. Results 252 274 Since the intrinsic viscosity depends on the particle orientation, which changes with time due to 275 the imposed shear ﬂow, it is important to reconstruct the particle dynamics. The rotational dynamics 276 of an irregularly shaped aggregate can be better understood by looking at its principal axes of inertia. 277 Speciﬁcally, let’s denote with P the principal axis corresponding to the smallest moment of inertia, 278 which for an elongated particle (small Df) corresponds to its ‘natural’ orientation. Figures 4 and 5 show 279 the time evolution of the Cartesian components of P and the angular velocity around it ωP = ωp · P, 280 for the aggregate reported in Figure 1b and two initial orientations. In both cases the orientational 281 dynamics is rather complex and neither a steady-state nor a simple periodic regime is achieved. Two 282 periods can be recognized, one shorter connected to the rotation around the vorticity axis z, and 283 one longer connected to the rotation around P. When the particle starts with an orientation close to 284 11 of 17 Version August 17, 2020 submitted to Micromachines Px -1 -0.5 0 0.5 1 Py -1 -0.5 0 0.5 1 Pz -1 -0.5 0 0.5 1 ωP 0 200 400 600 800 1000 -0.5 0 0.5 t Figure 5. Time evolution of the components of the particle smallest principal axis of inertia P and of the angular velocity around it ωP, for an initial orientation far from the vorticity axis z. The aggregate is the one reported in Figure 1b, the other parameters are Np = 20, Df = 1.5, and n = 1.0. Px -1 -0.5 0 0.5 1 Py -1 -0.5 0 0.5 1 Pz -1 -0.5 0 0.5 1 ωP 0 200 400 600 800 1000 -0.5 0 0.5 t Figure 5. Time evolution of the components of the particle smallest principal axis of inertia P and the angular velocity around it ωP, for an initial orientation far from the vorticity axis z. The aggrega Figure 5. Time evolution of the components of the particle smallest principal axis of inertia P and of the angular velocity around it ωP, for an initial orientation far from the vorticity axis z. The aggregate is the one reported in Figure 1b, the other parameters are Np = 20, Df = 1.5, and n = 1.0. 3. Results 252 B, B 4 5 6 7 8 9 10 11 a) B, B 0 200 400 600 800 1000 4 8 12 16 20 b) t Figure 6. (a) Time evolution of intrinsic viscosity B (blue curve) and time-average intrinsic viscosity B (red curve) for the trajectory reported in Figure 4. (b) The same as a for the trajectory in Figure 5. B, B 4 5 6 7 8 9 10 11 a) B, B 0 200 400 600 800 1000 4 8 12 16 20 b) t Figure 6. (a) Time evolution of intrinsic viscosity B (blue curve) and time-average intrinsic viscosity B (red curve) for the trajectory reported in Figure 4. (b) The same as a for the trajectory in Figure 5. Version August 17, 2020 submitted to Micromachines 12 of 17 12 of 17 Df = 1.5 P 0.1 0.2 0.3 t = 0 Df = 2.5 t = 0 P 0.1 0.2 0.3 t = 10 t = 10 P 0.1 0.2 0.3 t = 100 t = 1000 P 0 5 10 15 20 25 0.1 0.2 0.3 0.4 0.5 t = 5000 B 4 5 6 7 8 t = 30000 B Figure 7. Probability distribution of the time-average intrinsic viscosity B corresponding to the initial aggregate orientations used to build the look-up table and n = 1.0, for low (left) and high (right) fractal dimension, parametric in time. Df = 1.5 P 0.1 0.2 0.3 t = 0 P 0.1 0.2 0.3 t = 10 P 0.1 0.2 0.3 t = 100 P 0 5 10 15 20 25 0.1 0.2 0.3 0.4 0.5 t = 5000 B Df = 2.5 t = 0 t = 10 t = 1000 4 5 6 7 8 t = 30000 B Figure 7. Probability distribution of the time-average intrinsic viscosity B corresponding to the initial aggregate orientations used to build the look-up table and n = 1.0, for low (left) and high (right) fractal dimension, parametric in time. the z-axis (see Figure 4), it undertakes a kayaking-like dynamics with approximately elliptical orbits 285 around the z-axis, more elongated in the x-direction. On the other hand, when the initial orientation 286 is sufﬁciently far from the z-axis (see Figure 5), the particle tends to align with the shear plane, i.e., 287 Pz progressively reduces, similar to a tumbling motion. 3. Results 252 At the same time also ωP decreases, thus 288 determining an extension of the rotation period around P. The same qualitative behavior of the 289 aggregate dynamics is observed for shear-thinning ﬂuids. 290 Once the particle orientational dynamics is known, the time evolution of intrinsic viscosity B 291 and time-average intrinsic viscosity B can be reconstructed. Figure 6 shows the trend of B and B for 292 the trajectories seen in previous ﬁgures. Speciﬁcally, panel 6a refers to Figure 4 (where the particle 293 undertakes kayaking) and panel 6b refers to Figure 5 (where the particle tumbles close to the shear 294 plane). In both cases, while B continues to oscillate due to the aforementioned dynamics, after a certain 295 time, B reaches a steady state condition. Notice that such steady state values are different for the two 296 initial orientations considered. B is lower in Figure 6a since the aggregate is mainly aligned with its 297 longest axis towards the vorticity direction. Conversely, B is higher in Figure 6b since, during the 298 aggregate tumbling motion, P periodically passes close to the gradient direction, which corresponds 299 to the maximum intrinsic viscosity. 300 Version August 17, 2020 submitted to Micromachines 13 of 17 〈B〉m Np = 50 Np = 20 Np = 10 5 10 15 20 n = 1.0 Df = 1.5 〈B〉m 5 10 15 20 n = 0.7 Df = 2.0 〈B〉m 1.5 2 2.5 5 10 15 20 n = 0.4 Df 0.4 0.7 1 Df = 2.5 n Figure 8. Ensemble-average intrinsic viscosity as a function of fractal dimension, parametric in the number of aggregate primary particles and for different ﬂow indexes (left). Same data as a function of ﬂow index for different fractal dimensions (right). The data standard deviation and trend line are also reported. Df = 1.5 Df = 2.0 0.4 0.7 1 Df = 2.5 n Figure 8. Ensemble-average intrinsic viscosity as a function of fractal dimension, parametric in the number of aggregate primary particles and for different ﬂow indexes (left). Same data as a function of ﬂow index for different fractal dimensions (right). The data standard deviation and trend line are also reported. 3. Results 252 By repeating this procedure for many orientations, it is possible to obtain the evolution of the 301 probability distribution of the time-average intrinsic viscosity, as reported in Figure 7 for the usual 302 two aggregates and n = 1.0. Notice that the initial distributions are the same of Figure 2. As just seen 303 in the previous ﬁgure, for low fractal dimension more than one steady state value is present, with 304 the majority of initial orientations leading to dynamics like the one in Figure 6b with B ≈11.5. The 305 two lower peaks visible for Df = 1.5 at t = 5000 correspond to P approximately aligned with the 306 positive (shown in Figure 4) or negative (not shown) vorticity direction. These last two peaks are close 307 but not equal since the aggregate has no symmetries. A similar behavior can be observed at high 308 fractal dimension, but with a longer time needed to reach the steady state. The two peaks visible for 309 Df = 2.5 at t = 30000 are still related to the kayaking and tumbling motion. However, those related to 310 alignment of P to the vorticity direction or its opposite are not distinguishable because of the more 311 isotropic particle shape. 312 Finally, averaging on all the initial orientations and random seeds we get the ensemble-average 313 intrinsic viscosity deﬁned in Equation (22). Figure 8 reports the dependency of ⟨B⟩m on the fractal 314 dimension and ﬂow index, parametric in the number of primary particles, together with standard 315 deviation and trend line. In the range considered, ⟨B⟩m decreases with Df and increases with Np. The 316 decreasing trend of ⟨B⟩m with the fractal dimension, previously observed for Newtonian ﬂuids [10], is 317 related to the aggregate shape and can be justiﬁed recalling that the intrinsic viscosity of a suspension 318 of rod-like particles is higher than the one for a suspension of spheres [6]. For the same reason, the 319 14 of 17 Version August 17, 2020 submitted to Micromachines 〈B〉m,H 0 0.5 1 1.5 2 2.5 3 a) 〈B〉m,H / Bsph 1.5 2 2.5 0 0.5 1 1.5 b) Df Figure 9. (a) Ensemble-average intrinsic viscosity normalized with the aggregate hydrodynamic radius as in Equation (23). (b) Same data as panel a normalized by the intrinsic viscosity of a sphere in a power-law ﬂuid. 3. Results 252 In both panels, the symbols denote the number of primary particles composing the aggregate (lower triangle Np = 50, upper triangle Np = 20, and circle Np = 10) and the colors refer to the ﬂow index (blue n = 1.0, red n = 0.7, and green n = 0.4). Figure 9. (a) Ensemble-average intrinsic viscosity normalized with the aggregate hydrodynamic radius as in Equation (23). (b) Same data as panel a normalized by the intrinsic viscosity of a sphere in a power-law ﬂuid. In both panels, the symbols denote the number of primary particles composing the aggregate (lower triangle Np = 50, upper triangle Np = 20, and circle Np = 10) and the colors refer to the ﬂow index (blue n = 1.0, red n = 0.7, and green n = 0.4). number of primary particles forming the aggregate weakly affects the intrinsic viscosity at high fractal 320 dimension. On the contrary, Np has a strong inﬂuence on ⟨B⟩m for low values of Df. Indeed, for a 321 sphere-like aggregate the number of primary particles only deﬁnes its resolution, whereas for a rod-like 322 aggregate it is connected to the aspect ratio that, in turn, determines the viscosity of the suspension [6]. 323 As regarding the effect of the ﬂow index, in agreement with the previous literature for suspensions of 324 spherical and spheroidal particles [13,15,24], shear-thinning reduces the intrinsic viscosity. Speciﬁcally, 325 as visible on the right column of Figure 8, in the investigated range the intrinsic viscosity linearly 326 depends on the ﬂow index. As expected standard deviation is lower for high fractal dimensions, since 327 both initial orientation (see Figure 2) and random seed (see Figure 3) have a relatively minor effect. 328 The intrinsic viscosity shown in the previous ﬁgures accounts for the effective volume of the 329 aggregate that, as discussed in Section 2.1, is computed from the union of spheres and cylinders. An 330 alternative approach is to normalize the intrinsic viscosity by the aggregate hydrodynamic volume 331 that, for a set of spherical particles with radius a, is the volume of a sphere with radius RH a, where 332 RH is the hydrodynamic radius [10]. 4. Conclusions 353 The viscosity of a dilute suspension of fractal aggregates in a shear-thinning ﬂuid has been 354 investigated. The aggregate morphology is generated through a particle-cluster method combining 355 spherical particles with equal size in order to satisfy the fractal equation. The power-law constitutive 356 equation is used to model the ﬂuid. Finite element simulations are employed to solve the ﬂuid 357 dynamics problem of an aggregate in an unbounded shear ﬂow for a ﬁxed particle orientation. 358 The simulation gives the velocity and pressure ﬁelds, and the angular velocity of the aggregate. A 359 homogenization procedure is adopted to obtain the intrinsic viscosity. The simulations are run for 360 a uniform distribution of all the possible orientations, building a database of angular velocities and 361 intrinsic viscosities. The orbits followed by the aggregate are, then, reconstructed by integrating the 362 particle kinematic equation with angular velocity interpolated from the database. Finally, the intrinsic 363 viscosity computed along the orbit is averaged in time and over several initial orientations and seeds 364 used to build the morphology. 365 The rotational dynamics of the aggregate is rather complex, characterized by irregular oscillations 366 and more than one characteristic period. At long times, the aggregate approximately aligns with 367 one of its principal axes of inertia to the vorticity direction, performing a kayaking motion. Hence, 368 multiple regimes depending on the initial particle orientation are possible, thus leading to different 369 time-average intrinsic viscosities. The ensemble-average intrinsic viscosity decreases by increasing the 370 fractal dimension, i.e., from rod-like to sphere-like aggregates. For low values of the fractal dimension, 371 the number of particles forming the aggregate directly affects the aspect ratio and, in turn, leads to 372 relevant variations of the intrinsic viscosity. Shear-thinning reduces the intrinsic viscosity showing a 373 linear dependence with the ﬂow index in the investigated range. Finally, the intrinsic viscosity data, 374 normalized through the hydrodynamic volume and the intrinsic viscosity of a dilute suspension of 375 spheres in a power-law liquid, collapse on a single curve in terms of the fractal dimension. 376 The results presented in this work help to understand the effect of both complex particle shape 377 and non-Newtonian rheology on the intrinsic viscosity of suspensions in the absence of interparticle 378 hydrodynamic interactions. 3. Results 252 The hydrodynamic radius is deﬁned as the radius of a sphere 333 that gives the same drag force acting on the aggregate in a uniform ﬂow [33], and, in a Newtonian 334 ﬂuid, it is related to the eigenvalues of the translational mobility tensor. Neglecting the contribution of 335 the connecting cylinders, the ensemble-average intrinsic viscosity normalized with the hydrodynamic 336 volume is then: 337 ⟨B⟩m,H = Np R3 H ⟨B⟩m (23) (23) Figure 9a shows the trend of ⟨B⟩m,H, in which the values of RH are taken from the literature 338 [22,33]. Some remarks are in order: i) Equation (23) and the used hydrodynamic radii assume that 339 the aggregate is made of tangential spherical particles, ii) the values of RH are calculated for an 340 15 of 17 Version August 17, 2020 submitted to Micromachines aggregate suspended in a Newtonian ﬂuid (notice that the calculation of the hydrodynamic radius 341 for a power-law ﬂuid is not straightforward as, due to the non-linearity of the constitutive equation, 342 the mobility tensor cannot be used for its evaluation). Despite these approximations, the data scale 343 fairly well with respect to the number of primary particles (symbols with the same color in ﬁgure). 344 As a consequence of the previous normalization, at high fractal dimension ⟨B⟩m,H tends to the value 345 for a spherical particle (i.e., 2.5 in the Newtonian case) [10]. This motivates us to further normalize 346 the data with respect to the intrinsic viscosity of a dilute suspension of spheres in a power-law liquid, 347 given by Bsph = 0.383 + 2.117n [13,24,34]. As visible in Figure 9b, all the data collapse on a single 348 curve. Hence the viscosity of a dilute suspension of aggregates (with the same fractal parameters) in a 349 power-law ﬂuid is completely determined by the fractal dimension. Of course, the prediction of the 350 intrinsic viscosity form the master trend in Figure 9b requires the knowledge of the hydrodynamic 351 radius of the aggregate population. 352 Funding: This work was carried out in the context of the VIMMP project (www.vimmp.eu). The VIMMP project 382 has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 383 agreement No 760907. 384 Conﬂicts of Interest: The authors declare no conﬂict of interest. 385 References 386 1. Laun, H.M. 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https://openalex.org/W4389090191	https://www.frontiersin.org/articles/10.3389/fgene.2023.1304974/pdf?isPublishedV2=False	English	null	Bridging the gap: returning genetic results to indigenous communities in Latin America	Frontiers in genetics	2,023	cc-by	13,393	OPEN ACCESS OPEN ACCESS EDITED BY Ernesto Schwartz Marin, University of Exeter, United Kingdom REVIEWED BY Rana Dajani, Hashemite University, Jordan Maui Hudson, University of Waikato, New Zealand CORRESPONDENCE Epifanía Arango-Isaza, epifaniarango@gmail.com RECEIVED 30 September 2023 ACCEPTED 14 November 2023 PUBLISHED 28 November 2023 CITATION Arango-Isaza E, Aninao MJ, Campbell R, Martínez FI, Shimizu KK and Barbieri C (2023), Bridging the gap: returning genetic results to indigenous communities in Latin America. Front. Genet. 14:1304974. doi: 10.3389/fgene.2023.1304974 Epifanía Arango-Isaza1,2, María José Aninao3, Roberto Campbell 4, Felipe I. Martínez4,5, Kentaro K. Shimizu1,2 and Chiara Barbieri1,6 Epifanía Arango-Isaza1,2, María José Aninao3, Roberto Campbell 4, Felipe I. Martínez4,5, Kentaro K. Shimizu1,2 and Chiara Barbieri1,6 1Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland, 2Center for the Interdisciplinary Study of Language Evolution, University of Zurich, Zurich, Switzerland, 3Pontiﬁcal Catholic University of Peru, Lima, Peru, 4Escuela de Antropología, Facultad de Ciencias Sociales, Pontiﬁcia Universidad Católica de Chile, Santiago, Chile, 5Center for Intercultural and Indigenous Research, Santiago, Chile, 6Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy In response to inequality in access to genomics research, efforts are underway to include underrepresented minorities, but explicit (and enforcing) guidelines are mostly targeted toward the Global North. In this work, we elaborate on the need to return scientiﬁc results to indigenous communities, reporting the actions we have taken in a recent genomic study with Mapuche communities in Chile. Our approach acknowledged the social dynamics perpetuating colonial hierarchies. We framed genetic results to empower indigenous knowledge and communities’ history and identities. A fundamental step in our strategy has been sharing the results with the communities before publishing the scientiﬁc paper, which allowed us to incorporate community perspectives. We faced the challenge of translating genetic concepts like admixture, emphasizing the distinction between identity and biology. To reach a broad and diverse audience, we disseminated the study results to single community members, cultural representatives, and high schools, highlighting the importance of the history of the region before the European contact. To facilitate results dissemination, we prepared didactic material and a report in Spanish written in non-specialized language, targeting a wider Latin American readership. This work illustrates the beneﬁts of discussing scientiﬁc ﬁndings with indigenous communities, demonstrating that a collaborative and culturally sensitive approach fosters knowledge sharing and community empowerment and challenges power dynamics in genetic research. TYPE Policy and Practice Reviews PUBLISHED 28 November 2023 DOI 10.3389/fgene.2023.1304974 TYPE Policy and Practice Reviews PUBLISHED 28 November 2023 DOI 10.3389/fgene.2023.1304974 OPEN ACCESS Bridging the gap between academia and indigenous communities promotes equity and inclusion in scientiﬁc endeavors. COPYRIGHT © 2023 Arango-Isaza, Aninao, Campbell, Martínez, Shimizu and Barbieri. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS Latin America, indigenous communities, genomics, Global South, ethics, Chile COPYRIGHT © 2023 Arango-Isaza, Aninao, Campbell, Martínez, Shimizu and Barbieri. 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. frontiersin.org Bridging the gap: returning genetic results to indigenous communities in Latin America OPEN ACCESS EDITED BY Ernesto Schwartz Marin, University of Exeter, United Kingdom REVIEWED BY Rana Dajani, Hashemite University, Jordan Maui Hudson, University of Waikato, New Zealand CORRESPONDENCE Epifanía Arango-Isaza, epifaniarango@gmail.com RECEIVED 30 September 2023 ACCEPTED 14 November 2023 PUBLISHED 28 November 2023 CITATION Arango-Isaza E, Aninao MJ, Campbell R, Martínez FI, Shimizu KK and Barbieri C (2023), Bridging the gap: returning genetic results to indigenous communities in Latin America. Front. Genet. 14:1304974. doi: 10.3389/fgene.2023.1304974 KEYWORDS Latin America, indigenous communities, genomics, Global South, ethics, Chile 1 Introduction An earnest recognition of the historical and persistent marginalization experienced by these communities needs to proactively elevate the ethical standards governing research conducted within these regions (Zavala, 2013; Schwartz-Marin and Fiske, 2022; Silva et al., 2022). Conversely, genomic initiatives in Latin America contrast these forms of indigenous purism with a narrative referred to as the “mestizo rhetoric” (Séguin et al., 2008; Wade et al., 2014b; Kent et al., 2015). This rhetoric, aimed at constructing a uniﬁed political identity, has been explored by numerous scholars who seek to group diverse populations under the label of “mestizo”. However, this endeavor marginalizes other minority groups (Telles and Bailey, 2013; Wade et al., 2015; Rodríguez Mega, 2021; Silva Gallardo and González Zarzar, 2023). Paradoxically, this homogenizing effort has failed to eliminate disparities, even among those self-identiﬁed as mestizos. Individuals with mixed indigenous and non-indigenous heritage also bear the consequences of internalized colonialism, illustrating that the legacy of oppression extends beyond just minority communities but also those with diverse backgrounds (Fanon, 1961). The mestizo rhetoric idealizes a vision that fails to address socioeconomic disparities and conceals cultural and political privileges deriving from early colonialism (Cusicanqui, 2012). In several Latin American countries, there is a positive correlation between the percentage of European ancestry and socioeconomic status, in contrast to the Native American/Amerindian ancestry (Avena et al., 2012; Campbell et al., 2012; Bonilla et al., 2015; Barozet et al., 2021). This is a compelling illustration of how colonial historical legacies inﬂuence contemporary societal structures, shedding light on the intricate relationships between genetics, identity, and privilege in these regions. Addressing these multifaceted challenges requires comprehensive efforts to dismantle oppressive systems, promote inclusivity, and ensure equal rights and opportunities for all individuals, regardless of their background or ethnicity (González Casanova, 2006). It is worth noting that many Latin American countries have not had When researching within or about countries located in the Global South, scientists need to be aware of the local context to avoid past mistakes. These challenges are not exclusive to Latin America but are the focal point of this particular publication. In Latin America, indigenous communities and other marginalized groups, such as Afro-descendants, confront signiﬁcant challenges associated with land conﬂicts and structural racism. Power dynamics of political, economic, and cultural pressure reinforce existing inequality structures inherited by colonialism in what is referred to as Neocolonialism (Maldonado-Torres, 2016). 1 Introduction Genomic research has revolutionized our understanding of human history, ancestry, and the genetic basis of diseases. However, it is essential to acknowledge that this ﬁeld has not been exempt from the pervasive issue of inequality. The past and present of genomic research urgently need to address disparities and foster a more inclusive approach (Popejoy and Fullerton, 2016; Atkinson et al., 2022; Fatumo et al., 2022; Villanea and Witt, 2022). After decades of analyzing cohorts of (almost exclusively) European descent, geneticists Frontiers in Genetics 01 frontiersin.org frontiersin.org Arango-Isaza et al. 10.3389/fgene.2023.1304974 misconception of their static nature (Makaran and Gaussens, 2020). Cultural and political discourse has often portrayed indigenous groups as static, rural communities serving as custodians of the land and traditional knowledge. However, this narrow depiction fails to capture the true diversity of indigenous identities today and, notably, overlooks a signiﬁcant demographic: descendants of indigenous people who have emigrated into urban centers. By reinforcing the stereotype of distant “others”, the prevailing narrative excludes the majority of the indigenous population and decreases their capacity for substantial inﬂuence and the potential to shape the trajectory of the state (Cusicanqui, 2012). While there is no precise data on the indigenous population in urban areas, it is estimated that around 50% of the indigenous population lived in Latin American cities by 2010 (Del Popolo, 2018). This urban migration often results in individuals of indigenous descent occupying the lowest social strata and engaging in informal labor (Oliva Martínez, 2022). recognized the importance of increasing diversity and representation in genomic studies, particularly among minorities and indigenous communities (Oh et al., 2015; Peterson et al., 2019; Dajani et al., 2022). Initiatives are underway to expand the sampling pool and include populations historically underrepresented in research efforts. However, these initiatives often face unique challenges and complexities, starting from the position of indigenous communities who harbor mistrust towards the academic world due to past practices of exploitation that are still vivid in their collective memory (Garrison et al., 2019; Mc Cartney et al., 2022). 1 Introduction While ethical guidelines regarding genomic research have gained considerable attention in high-income countries like the United States, New Zealand, and Australia (Claw et al., 2018; Collier-Robinson et al., 2019; Garrison et al., 2019; 2020; Caron et al., 2020; Hudson et al., 2020; Tsosie et al., 2020), a striking lack of literature addresses the speciﬁc context and challenges researchers encounter in the Global South (Zavala, 2013; Australian Institute of Aboriginal and Torres Strait Islander Studies, 2020; Silva et al., 2022; Ávila-Arcos et al., 2022). In a recently published study on the genetic history of Mapuche groups in Chile (Arango-Isaza et al., 2023), we incorporated several elements to foster inclusivity, transparency, and best ethical practices when working with indigenous groups. Reporting on particular challenges and procedures from speciﬁc research studies provides solutions to implement and adapt to the circumstances of other similar research studies (Rodriguez et al., 2022). With the present paper, we want to describe our research strategies, reﬂecting on actions that positively impacted the communities, the genetic study, and aspects that could be improved. misconception of their static nature (Makaran and Gaussens, 2020). Cultural and political discourse has often portrayed indigenous groups as static, rural communities serving as custodians of the land and traditional knowledge. However, this narrow depiction fails to capture the true diversity of indigenous identities today and, notably, overlooks a signiﬁcant demographic: descendants of indigenous people who have emigrated into urban centers. By reinforcing the stereotype of distant “others”, the prevailing narrative excludes the majority of the indigenous population and decreases their capacity for substantial inﬂuence and the potential to shape the trajectory of the state (Cusicanqui, 2012). While there is no precise data on the indigenous population in urban areas, it is estimated that around 50% of the indigenous population lived in Latin American cities by 2010 (Del Popolo, 2018). This urban migration often results in individuals of indigenous descent occupying the lowest social strata and engaging in informal labor (Oliva Martínez, 2022). The trajectory towards ethical and considerate conduct in genomic research hinges on the scientiﬁc community’s approach. Scientists are called to embrace cultural humility, meticulously eschewing broad generalizations and detrimental stereotypes that perpetuate damaging narratives or undermine the autonomous agency of indigenous peoples and their descendants, irrespective of their residential context (Pappas, 2020). Frontiers in Genetics frontiersin.org 1 Introduction The involvement of participants and the community in a broader sense is essential to guarantee respect for cultural settings, beneﬁt for the participants, and enrichment of the scientiﬁc study. The need for the involvement of participants has been highlighted by indigenous representatives and indigenous geneticists and recognized by scientists from Western institutions (Claw et al., 2018; Garrison et al., 2019). This can be done from the beginning of the study design to the discussion of the scientiﬁc results. Returning the genetic result of a study has been largely neglected in the past. Until today, it is often conducted out of the spontaneous initiative of the geneticists responsible for the project and is not enforced by the institute’s best scientiﬁc practice. In recent years, there have been examples of studies that included the return and discussion of the results prior to the drafting of the scientiﬁc publication as a requirement in the research agreement (Rodriguez et al., 2022). The 1980 constitution in Chile does not recognize the existence of indigenous people. Currently, the sole legal framework offering limited protection is the Indigenous Law from 1993 (Law 19253). This legislation paved the way for the establishment of CONADI (Corporación Nacional de Desarrollo de los Pueblos Indígenas), responsible for implementing policies and programs to promote the development and wellbeing of indigenous peoples in Chile. Between 2001 and 2003, the “Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas “(Historical Truth and New Deal with Indigenous Peoples Commission) aimed to address historical issues and demands of indigenous peoples. This commission recognized the diversity of indigenous cultures and identiﬁed past injustices. Its recommendations aimed for a new relationship between the Chilean state and indigenous people, including land restitution, cultural recognition, and consultation on matters affecting indigenous rights (Aylwin Azócar et al., 2003). In 2004, the Indigenous Law was completed by introducing the “New Treatment politics”, an attempt to establish a new relationship with the indigenous communities. Although Chile has adhered to the UN Declaration on the Rights of Indigenous Peoples and the International Labour Organization 169 Convention since 2009, evidence suggests inadequate compliance with these international laws (Lucic and Guzmán, 2018). At the moment, none of these laws (or the creation of CONADI) have managed to solve the territorial conﬂict in Chile. The land claims remain one of the priorities for the indigenous political movement in Chile (Oliva Martínez, 2022). 1 Introduction Many of these areas have been converted into monocultures, particularly in the Biobío region, with non-native plants like eucalyptus and radiata pine replacing the local forests (Lara et al., 2012; Martínez Navarrete, 2021). (with an 11th group in the process of being recognized), of which the Mapuche constitute the largest group (Sandoval et al., 2023). However, despite their signiﬁcant presence, indigenous communities, including the Mapuche, face socio-economic challenges such as higher poverty levels, indigence, and lower life expectancy than non-indigenous populations (Agostini et al., 2010; Sandoval et al., 2022). During the 20th century, the Mapuche population was involved in an intense internal migration from the rural areas to the cities (Antileo, 2014). Of the Mapuche population, 62.4% live in the cities, of which around 30% are in the capital city of Santiago (Reynoso and Sánchez, 2020). While the majority of Mapuche people and their descendants reside in Santiago, their original territory, known as Wallmapu, encompassed central and southern Chile, spanning from the Atlantic to the Paciﬁc coast in Argentina (Zúñiga, 2006; Vitar, 2010). Since the 19th century, with the occupation of Araucanía, this territory has been threatened by the Chilean state with a consistent pattern of land appropriation that closely mirrors settler colonialism’s historical practices (Aylwin Azócar et al., 2003; Wolfe, 2006; Nahuelpan Moreno and Antimil Caniupán, 2019; Correa Cabrera, 2021; Martínez Navarrete, 2021). Over the past 3 decades, Mapuche land has been reduced by approximately 510,000 ha due to the privatization of indigenous territories since 1973 (Foerster, 2002; Klubock, 2014; Gómez-Barris, 2017; Nahuelpan Moreno and Antimil Caniupán, 2019). Many of these areas have been converted into monocultures, particularly in the Biobío region, with non-native plants like eucalyptus and radiata pine replacing the local forests (Lara et al., 2012; Martínez Navarrete, 2021). meaningful conversations regarding their stance on genetic research (Wade et al., 2015). The discussion surrounding genomic research in the Latin American context, encompassing the mestizo rhetoric and indigenous purism, must be examined alongside the pressing issues faced by indigenous communities, Afro-descendants, and mestizos. By considering the historical and current challenges and prioritizing ethical considerations and inclusivity, researchers can work towards meaningful and equitable engagement with these communities in genetic research endeavors (Silva et al., 2022). A key factor in opening scientiﬁc research to indigenous representation and abandoning neocolonialist practices is transparency in research protocols and community participation. 1 Introduction This paper illustrates and contextualizes our experience conducting a project on the genetic history of Mapuche populations in Chile. The majority of the researchers involved in the primary genetic study and the main authors of the current paper are located in the Global North; this factor is recognized as a limitation and potential source of bias, as our positionality and cultural background can inﬂuence our perspectives and interpretations. Our speciﬁc goal is to ascertain the qualitative impact of our collaborative and participatory efforts, illustrating the potential for constructive engagement with local communities. By recognizing the importance of community engagement and meaningful participation, we aimed to make research ﬁndings accessible, culturally sensitive, and beneﬁcial to the community. Moreover, we focus on how these collaborative endeavors have impacted the scientiﬁc knowledge generated by our research. Finally, we provide a list of actions and recommendations in line with developing guidelines for genetic research in Latin America. Through sharing our experiences and lessons learned, we hope to foster dialogue, collaboration, and critical reﬂection within the scientiﬁc community, ultimately contributing to more inclusive and respectful research practice. Frontiers in Genetics 1 Introduction Estimates derived from a census in 2015 indicate that self-identiﬁed indigenous people constituted approximately 8%–10% of the population in the Caribbean and Latin America by 2018, totaling around 60 million people, a consistently growing number (CEPAL, 2020; Oliva Martínez, 2022; Flores et al., 2023). These indigenous groups endure ongoing social, economic, and political marginalization rooted in historical power imbalances and systemic inequalities (Cusicanqui, 2012). The scientiﬁc discourse has inadvertently contributed to this exoticization of indigenous groups by perpetuating the idea of a “pristine” indigenous identity (indigenous purism) and linking it to speciﬁc genetic proﬁles (an aspect of essentialism or genetic determinism). This concept erroneously connects indigenous identity or tribal afﬁliation to DNA and implies that one’s genome dictates one’s characteristics as an individual or collective (Nordgren and Juengst, 2009; Blanchard et al., 2019). Indigenous communities are diverse and dynamic, comprising various cultures, languages, and traditions (Ryan-Davis and Scalice, 2022). Like the broader society, indigenous communities have changed over time, challenging the 02 frontiersin.org 10.3389/fgene.2023.1304974 Arango-Isaza et al. (with an 11th group in the process of being recognized), of which the Mapuche constitute the largest group (Sandoval et al., 2023). However, despite their signiﬁcant presence, indigenous communities, including the Mapuche, face socio-economic challenges such as higher poverty levels, indigence, and lower life expectancy than non-indigenous populations (Agostini et al., 2010; Sandoval et al., 2022). During the 20th century, the Mapuche population was involved in an intense internal migration from the rural areas to the cities (Antileo, 2014). Of the Mapuche population, 62.4% live in the cities, of which around 30% are in the capital city of Santiago (Reynoso and Sánchez, 2020). While the majority of Mapuche people and their descendants reside in Santiago, their original territory, known as Wallmapu, encompassed central and southern Chile, spanning from the Atlantic to the Paciﬁc coast in Argentina (Zúñiga, 2006; Vitar, 2010). Since the 19th century, with the occupation of Araucanía, this territory has been threatened by the Chilean state with a consistent pattern of land appropriation that closely mirrors settler colonialism’s historical practices (Aylwin Azócar et al., 2003; Wolfe, 2006; Nahuelpan Moreno and Antimil Caniupán, 2019; Correa Cabrera, 2021; Martínez Navarrete, 2021). Over the past 3 decades, Mapuche land has been reduced by approximately 510,000 ha due to the privatization of indigenous territories since 1973 (Foerster, 2002; Klubock, 2014; Gómez-Barris, 2017; Nahuelpan Moreno and Antimil Caniupán, 2019). frontiersin.org 3.1 Data collection and availability The genetic study of the Mapuche ancestry in Southern Chile was framed with interdisciplinary perspectives from anthropology, archaeology, and linguistics. For the study, genetic data was retrieved with a new sample collection from individuals who either self-identiﬁed as having Mapuche ancestry or resided in regions with a historically attested Mapuche presence. The research project and sample collection were approved by the Unidad de Ética y Seguridad de Investigación of the Pontiﬁcia Universidad Católica de Chile’s Institutional Review Board (project #171009001, decree #1520863561038). All project stages adhered to the principles of the Declaration of Helsinki Association (World Medical Association, 2013). Sampling was conducted in early 2019 by two project members, a geneticist and a linguist of Mapuche descent. The sampling trip took place in the Araucanía region and on the island of Chiloé. Before and/or during the sampling process, consultation took place with local authorities such as municipalities, cultural centers, and lonkos (traditional leaders of Mapuche communities). This collaborative approach ensured respectful engagement and adherence to local protocols. The precise sampling locations were not disclosed to protect the privacy of participants. Extensive time (minimum one hour, usually three to four hours) was dedicated to explain the project to a potential participant and building reciprocal trust. The conversations were held in Spanish, with the two project members proposing a colloquial atmosphere. The potential participants were not pressured into participating in the project. After receiving extensive explanations of the project’s aims and conditions, the 67 participants who voluntarily agreed to donate a sample could sign an informed consent and receive a copy for them to keep. Samples consisted of 2 mL of saliva collected in Oragene tubes from DNAgenotek and stored with an anonymous code. A second random anonymization step was carried out before processing the samples in the laboratory so neither the participants nor the geneticist who collected the sample could link the sample code to a speciﬁc person. This extra step was performed in case they could remember the sample code associated with a participant. The COVID-19 pandemic signiﬁcantly impacted our plans. Our ability to travel and engage with people and communities was restricted, and our actions were contingent on ever-changing COVID-19 reports. 3.2 Returning the genetic results After ﬁnalizing the data analysis, we returned the research results to the participants and the broader local population. We took several steps to disseminate the results effectively, making the ﬁndings accessible to both the participants and the general public in an inclusive manner and distancing from the exclusiveness of the scientiﬁc publication in English, written in academic jargon. 2 Challenges and threats to indigenous identity and genetic legislation in Chile The Chilean population, estimated at 19.5 million, is predominantly of mixed genetic origin, like the population of most Latin American countries. Genetic analysis methods recognize two main ancestral components: the Amerindian, usually referred to as “Native American”, and the European (Fuentes et al., 2014; Barozet et al., 2021). Current legislation recognizes 10 Indigenous groups in Chile In the ﬁeld of genetic research, studies conducted in Chile have contributed to understanding the genetic history and population dynamics of various groups, including the Mapuche (Acuña P. et al., 2000; De Saint Pierre et al., 2012; Fuentes et al., 2014; De la Fuente et al., 2015; Eyheramendy et al., 2015; De la Fuente et al., 2018; 03 frontiersin.org Arango-Isaza et al. 10.3389/fgene.2023.1304974 Arango-Isaza et al. Verdugo et al., 2020; Barozet et al., 2021; Arango-Isaza et al., 2023). The existing Law 20.120, in force since September 2006, focuses on scientiﬁc research in human beings and their genome while prohibiting human cloning. This law requires authorization from the relevant authorities and a favorable report from an accredited ethical scientiﬁc committee before conducting the study. It also established the National Bioethics Commission and emphasized the importance of informed consent (Vargas Catalán and Millán Klüsse, 2016). Nevertheless, the current legal framework does not explicitly and speciﬁcally safeguard indigenous communities’ genetic privacy and rights (Silva et al., 2022). from the University of Zurich and the Universidad Católica de Chile, subject to the conditions outlined in the Data Access Agreement Form, which is available upon request. Frontiers in Genetics 3 Methodology To begin with, we organized a return expedition trip in early 2022 with three project members: the Ph.D. student responsible for the genetic analysis, the genetic principal investigator who conceptualized the study, and the main local researcher with extensive ﬁeld experience in the area and a vital local network, being the last two also the ones who collected the data in 2019. During this expedition, our main objective was to ensure effective communication. We recognized the importance of Mapudungun, the local Mapuche language, which is the subject of revitalization practices with dedicated courses at school, but we realized that Spanish is universally spoken in the region, so we decided to use Spanish to translate the scientiﬁc results from English, the language of the scientiﬁc publication. The results were organized in a slide presentation, a common medium for scientiﬁc dissemination. Recognizing that not everyone had access to screens or video projectors, and to add material value to our dissemination aids, we turned the slide display into a printed version in A3 format that could be conveniently displayed and understood without additional technology. The slides are available as Supplementary Material S1. 16 meetings were organized to present the results; in each session, a minimum of one person and a maximum of ~15 people were present. In the meetings, we worked to include a very diverse audience: for example, active members of the communities engaged in keeping the culture alive, teachers, people with doctoral degrees as well as people with no state education, women and men, and children of various ages. Moreover, we actively engaged with local schools in each study area. We conducted three lectures tailored for students and teachers, one in each community area, ensuring that our ﬁndings reached the younger generation and educational professionals who play a crucial role in knowledge dissemination. For the presentations in high schools, we modiﬁed and expanded the material to provide a more comprehensive insight into genetics and stress the implications of our results for rooting the region’s history within the community. frontiersin.org Arango-Isaza et al. 3.1 Data collection and availability Not only did we adhere to the quarantine periods mandated by the government upon arrival, but we also proactively implemented additional self-determined safety measures to safeguard the wellbeing of the participants, like wearing FP2 masks and carrying rapid diagnostic testing every 3 days. The data generated (SNP chip genotypes, with ~600,000 SNPs typed per individual) were deposited in the European Genome- phenome Archive (EGA; https://ega-archive.org/) with the accession number EGA: EGAS00001007200. The data is not publicly available but under access control, restricted to research purposes related to human genetic history. Access to the data is granted by a Data Access Committee composed of principal authors Our collaboration and continuous dialogue with local stakeholders proved invaluable throughout the process. Their insights and perspectives were instrumental in framing our research questions, presenting our results in culturally appropriate ways, and maintaining sensitivity to the cultural and 04 frontiersin.org Arango-Isaza et al. 10.3389/fgene.2023.1304974 FIGURE 1 Colored corn analogy for explaining heritability from Supplementary Material S1. An analogy using colored corn was employed during our engagement with Mapuche Indigenous communities to explain heritability. Each colored kernel represented observable traits inﬂuenced by genetics, demonstrating how traits are passed down from parents to offspring. FIGURE 1 Colored corn analogy for explaining heritability from Supplementary Material S1. An analogy using colored corn was employed during our engagement with Mapuche Indigenous communities to explain heritability. Each colored kernel represented observable traits inﬂuenced by genetics, demonstrating how traits are passed down from parents to offspring. social context of the study areas. Notably, the return expedition was conducted before writing the genomic manuscript, enabling us to incorporate participants’ suggestions and feedback into the ﬁnal publication. This approach ensured that their voices and contributions were respectfully acknowledged. As mentioned above, this particular step is only sometimes applied to genetic studies, with relevant exceptions (Rodriguez et al., 2022). general audience exhibit diverse knowledge on biology, we adapted the language use to communicate the study ﬁndings. Our communication strategies aimed at maintain clarity without compromising the quality of the message (Hintz and Dean, 2020). We insisted on analogies, a well-established science teaching strategy to bridge the gap between complex scientiﬁc concepts and everyday language (Aubusson et al., 2006). For example, we used the analogy of the DNA molecule as a book that stores information or the use of colored corn to explain genetic diversity and heritability (Figure 1). 3.1 Data collection and availability These analogies were relatable and accessible entry points for explaining key biological concepts to the participants. After the publication of the genomic manuscript (Arango-Isaza et al., 2023), we prepared a comprehensive report in Spanish that was distributed to both the participants and can be diffused online to the general Latin American public (Supplementary Material S2). This report serves as a platform for scientiﬁc dissemination and provides a broad understanding of molecular biology, population genetics, and the key genetic results. During ﬁeldwork, we encountered widespread misconceptions about genetic ancestry, and a tendency towards genetic essentialism (Graves Jr, 2002; Donovan, 2017; Donovan et al., 2019). Even among geneticists, there is a misuse of certain terms that inadvertently reinforce social and genetic proﬁles, suggesting a genetic basis for racial categories - even if no biological support exists for those (Wade et al., 2015; Silva Gallardo and González Zarzar, 2023). The terms “race”, ethnicity, and genetic ancestry are often interpreted as overlapping concepts in scientiﬁc and popular literature (Yudell et al., 2016). For example, our interlocutors wondered if their genetic ancestry determines physical traits and unique cultural practices and traditions, revealing a subtle yet prevalent tendency towards genetic essentialism. We explicitly addressed this misconception by explaining that our identity is not rooted in our genetic ancestry (see further notes in Section 4.4). We also showed that the genetic diversity among any human individuals is limited, and that genetic diversity is primarily found among individuals rather than between 4 Engaging mapuche indigenous communities: challenges and considerations in returning genetic scientiﬁc results in the Latin American context Frontiers in Genetics 4.2 The relevance of local collaborators: ensuring cultural translation in genetic research populations, as illustrated in Figure 2, from the scientiﬁc report (Supplementary Material S2) (Lewontin, 1972; Hunley et al., 2016). Additionally, we explained the process of DNA sequencing in simple terms and presented the scale of human migrations in the world and South America, highlighting the power of genetics in understanding human history (Supplementary Material S2). The research project was enriched through the involvement of local collaborators and stakeholders, most of whom identiﬁed as Mapuche, while some did not (winka is the Mapudungun word for non-Mapuche people). As highlighted by other scholars, the local population’s participation beneﬁts both academia and the community (Copete et al., 2023). These connections were established during our trip and through the extensive network of the main local researcher. Their contributions were instrumental across multiple facets of the project, from study design and data collection to the critical aspects of the results dissemination and manuscript preparation. Their involvement enabled us to navigate the intricacies of cultural translation, which extended beyond language proﬁciency alone. Importantly, the presence of a Mapuche researcher did not exempt the rest of the team from dedicating effort to acquiring cultural competence. By listening to the concerns and perspectives of different community members during the two ﬁeld trips, the team adapted terminology, phrasing, research angles, and general sensitivity to the local history and culture, cultural norms, non-spoken communication, and political dynamics to ensure effective cross-cultural communication and engagement (Hudson et al., 2020). In the ﬁeld, researchers can face the initial challenge of establishing connections with participants before introducing the project’s aims and scope. Local collaborators were vital as cultural intermediaries, providing valuable insights into community dynamics, customs, and traditions that might elude outsiders. Their assistance facilitated the exchange of knowledge, ideas, and concerns between researchers and the community members, ensuring that dialogue was respectful, reciprocal, and culturally sensitive. This understanding was crucial in ensuring that the dialogue was conducted respecting the local context and contributing positively to the community’s wellbeing. Creating an environment that fostered participant engagement was crucial. We encouraged questions during and after our presentations, recognizing that open dialogue and oral communication are central to Mapuche communities (Bañales- Seguel et al., 2020). We also acknowledged that certain genetic ﬁndings might challenge traditional or religious community beliefs or perspectives (Ávila-Arcos et al., 2022). To address this sensitivity, we included a dedicated slide (Supplementary Material S1. 4.1 Communicating scientiﬁc results: how to make scientiﬁc language accessible to a broad audience It is a fundamental right for both the participants and the broader region audience to have access to the study results (Hudson et al., 2020). As the participants in our project and the 05 frontiersin.org Arango-Isaza et al. 10.3389/fgene.2023.1304974 FIGURE 2 Distribution of genetic diversity among humans. This ﬁgure shows how genetic diversity is distributed among human populations. Part of Supplementary Material S2 (Lewontin, 1972; Lander et al., 2001; Hunley et al., 2016; Novembre, 2022). FIGURE 2 Distribution of genetic diversity among humans. This ﬁgure shows how genetic diversity is distributed among human populations. Part of Supplementary Material S2 (Lewontin, 1972; Lander et al., 2001; Hunley et al., 2016; Novembre, 2022). Frontiers in Genetics frontiersin.org 4.2 The relevance of local collaborators: ensuring cultural translation in genetic research Slide 6) and paragraph in the report (Supplementary Material S2) emphasizing that scientiﬁc knowledge is just one facet of overall knowledge and should not be seen as dominant or absolute truth. “Knowledge is vast and includes other ways of understanding the world, such as experience, tradition, legends and myths, among others. These types of knowledge are not mutually exclusive but can complement each other to obtain a broader vision of reality. Science is simply one of the ways of acquiring knowledge about the world.” We underscored the importance of fostering dialogue between knowledge systems (Escobar, 2016; Bañales-Seguel et al., 2020). During our discussions and conversations with community members, we posed questions such as “What are your thoughts on this ﬁnding?” and “Does it align with your previous notions?” or even “How do you feel about these results?” These questions encouraged open dialogue and created an inclusive atmosphere for diverse viewpoints. As we delivered and discussed scientiﬁc knowledge, participants could make informed interpretations and engage in meaningful discussions on the research ﬁndings. Through these efforts, we aimed to foster scientiﬁc literacy and promote a more informed participation in genetic research. It is essential to acknowledge that our research unfolded within a challenging political context. Mapuche communities have been living in a conﬂictual dynamic with the state in relationship to 06 frontiersin.org Arango-Isaza et al. 10.3389/fgene.2023.1304974 opportunity to connect with the heritage of their community (Manning and Harrison, 2018). This erasure created a tense dynamic between teachers and indigenous students, stemming from the educators’ lack of knowledge about local and cultural history and their limited understanding of indigenous education. These factors restricted education to a nationwide monocultural and homogeneous curriculum, perpetuating prejudices against the indigenous population (Arias-Ortega et al., 2023). Even today, the educational system has not met the local populations’ educational, social, cultural, and linguistic needs (Milne and Wotherspoon, 2023). Schools perpetuated symbolic and cultural domination over the indigenous population, removing students from their social, cultural, and spiritual foundations (Arias- Ortega and Quintriqueo, 2021). During our ﬁeldwork experience, we had conversations with several elderly Mapuche people who explained how they consciously chose not to teach Mapudungun to their children, to protect them from discrimination. These interactions show how deeply rooted prejudice and cultural marginalization have affected Mapuche communities for generations. land ownership and recognition of indigenous culture and rights (Aylwin Azócar et al., 2003). 4.2 The relevance of local collaborators: ensuring cultural translation in genetic research Given the sensitive nature of our work, some local researchers and stakeholders, although deeply committed to our research goal, declined to participate as DNA donors or preferred not to take the role of ofﬁcial coauthors. Instead, they actively engaged in discussions, providing invaluable insights and logistic support while maintaining a degree of anonymity. In a couple of circumstances, during sample collection, community members displayed hostility towards the presence of winkas performing genetic studies with Mapuche communities, reporting on known cases of scientiﬁc misconduct with the handling of sensitive genetic data from indigenous groups that occurred in the late 90s and early 00s (see Malhi, 2009), or negative experiences of the type “other academics came to study us, but never came back”. In these circumstances, we respected the individual perspectives and left the region or the household without collecting any data. Despite these complexities, the people involved in the step of returning the results overall expressed satisfaction with our collaborative approach, as reﬂected by the positive feedback obtained during all our 16 meetings. This trust was not solely due to the presence of a Mapuche researcher but also because of our team’s commitment to earning their conﬁdence. We valued the communities’ trust and recognized that it was essential in our research. This collaborative approach allowed us to achieve our research goals effectively. opportunity to connect with the heritage of their community (Manning and Harrison, 2018). This erasure created a tense dynamic between teachers and indigenous students, stemming from the educators’ lack of knowledge about local and cultural history and their limited understanding of indigenous education. These factors restricted education to a nationwide monocultural and homogeneous curriculum, perpetuating prejudices against the indigenous population (Arias-Ortega et al., 2023). Even today, the educational system has not met the local populations’ educational, social, cultural, and linguistic needs (Milne and Wotherspoon, 2023). Schools perpetuated symbolic and cultural domination over the indigenous population, removing students from their social, cultural, and spiritual foundations (Arias- Ortega and Quintriqueo, 2021). During our ﬁeldwork experience, we had conversations with several elderly Mapuche people who explained how they consciously chose not to teach Mapudungun to their children, to protect them from discrimination. These interactions show how deeply rooted prejudice and cultural marginalization have affected Mapuche communities for generations. 4.2 The relevance of local collaborators: ensuring cultural translation in genetic research In more recent times, an increasing awareness of the value of cultural and linguistic diversity, coupled with rising attention to human and indigenous rights, has enhanced the way for the implementation of intercultural and bilingual education programs in Chile such as the “Programa Orígenes” and the program for Intercultural Bilingual Education (Figueroa Burdiles et al., 2018; Aguayo et al., 2022). These initiatives have primarily been applied in rural areas and, while they still require reﬁnement, have contributed to a stronger sense of indigenous identity among the students (Cisternas Irarrázabal, 2018). 4.3 Engaging with schools: a focus on pre- European-contact history Particularly noteworthy was the positive response from “traditional educators,” who specialize in teaching basic Mapudungun language and Mapuche indigenous traditions. Their expertise and deep connection to indigenous cultures made them particularly receptive to including prehispanic history in the curriculum. Finally, the classes have also been positively received by students becoming more aware of indigenous traditions, either as self-identifying as Mapuche, or recognizing having Mapuche ancestry, or with students who live in contact with Mapuche peers. By acknowledging the prehispanic history and cultural diversity in the Americas, we encouraged a more inclusive and accurate narrative encompassing indigenous communities’ rich heritage, in line with the claims of diverse indigenous movements (Oliva Martínez, 2022). This approach broadened students’ perspectives, allowing them to develop a more comprehensive understanding of the continent’s history and cultural dynamics. From the beginning of the colonial period, after the independence of Chile and the subsequent occupation of the territories south of the Biobío river, which were managed by Mapuche communities, Mapuche children in schools and institutions were prohibited from speaking their language and subjected to various forms of physical and psychological abuse (Reynoso and Sánchez, 2020; Dillon et al., 2022). The curriculum effectively erased indigenous history, denying Mapuche students the 4.3 Engaging with schools: a focus on pre- European-contact history ( ) Our presentations received signiﬁcant appreciation from the teaching staff at the schools we visited. History teachers, in particular, positively received our research angle on genetic human history (and prehistory) as it provided a more comprehensive understanding of the region’s historical roots. Particularly noteworthy was the positive response from “traditional educators,” who specialize in teaching basic Mapudungun language and Mapuche indigenous traditions. Their expertise and deep connection to indigenous cultures made them particularly receptive to including prehispanic history in the curriculum. Finally, the classes have also been positively received by students becoming more aware of indigenous traditions, either as self-identifying as Mapuche, or recognizing having Mapuche ancestry, or with students who live in contact with Mapuche peers. By acknowledging the prehispanic history and cultural diversity in the Americas, we encouraged a more inclusive and accurate narrative encompassing indigenous communities’ rich heritage, in line with the claims of diverse indigenous movements (Oliva Martínez, 2022). This approach broadened students’ perspectives, allowing them to develop a more comprehensive understanding of the continent’s history and cultural dynamics. The history curriculum in Latin America has predominantly focused on the last ﬁve centuries from the Spanish occupation, often neglecting the continent’s rich and diverse prehispanic heritage. There is an awareness of the need for a representation of the continent’s history that gives justice to its cultural roots before the arrival of the Europeans (Cisternas Irarrázabal, 2018). To address this historical imbalance, we emphasized how the Americas were populated long before the Spanish colonization. Throughout our ﬁeld presentations, we took chances to highlight the cultural and ethnic diversity of the continent’s populations before Europeans arrived. It is important to consider how educational institutions during the 20th century served as “civilizing” entities under the pressure of Chilean elites, who promoted the narrative of Mapuche people as “second-class” citizens (Merino et al., 2007; Nahuelpan Moreno and Antimil Caniupán, 2019). Additionally, assimilationist educational policies have dominated national and international contexts. Indigenous children worldwide experienced the active eradication of their cultural identity through humiliation, psychological violence, and even physical abuse, which were part of what has been termed “cultural genocide” (Commission de Vérité et Réconciliation du Canada, 2015). Our presentations received signiﬁcant appreciation from the teaching staff at the schools we visited. History teachers, in particular, positively received our research angle on genetic human history (and prehistory) as it provided a more comprehensive understanding of the region’s historical roots. Frontiers in Genetics frontiersin.org 4.4 Addressing misconceptions and sensitive topics in genetics During our ﬁeldwork with indigenous communities, we encountered several complex and delicate topics that required 07 frontiersin.org Arango-Isaza et al. 10.3389/fgene.2023.1304974 careful consideration and preparation by our team. We prepared in particular for two aspects of the genetic analysis that traditionally meet sensitive reactions: the concept of Native American and European genetic admixture, and the concept of indigenous identity and genetic determinism. Despite our primary focus being on prehispanic history, our genetic analysis included an overview of the degree and timing of European admixture in the region and the continent. This type of analysis is common practice in a population genetics study and serves to contextualize the rest of the analysis, which focuses on Native American ancestry and prehispanic history. The genetic concept of European admixture is not exempt from the power dynamics and exploitation that brought European lineages into the region. First of all, it was essential to navigate the political connotations associated with terms like “genetic admixture” or “mestizaje”, not only confronting the scientiﬁc jargon to the understanding of the layperson but also confronting the use of these terms in English and their equivalents in Spanish. Given the signiﬁcance of word choice, we used the term “genetic ﬂow” instead, as it carried a less loaded meaning and conveyed our understanding without evoking negative or positive connotations. For instance, we used sentences like “The different populations exhibit a wide degree of European genetic ﬂow.” In our on-site presentations (Supplementary Material S1) and in the ﬁnal report (Supplementary Material S2), we explained that this observation had no positive or negative connotation. Instead, it simply reﬂected a part of the historical dynamics that Mapuche communities encountered. and González Zarzar, 2023). Research has shown how identities can be ﬂuid and even intersectional (Puar, 2012). Identity should not be tied to biological or phenotypical factors but to sociological and cultural elements and the subjective dimension of the self- identiﬁcation (Oliva Martínez, 2022). Also, we commented on the widespread notion that indigenous people are “less advanced and not evolved”. Our historical, cultural, and biological approach provided tools to reframe colonialist practices in this sense. We stressed that such notions of progress come from power dynamics deeply rooted in the scientiﬁc discourse of the Western world and have been confronted in Anthropology since the early 20th century. 4.4 Addressing misconceptions and sensitive topics in genetics “Another important point is that claiming that one population is more advanced or evolved than another contradicts the principles of biology and is incorrect. Each human population has evolved or developed in a unique way in response to the environment and historical circumstances, which has given rise to today’s cultural diversity. No linear scale of progress or superiority can be established among human populations, as all have their own valuable knowledge, traditions, and ways of life.” In approaching these delicate topics, we intended to foster respectful and meaningful dialogue while being mindful of the complexities and sensitivities involved, without avoiding problematic topics. We carefully selected our terminology and focused on broader population dynamics to create a space for understanding and appreciation without diminishing community members’ individual experiences and self-identiﬁcations. “Furthermore, it is important to emphasize that gene ﬂow between the settlers and the Mapuche populations should not be interpreted as a measure of superiority or inferiority. Rather, it reﬂects the complex interactions and cultural mixtures that occurred during this historical period. These genetic interactions are a testimony to the resilience and adaptability of the Mapuche people and their culture, despite colonial inﬂuence.” 4.5 Moving away from neocolonial science: mitigating biocolonialism and scientiﬁc extractivism We are aware of the limitations inherent in our study and acknowledge the inﬂuence of our cultural backgrounds on the research process. To mitigate neocolonial practices, we adopted a collaborative approach involving local scientists from diverse disciplines, ensuring inclusivity and cultural sensitivity throughout the research process. Community engagement and reciprocity played a central role, focusing on sharing the knowledge we generated with the involved communities, thereby respecting participants’ ownership (Hintz and Dean, 2020). We carefully explained our informed consent document, emphasizing clear explanations of the research purpose, potential beneﬁts, risks, and handling of genetic data. These practices have been long enforced in basic scientiﬁc practice when dealing with human participants and are highlighted in the Declaration of Helsinki (World Medical Association, 2013). While drafting a complete and appropriate informed consent is mandatory, extra effort should be placed to ensure that the participant fully understands all these aspects. We found challenges in establishing a fully horizontal dialogue with potential participants as they were often introduced for the ﬁrst time on the concept of genetic data, and they understandably lacked the foundational knowledge necessary to engage fully, especially concerning data storage and sharing. This challenge is not only due to a disparity in knowledge but also to the perception of our team as “authorities” in the ﬁeld, inadvertently creating a hierarchical dynamic. Genetic estimates of indigenous ancestry have been associated with questions of identity, even in legal terms, as some indigenous representatives require any person who would like to join a community to apply with a DNA ancestry test to prove their indigenous ancestry (TallBear, 2013; Blanchard et al., 2019). The very delicate question “How indigenous am I?” is not uncommon and bears traces of a genetic deterministic approach that connects cultural identities to speciﬁc biological markers. To address this sensitively, we implemented two strategies. Firstly, we provided population-based results rather than individualized ones, ensuring that the focus remained on the broader context rather than individual identity. Secondly, we proactively explored the space between biology and identity with our interlocutors to reﬂect together on the limitations of such an approach and the nuanced relationship between these concepts (Supplementary Material S2). “Genetic ancestry is not equivalent to identity. Frontiers in Genetics frontiersin.org 5 Discussion To create ethical and sustainable research in the Global South, it is essential to understand the different layers of inequality and the social reality of Latin American societies. In general, academic research is inserted in power dynamics where economic and epistemological asymmetries reproduce colonial attitudes (Argüelles et al., 2022). Addressing epistemic extractivism is crucial, as it perpetuates a power dynamic that takes data and knowledge from marginalized communities without equitable collaboration or beneﬁt-sharing, reinforcing existing inequalities and undermining autonomy (Mc Cartney et al., 2022). On the other hand, academic research can be an agent of positive change, empowering indigenous communities in countries with little or no recognition from the state (Silva et al., 2022). Genuine collaboration and meaningful engagement with local communities increases the value of local knowledge and perspectives in the research process. Creating long-term partnerships and capacity-building initiatives that empower local researchers is essential (Hudson et al., 2020). We acknowledged the limitations of our research approach, as Community-Based Participatory Research should involve continuous, long-term engagement with the community (TallBear, 2014). While our engagement with the indigenous communities was impactful, it lacked a long-term vision, a limitation often encountered by other scholars (TallBear, 2014; Tsosie et al., 2020). Teaching technical knowledge about genetics was challenging, especially in the absence of established structures for indigenous genomic projects in the Global South. Nonetheless, we aimed to improve the current research model, recognizing that achieving a gold standard was not feasible. Though resources for community-driven efforts have been traditionally scarce or non- existent for most institutions and research funding bodies, our approach was uniquely positioned due to the relatively substantial resources available to us, thanks to the signiﬁcant funding of our institution (University of Zurich). These resources allowed us to involve local researchers and experts while including cultural protocols and sensitivities. We were able to provide schematic graphics and transparent project steps to develop open and accountable research practices. We could also take time to organize a dedicated trip and undertake the described actions towards the communities. Integrating community engagement as an intrinsic component of the original research proposal should take a higher value in our academic curricula and be supported by an appropriate timeline and budget approved by the institution’s boards. Our experience underscores the need for grant agencies to recognize the importance of such engagement initiatives and allocate resources accordingly. 4.5 Moving away from neocolonial science: mitigating biocolonialism and scientiﬁc extractivism However, creating long-term partnerships and facilitating capacity building presented challenges, primarily attributable to the structure of scientiﬁc funding and the relative early-career stage of the researchers involved. Frontiers in Genetics 4.5 Moving away from neocolonial science: mitigating biocolonialism and scientiﬁc extractivism Identity is constructed through variable elements such as culture, sense of belonging and other factors, and is not determined by biology, or at least not solely.” We suggest that geneticists can and should speak up to avoid reducing indigenous identity to mere genetic factors (Silva Gallardo 08 frontiersin.org 10.3389/fgene.2023.1304974 10.3389/fgene.2023.1304974 Arango-Isaza et al. 10.3389/fgene.2023.1304974 Arango-Isaza et al. To help build a deeper understanding of the methodology in data generation, analysis, and storage, scientists could work on building long-term partnerships with the communities. Yet, practical barriers such as budget constraints, time limitations, and the disruptive impact of the COVID-19 pandemic impeded the full realization of such a long-term collaboration. One way to build long-term partnerships would be through a consortium to facilitate preliminary education, provide educational materials and resources to community members on topics related to genetics and the research process, and develop capacity building for access to research activity. This educational initiative could include workshops, seminars, and community meetings. Additionally, the sessions would emphasize the potential beneﬁts and risks associated with the research and involve members of other communities not involved in the genomic project to appreciate the broad reach of such genetic studies. Finally, the consortium could elaborate on technical options related to data sharing, storage, and sequencing technology. Similar initiatives are not yet developed in the Global South but are designed as a gold standard in some countries, including the United States, New Zealand, Canada, and Australia (Claw et al., 2018; Garrison et al., 2019; Hudson et al., 2020; Tsosie et al., 2020). In Chile, an example of a long-term academic initiative fostering ethical and inclusive practice on genetic research in indigenous communities is the Grupo de Estudio Ciencia y Comunidades Originarias organized by the Center for Intercultural and Indigenous Research and the Pontiﬁcia Universidad Católica de Chile (Silva et al., 2022). “And now, what else?”. The participants’ eagerness to research further aspects demonstrated the potential for a broader engagement aligned with their interests and questions. However, creating long-term partnerships and facilitating capacity building presented challenges, primarily attributable to the structure of scientiﬁc funding and the relative early-career stage of the researchers involved. “And now, what else?”. The participants’ eagerness to research further aspects demonstrated the potential for a broader engagement aligned with their interests and questions. Arango-Isaza et al. 5 Discussion This recognition ensures that the beneﬁts and insights from collaborative endeavors can be maximized for both scientiﬁc advances and community empowerment. More speciﬁcally, genetic research in the Global South can have repercussions on the local society and identity. It contributed to stigmatization and perpetuated harmful perspectives, such as mestizo rhetoric or indigenous purism (Garrison et al., 2019; Silva et al., 2022). Genetic admixture from different ethnic backgrounds is an undeniable reality in Latin America. However, it was not a result of voluntary choices but a violent process accompanied by subjugation and stigma, often hidden from the ofﬁcial historical narrative (Galindo, 2020). To move away from these harmful perspectives, we must embrace a more nuanced understanding of genetic admixture in the region (Wade et al., 2014a). Consultation with state institutions and the “mestizo” population is insufﬁcient and will reproduce historical harm (Ávila-Arcos et al., 2022). National or international efforts in genomic research should consider the speciﬁc contexts of countries with limited investments and respect indigenous governance structures. Ethical science should be central to budget distribution systems for granting agencies, and researchers, research institutions, and indigenous communities should collaboratively develop guidelines (Argüelles et al., 2022). There is a growing number of genomic projects in Latin American countries managed by local researchers, like Latin Cells, CANDELA, DNAdoBrasil, INMEGEN, and ChileGenómico, and a growing interest in the indigenous history of the continent. However, indigenous participation is still scarce compared to North America (Claw et al., 2018; Tsosie et al., 2020). Interestingly, the participants often expressed curiosity and enthusiasm for expanding the scope of the study into other areas. A recurring sentiment was encapsulated in the common question, 09 frontiersin.org Arango-Isaza et al. Arango-Isaza et al. 10.3389/fgene.2023.1304974 • Create a safe and inclusive environment for discussion and exchange, taking into account the community’s communication styles and preferred methods of debate. • Create a safe and inclusive environment for discussion and exchange, taking into account the community’s communication styles and preferred methods of debate. Community engagement has been our primary focus for supporting ethical and inclusive research in our genetic study of Mapuche ancestry in Chile (Arango-Isaza et al., 2023). Here, we presented our strategies to report research to the Mapuche communities and involve their perspectives in the genetic study. Funding • In the Latin American context, where colonial structures often persist, a non-indigenous university board’s approval of a research permit alone will not exempt a researcher from validation with local communities and stakeholders. Mere acceptance by the mestizo population is not an adequate measure of ethical engagement. The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. CB and KS were supported by the URPP ‘‘Evolution in Action’’ of the University of Zurich and the NCCR Evolving Language, Swiss National Science Foundation Agreement #51NF40_180888. CB, EA-I, and KS were supported by the SNSF Sinergia project ‘‘Out of Asia’’ (grant number 183578). For the return expedition, the University of Zurich Graduate Campus (GRC) supported EA-I, and the Zurich Latin American Center (LZZ) supported both EA-I and CB. FM is funded by CIIR-FONDAP 15110006. • Emphasize the importance of speaking the local language or the language most commonly used within the community. • Provide comprehensive explanations of biological concepts during data collection and results dissemination to ensure a thorough understanding of the implications and potential risks involved. Develop teaching and scientiﬁc outreach strategies. • Work towards long-term partnerships and empowerment of the communities. 5 Discussion Garrison et al., 2019 emphasized the importance of giving back results in genomic research when involving indigenous communities or minorities, but this action is not guaranteed enough in scientiﬁc studies. These dialogues were designed to bridge the gap between different epistemologies and change societal perspectives on issues like racism (Haverkort, 2013; Wade et al., 2015). We also evaluated the qualitative impact of our collaborative and participatory efforts to demonstrate the potential for meaningful and more reciprocal engagement with the local communities. By incorporating comments and suggestions, the scientiﬁc paper beneﬁtted from more rigorous data interpretation. Furthermore, we addressed economic and epistemological asymmetries to build a more horizontal collaboration and enhance the integrity of the scientiﬁc knowledge production process. • Be prepared for potential negative responses and the possibility that the community may not wish for the research to be published or to disclose certain aspects of it. • Be prepared for potential negative responses and the possibility that the community may not wish for the research to be published or to disclose certain aspects of it. Approach framing and dissemination of results with sensitivity and respect. Approach framing and dissemination of results with sensitivity and respect. • Establish mechanisms to deliver research ﬁndings to direct participants and a broader audience, including schools, cultural centers, or organized communities. • Prioritize sharing research results with the community before the publication of the manuscript to ensure transparency and mutual understanding. In conclusion, there are still several concerns for which the scientiﬁc community must actively improve the ethical standards of research, especially in the Global South. This includes fostering collaborations with local researchers, meaningful community consultation, and respecting principles of informed consent and data sovereignty. By involving indigenous communities as active partners and incorporating their perspectives, researchers can conduct genomic research that respects the communities’ rights, values, and aspirations. The international community, especially in the Global North, is responsible for approaching research respectfully, considering their inherited privilege and background. Our experience of engagement with the indigenous communities can contribute to the challenging task of establishing guidelines for genomic research in Latin America. Based on the challenges we faced and on the feedback received, we can share our recommendations for effective and sustainable community partnerships. • Understand the country’s current and past social and political context, not only from an anthropological or genetic point of view. Author contributions • Comprehend the social and power dynamics between the diverse demographics in Latin America, which may not directly align with the indigenous communities. EA-I: Investigation, Writing–original draft, Writing–review and editing, Visualization. MA: Supervision, Validation, Writing–review and editing. RC: Writing–review and editing. FM: Writing–review and editing. KS: Resources, Writing–review and editing, Supervision, Funding acquisition. CB: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing–review and editing. • Educate yourself about different knowledge systems, including indigenous ones, to appreciate the diverse perspectives and worldviews within the community. • Develop a deep understanding of the pressing issues the communities face, such as challenges related to education, land rights, and access to essential services, to ensure that the research is contextually relevant and beneﬁcial to the community. Frontiers in Genetics frontiersin.org References Acuña, P. M., Llop, R. E., and Rothhammer, E. F. (2000). Genetic composition of Chilean population: rural communities of Elqui, Limari and Choapa valleys. 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A., and Raghavan, M. (2022). 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. Conﬂict of interest The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2023.1304974/ full#supplementary-material 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. Acknowledgments • Acknowledge common misconceptions and prejudices to provide an educated response if these issues arise during conversation. We thank all the voluntary participants in this study from Araucanía and the Island of Chiloé, and all the people who discussed the genetic results and gave feedback during the • Recognize the signiﬁcance of local collaborators and stakeholders in ensuring culturally sensitive and meaningful research engagement. • Recognize the signiﬁcance of local collaborators and stakeholders in ensuring culturally sensitive and meaningful research engagement. 10 frontiersin.org Arango-Isaza et al. 10.3389/fgene.2023.1304974 10.3389/fgene.2023.1304974 expedition in March–April 2022 (Jaqueline Caniguan, Jaime Haro, Carlos Catrileo, Juan Manuel Huentelican, Carmen Cayun, Julio Chewin, María Isabel Díaz, Carolina Aillapán, Awunwuenu Aillapán, Sixto Cuyul, Juan Carlos Domihual, Romero family, Sonia Catepillan, Guido Brevis, Colegio Adenauer in Melipeuco, Liceo Público Reino de Suecia in Puerto Saavedra, Liceo Galvarino Riveros Cárdenas in Castro Chiloé, Nicolás Montalva, Marcelo González, Fernando Pairican, Piergiorgio Di Giminiani, Alejandra Vidal, Verónica Silva, and Eduardo Barrientos). 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https://openalex.org/W2888187041	https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/234086/1/journal.pone.0197719.pdf	English	null	Morphological and ecological adaptation of limpet-shaped top shells (Gastropoda: Trochidae: Fossarininae) to wave-swept rock reef habitats	PloS one	2,018	cc-by	7,246	RESEARCH ARTICLE Morphological and ecological adaptation of limpet-shaped top shells (Gastropoda: Trochidae: Fossarininae) to wave-swept rock reef habitats Luna Yamamori, Makoto Kato Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto, Japan Luna Yamamori, Makoto Kato Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto, Japan * strobilation980@gmail.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Geerat J. Vermeij, University of California, UNITED STATES Received: May 7, 2018 Accepted: July 17, 2018 Published: August 22, 2018 Copyright: © 2018 Yamamori, Kato. 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: © 2018 Yamamori, Kato. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. OPEN ACCESS Citation: Yamamori L, Kato M (2018) Morphological and ecological adaptation of limpet- shaped top shells (Gastropoda: Trochidae: Fossarininae) to wave-swept rock reef habitats. PLoS ONE 13(8): e0197719. https://doi.org/ 10.1371/journal.pone.0197719 Citation: Yamamori L, Kato M (2018) Morphological and ecological adaptation of limpet- shaped top shells (Gastropoda: Trochidae: Fossarininae) to wave-swept rock reef habitats. PLoS ONE 13(8): e0197719. https://doi.org/ 10.1371/journal.pone.0197719 Editor: Geerat J. Vermeij, University of California, UNITED STATES Abstract Flattening of coiled shells has occurred in several gastropod lineages, while the evolutionary process of shell flattening is little known. The subfamily Fossarininae of the top shell family (Trochidae) is unique, because it includes four genera at various stages of shell flattening. Broderipia and Roya, have zygomorphic shells that has lost coiling, while the sister genera, Fossarina and Synaptocochlea, have respectively turbiniform and auriform shells. There- fore, comparisons of biology, habitats and detailed morphology among these four genera may help us to detect selection pressure driving shell flattening and loss of coiling. Although Broderipia has recently been identified as living symbiotically in the pits of sea urchins, the habitats and biology of the other three Fossarininae species, especially Roya are poorly known. After an extensive search on rocky shores of the Japanese Archipelago, we found live Roya eximia snails on intertidal/subtidal rock surfaces exposed to strong waves. Roya snails crept on the bare rock surface to graze periphyton at low tide, and fled into vacant bar- nacle shells at high tide. Comparison of the morphology of soft bodies in Fossarininae revealed that the columellar muscle of flattened species has been drastically elongated and arranged in posterior semi-outer edge of the flattened shell as observed in true limpets. The flattering and loss of coiling of the shell in Roya caused acquisition of a zygomorphic flat body, retraction of coiled visceral mass, and expansion of the foot sole. All of these changes improved tolerance against strong waves and the ability to cling to rock surfaces, and thus enabled a lifestyle utilizing both wave-swept rock surfaces and the inside of vacant barnacle shells. Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fissurellidae, Phenacolepadidae, Hipponicidae, Calyptraeidae, Umbraculidae, Trimusculidae, Siphonariidae, Ancylidae, some Capulidae, Thyca crystallina (Eulimidae), Amathina (Amathi- nidae), and a portion of Fossarininae (Trochidae), among other groups [3]. analysis, decision to publish, or preparation of the manuscript. analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Because limpet-shape is thought to be not well adapted to intense competition and preda- tion, it is thought to be originated in refugial habitats, however, there are some advantages of limpet-shape, depending on their habitats and feeding types [4]. For example, because of the structurally compact body, limpets potentially have large respiratory surface. This large respi- ratory surface is advantageous not only for oxygen-poor deep sea, but also for suspension feed- ing as seen in Calyptraeidae [5]. Other major advantages of the limpet-shaped shells are hypothesized to be reduction of the risk to be pulled off by strong waves, improvement of adhering strength, and rapid locomotion, which are enabled by low-conical shell and large foot [3]. Although limpet-shaped shells show various ecologies, the factors that promote the evolution of limpet-shaped shells have not yet been identified, because most members of the aforementioned families have limpet-shaped shells and species in transition from coiled to non-coiled shells are rare. The top-shell family Trochidae is characterized by conical, coiled shells and an alga-grazing habit, although some linages contain filter feeders (Umboniinae). In Trochidae, the two sub- families (Fossarininae and Stomatellinae) contain species with flattened shells. The phyloge- netic tree of Fossarininae [6] suggests that shell flattening has occurred from a turbiniform shell to an auriform shell, and subsequently to a cap-shaped shell (Fig 1). Because the four gen- era of Fossarininae are currently at various stages in the evolutionary process of shell flatten- ing, comparisons of their biology and habitats will help us to detect selection pressure driving shell-flattening and loss of coiling. However, only limited information is available about the ecology of Fossarininae species. Broderipia, which has an extremely flat shell, has recently been revealed to be symbiotic in the pits of sea urchins, and its flat limpet-shaped shell is apparently adaptive to life in the narrow open space of the pits [7]. Introduction Funding: This study was supported by a Japan Ministry of Education, Culture, Science, Sports, and Technology Grant-in-Aid for Scientific Research (15H02420; https://www.jsps.go.jp/english/index. html) and The Sasakawa Scientific Research Grant (29-537; http://www.jss.or.jp/en/). The funders had no role in study design, data collection and Molluscs exhibit a wide range of shell forms as adaptations to surrounding environmental con- ditions [1], and for defense against predators [2]. In the history of shell-shape evolution, flat- tening of the coiled shell is one of the most common strategies; some of these flattened shells subsequently lost their coiling, forming limpet-shaped shells. Limpet-shaped shells are adopted in diverse gastropod lineages, e.g., Patellogastropoda, Cocculiniformia, Lepetodriloidea, html) and The Sasakawa Scientific Research Grant (29-537; http://www.jss.or.jp/en/). The funders had no role in study design, data collection and 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Study sites This study was conducted on rocky shores in the Japanese Archipelago, which are influenced by the warm Kuroshio Current. Through a preliminary search for the rare limpet-shaped tro- chid snail R. eximia, five populations were identified on the southern coasts of the Kii Penin- sula and Shikoku Island (sites A–E in Fig 2). No specific permissions were required for these locations, and neither endangered nor protected species were involved in this field study. At all sites, the maximum tidal range during spring tide is around 2.0 m. Because the habitat of R. eximia is constantly exposed to violent waves, accessible sites were rare. The only accessible coastal sites were sites A (33˚6905100N, 135˚3305800E) and E (32˚46’00"N, 132˚37’18"E). Site A is a small near-shore island called Toshima, the west coast of which faces the Kii Channel and is exposed to strong waves. The rock bed is conglomerate mixed with brittle sandstone, and the surface remains rough due to constant erosion from waves (Fig 3A and 3B). Site E is a rocky and boulder-filled shore of Kashiwajima Island, which is constantly exposed to strong waves (Fig 3C and 3D). The large boulders accumulated on the shore are made up of hard and smooth igneous rock [8]. The habitat and biology of other Fos- sarininae species, especially Roya are still poorly known. To detect selective pressures acting on shell flattening and the loss of coiling in Fossarini- nae, we first conducted an extensive search for habitats of the key genus, Roya. Because Roya snails are found in low intertidal areas of wave-swept rocky reefs, we carried out a field survey of the macrobenthic and macrophytic communities on various types of rocky reefs surround- ing Roya snails, and observed the diurnal behavior of the snails. To determine their feeding biology, radula and gut contents of Roya snails were also examined. By superimposing the Fig 1. The phylogenetic tree of subfamily Fossarininae (Williams et al. 2010, modified). https://doi.org/10.1371/journal.pone.0197719.g001 Fig 1. The phylogenetic tree of subfamily Fossarininae (Williams et al. 2010, modified) Fig 1. The phylogenetic tree of subfamily Fossarininae (Williams et al. 2010, modified). https://doi.org/10.1371/journal.pone.0197719.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 2 / 13 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fig 2. Locations of the study sites. A: Toshima in Shirahama, Wakayama Prefecture, B: Muroto Cape, C: Goshikihama, D: Chihiro Cape, E: Kashiwajima Island in Kochi Prefecture. https://doi.org/10.1371/journal.pone.0197719.g002 Fig 2. Locations of the study sites. A: Toshima in Shirahama, Wakayama Prefecture, B: Muroto Cape, C: Goshikihama, D: Chihiro Cape, E: Kashiwajima Island in Kochi Prefecture. https://doi.org/10.1371/journal.pone.0197719.g002 https://doi.org/10.1371/journal.pone.0197719.g002 obtained data on a cladogram of Fossarininae, we discussed the evolution of shell flattening and loss of coiling in gastropods. obtained data on a cladogram of Fossarininae, we discussed the evolution of shell flattening and loss of coiling in gastropods. Morphology and diet of Fossarininae The shell, soft body and radula morphologies of the four Fossarininae species were compared. Firstly, the side view of the living snail and side view and aperture view of their shells were photographed. Secondly, to examine the soft bodies, snails were anesthetized in 8% MgCl2 solution for 1 h, and fixed in 4% formalin solution for about 12 h. After fixation, snails were removed from the formalin solution, rinsed with flowing fresh water for 30 minutes, and transferred to 70% Ethanol to examine soft bodies under optical microscope. Radulae were removed from each snail and examined using an electron microscope. Lastly, to determine the diet of Fossarininae snails, the stomach and intestinal contents were examined. Fossarininae snails were collected at site A and immediately fixed in 4% formalin solution for 12 h, and rinsed with water. The stomach and intestine of each Fossarininae snail were removed and opened in water; approximately 1/5 of the contents were observed and its organic matters were counted using an optical microscope. Census on macroalgae and macrobenthos In the lower intertidal zones of sites A and E, two different settings of rock surfaces were cho- sen, which were exposed to and protected from strong waves. We refer to the former and latter PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 3 / 13 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fig 3. Study sites and habitats of snail species belonging to Fossarininae. a: Exposed reef at site A; arrowhead shows the tidal level inhabited by R. eximia. b: Protected reef at site A. c: Exposed reef at site E; arrowhead shows the rock inhabited by R. eximia. d: Protected reef at site E. https://doi.org/10.1371/journal.pone.0197719.g003 Fig 3. Study sites and habitats of snail species belonging to Fossarininae. a: Exposed reef at site A; arrowhead shows the tidal level inhabited by R. eximia. b: Protected reef at site A. c: Exposed reef at site E; arrowhead shows the rock inhabited by R. eximia. d: Protected reef at site E. https://doi.org/10.1371/journal.pone.0197719.g003 Fig 3. Study sites and habitats of snail species belonging to Fossarininae. a: Exposed reef at site A; arrowhead shows the tidal level inhabited by R. eximia. b: Protected reef at site A. c: Exposed reef at site E; arrowhead shows the rock inhabited by R. eximia. d: Protected reef at site E. https://doi.org/10.1371/journal.pone.0197719.g003 settings as exposed and protected reefs, respectively. At first, we evaluated the intensity of waves by measuring the wave beat frequency and the average/maximum wave height (i.e., ver- tical range to which a wave dashes up on rock reef) in a minute by checking the video of waves at low tides at each site. In both rock surface settings, 10 quadrats (10 × 10 cm) were set, and all macroalgae and macrobenthos in each quadrat were examined in terms of species, abun- dance (for macrobenthos) and coverage (for macroalgae) during a spring tide in April 2017. Rock-surface environment and macrobenthic community at each site As the wave intensity, frequencies and the average and maximum heights of waves per min are shown in Table 1. The wave intensities were higher in exposed than protected rock reef y, q g g p are shown in Table 1. The wave intensities were higher in exposed than protected rock reefs. The surfaces of the exposed reefs in the lower intertidal zones of sites A and E were covered with 13 species of encrusting and branching algae. The proportions of bare surface, i.e., areas without cover of macroalgae or sessile organisms, of exposed and protected rock surfaces were 8.86% and 47.1% at site A, and 24.3% and 20.8% at site E, respectively. Most rock surfaces were not covered with sand, although the protected rock surfaces of site A were partly covered with sand. This low algal coverage of protected reefs may be caused by sedimentation on rock surfaces, as the growth of branching algae is hindered by sedimentation [9–10]. The most frequent and dominant macroalgae were the encrusting coralline red algae Litho- phyllum spp. and encrusting non-coralline red alga Hildenbrandia rubra (S1 Table). The branched coralline algae Amphiroa beauvoisii and Serraticardia maxima were found only at site A. The assemblages of brown algae differed between the two sites, as well as between exposed and protected microhabitats. At site A, the rock surfaces exposed to strong waves were dominated by the large brown algae Sargassum fusiforme and S. patens, while more pro- tected rock surfaces were often inhabited by the brown alga Palisada intermedia. On the other hand, at site E, exposed rock surfaces were inhabited by P. intermedia and Ishige okamurae, while no brown algae were observed on protected rock surfaces. The coverages of macroalgae and sessile organisms at sites A and E on exposed and protected rock surfaces are shown in Fig 4. The coverage of branching red algae on exposed rock surfaces was greater at site A than site E (t-test, p = 0.011), whereas the coverage of encrusting red algae on protected rock surfaces was greater at site E than site A (t-test, p = 0.0047). Coverages of encrusting red algae and branching brown algae at site A were significantly greater on exposed rock surfaces than on protected rock surfaces (t-test: p = 0.033 for encrusting red algae; p = 0.047 for branching brown algae). Diurnal behavior of R. eximia At site A, diurnal changes in the distribution of R. eximia snails were surveyed in the intertidal area at high tide, the middle of the ebb tide (awash time of their habitat tidal level) and low tide in the daytime, and at low tide in the nighttime on 15th April 2017. In each census, R. eximia snails were sought out not only on the rock surface, but also in the interspaces of sessile organ- isms and inside vacant shells of barnacles; their behavior was observed. 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Table 1. The wave intensities at protected and exposed rock reefs in site A and E. frequency (± sd) (beats / min.) average (± sd) height in a minute (cm) maximum (± sd) height in a minute (cm) site A protected 22.7 ± 5.6 2.3 ± 1.0 5.5 ± 1.1 exposed 21.3 ± 6.1 112.4 ± 12.1 223.9 ± 25.5 site E protected 35.8 ± 4.2 4.9 ± 1.3 8.7 ± 2.1 exposed 14.4 ±3.3 122.7 ± 21.1 284.9 ± 18.9 https://doi.org/10.1371/journal.pone.0197719.t001 Table 1. The wave intensities at protected and exposed rock reefs in site A and E. (Fig 6A) and inside crevices and vacant shells of barnacles on the exposed reef (Fig 6B and 6C). Synaptocochlea pulchella was found in crevices on rocks in the exposed reef (Fig 6D and 6E). Broderipia iridescens was observed exclusively inside the pits or crevices inhabited by sea urchins (Fig 6F and 6G). R. eximia was found on bare rock surfaces around barnacle colonies in exposed area (Fig 6H). The density of R. eximia at site E was significantly greater than at site A (t-test, p = 0.033). It is important to note that Montfortula picta is a snail in the coil-less family Fissurellidae (Vetigastropoda), with a shell that bears a remarkable resemblance to that of R. eximia. M. It is important to note that Montfortula picta is a snail in the coil-less family Fissurellidae (Vetigastropoda), with a shell that bears a remarkable resemblance to that of R. eximia. M. picta was observed only on the exposed reef, and not on the protected reef, at both sites A and (Vetigastropoda), with a shell that bears a remarkable resemblance to that of R. eximia. M. picta was observed only on the exposed reef, and not on the protected reef, at both sites A and B, as was also observed for R. eximia. The coexistence of morphologically similar, phylogeneti- cally distant snail species suggests that the limpet-shaped shell morphology is a product of con- vergence due to adaptation to life on exposed rock reefs. Rock-surface environment and macrobenthic community at each site In addition to these macroalgae, the rock surfaces of the intertidal zones were covered with sessile organisms, such as barnacles and a sessile vermetid snail, Serpulorbis imbricatus. At site A, three large barnacle species, Megabalanus volcano, Tetraclita japonica and T. squamosa were observed on exposed rock surfaces, while no barnacles were found on protected rock surfaces. At site E, four barnacle species were observed. Two species of small barnacles, Fistulobalanus albicostatus and Chthamalus challengeri, inhabited the protected rock surfaces, while three species, including the two large tetraclitid speciesT. japonica and T. squa- mosa, inhabited exposed rock surfaces (S1 Table). Coverage of sessile organisms on exposed rock surfaces was greater at site E than site A (t-test, p = 0.00050); at site E, coverage was greater on exposed rock surfaces than protected rock surfaces (t-test, p = 0.00032). The avail- ability of plankton is affected by wave action [11], therefore, it is assumed that large barnacles inhabit exposed rock surfaces where plenty of planktons are provided by strong waves. The composition of mobile fauna was also influenced by wave strength. From the compari- son of species richness and density between exposed and protected reefs (Fig 5), species rich- ness on exposed habitats was higher at site A than at site E (t-test, p = 0.018), and species richness on the exposed reef was higher than on the protected reef at both sites A and E (t-test: p = 0.000011 for site A, p = 0.00040 for site E). The density of macrobenthos on the exposed reef was also greater than that on the protected reef (t-test: p = 0.024 for site A, p = 0.006 for site E). At both sites, four Fossarininae species were observed in four microhabitats: open rock sur- faces, crevices, sea urchin pits and vacant barnacle shells (Table 2). Fossarina picta was found on both protected and exposed reefs, specifically on open rock surfaces of the protected reef 5 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Diurnal behavior of Roya eximia Species richness and density of the macrobenthos in 10 × 10 cm quadrats (n = 10) set on exposed and protected rock surfaces at sites A and E. https://doi org/10 1371/journal pone 0197719 g005 Fig 5. Species richness and density of the macrobenthos in 10 × 10 cm quadrats (n = 10) set on exposed and protected rock surfaces at sites A and E. https://doi.org/10.1371/journal.pone.0197719.g005 https://doi.org/10.1371/journal.pone.0197719.g005 This behavioral pattern contrasts with many non-homing limpets that follow the wash zone up and down. This vertical movement is considered to prevent attacks from aquatic predators, and simultaneously to minimize desiccation stress caused by exposure to air [12–13]. In con- trast, Roya snails minimize predation pressure by moving over wave-swept rock surfaces at mid-low tide and hiding in vacant shells of barnacles at high tide, rather than vertically migrat- ing. Because wave-swept rock surfaces are scarcely inhabited by carnivorous muricid snails, Roya snails are largely free from predation. Although submerged rock surfaces are potentially vulnerable to carnivorous fishes at high tide, the vacant shells of barnacles act as refugia from predatory fishes. Diurnal behavior of Roya eximia Diurnal changes in the number of R. eximia snails found at site A are shown in Fig 7. The habi- tat of R. eximia was within 50 cm of the ebb tide line. This habitat emerges from the water for about 1 hour during the spring tide, and is constantly splashed during its emergence. During the daytime high tide, four R. eximia were found inside vacant shells of the barnacle Megabala- nus volcano, completely covered with water (Fig 6I), and all four snails had tightly adhered their bodies to the inner surface of the barnacle shells, and did not move. At mid-ebb tide in the daytime, on the other hand, the snails swiftly crept over rock surfaces awash with seawater and grazed. When the tide was low during the daytime, six snails were observed on the wet surface of emergent rocks. Two snails slowly and intermittently moved over the emergent zone and grazed, while the other four snails remained motionless, with their bodies tightly adhered to the rock surface. At the nighttime low tide, three snails were observed remaining motionless on the wet rock surface. Fig 4. Mean coverage (cm2) of various algae and sessile organisms in 10 × 10 cm quadrats (n = 10) set on exposed and protected rock surfaces at sites A and E. ER: encrusting Rhodophyta, BR: branching Rhodophyta BO: branching Ochrophyta. S: sessile invertebrates, bare: bare surface. https://doi.org/10.1371/journal.pone.0197719.g004 Fig 4. Mean coverage (cm2) of various algae and sessile organisms in 10 × 10 cm quadrats (n = 10) set on exposed and protected rock surfaces at sites A and E. ER: encrusting Rhodophyta, BR: branching Rhodophyta BO: branching Ochrophyta. S: sessile invertebrates, bare: bare surface. https://doi.org/10.1371/journal.pone.0197719.g004 Fig 4. Mean coverage (cm2) of various algae and sessile organisms in 10 × 10 cm quadrats (n = 10) set on exposed and protected rock surfaces at sites A and E. ER: encrusting Rhodophyta, BR: branching Rhodophyta BO: branching Ochrophyta. S: sessile invertebrates, bare: bare surface. https://doi.org/10.1371/journal.pone.0197719.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 6 / 13 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fig 5. Species richness and density of the macrobenthos in 10 × 10 cm quadrats (n = 10) set on exposed and protected rock surfaces at sites A and E. https://doi org/10 1371/journal pone 0197719 g005 Fig 5. Morphology of the shell and soft body of Fossarininae The side views of a living snail, and side and aperture views of shells of four Fossarinae species are shown in Fig 8. F. picta has a turbiniform shell and four pairs of epipodial tentacles (Fig 8A, 8E and 8I). S. pulchella has an auriform shell and four pairs of epipodial tentacles (Fig 8B, 8F and 8J). B. iridescens has a depressed limpet-shaped shell and three pairs of epipodial tenta- cles (Fig 8C, 8G and 8K). R. eximia (Fig 8D, 8H and 8L) has a higher limpet-shaped shell armed with radial ribs, and three pairs of epipodial tentacles. The shell morphology of Roya is advantageous for survival in wave-swept habitats for the following reasons. First, limpet- shaped shells are more tolerant of strong waves than coiled shells [14]. Second, limpet-shaped shells with large apertures promote the development of a highly contracted soft body with a large foot sole, which would confer strong clinging ability [15, 16]. Third, the radial ribs of the shell (Fig 8H) may reinforce structural integrity, defensive capability against carnivores or light interception. Limpets living in areas exposed to direct sunlight tend to have more ribs and nodules on the shell than those living in shaded areas, which serve to radiate heat [17], while shell ornamentation, including spines and ribs, offers protection against drilling carni- vores [18–19]. The first and second epipodial tentacles of B. iridescens are longer than those of R. eximia. The lengths of the epipodial tentacles were 2.7, 2.2 and 1.7 mm for B. iridescens (8.2 mm shell length; from anterior to posterior ones), and 1.7, 1.7, and 1.6 mm for R. eximia (9.5 mm shell length). These long tentacles may be useful for monitoring the position of their host Table 2. The numbers of individuals of the Fossarininae species observed in each habitat at low tide (from 10 quadrats in both site A and E, 200cm2 in total). Open rock surface Crevice Sea urchin’s pit Vacant barnacle shell Total F. picta 6 2 0 3 11 S. pulchella 0 2 0 0 2 B. iridescens 0 0 39 0 39 R. eximia 23 0 0 0 23 https://doi.org/10.1371/journal.pone.0197719.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 7 / 13 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fig 6. Microhabitats of Fossarininae snails. a−c: F. Morphology of the shell and soft body of Fossarininae picta in rock crevices of protected (a) and exposed (b) rock reefs, and in vacant shells of the barnacle Megabalanus volcano on exposed reefs (c); sandy sediments are sometimes accumulated in crevices, as shown in a. d−e: S. pulchella in a rock crevice. f−g: B. iridescens in sea urchin pits. h−i: R. eximia found on wave-swept rock surfaces at low tide (h), and in vacant shells of the barnacle M. volcano at high tide (i). Fig 6. Microhabitats of Fossarininae snails. a−c: F. picta in rock crevices of protected (a) and exposed (b) rock reefs, and in vacant shells of the barnacle Megabalanus volcano on exposed reefs (c); sandy sediments are sometimes accumulated in crevices, as shown in a. d−e: S. pulchella in a rock crevice. f−g: B. iridescens in sea urchin pits. h−i: R. eximia found on wave-swept rock surfaces at low tide (h), and in vacant shells of the barnacle M. volcano at high tide (i). https://doi.org/10.1371/journal.pone.0197719.g006 https://doi.org/10.1371/journal.pone.0197719.g006 sea urchin. The presence of opercula varied among Fossarinae species: F. picta has an opercu- lum which can close up the aperture tightly (Fig 8M and 8Q). In S. pulchella, however, the operculum is vestigial and too small to close up the aperture (Fig 8N and 8R). In B. iridescens and R. eximia, the opercula are completely absent (Fig 8O and 8P). Most gastropods with lim- pet-shaped shells or loosely coiled auriform shells invariably live clinging to hard substrates, and originally or secondarily lack an operculum [20]. Our results suggest that reduction and loss of the operculum has occurred during the evolution of shell-flattering in Fossarininae. g g In Trochidae, Similar depression of shells are seen in Stomatellinae; basal clade Calliotro- chus has turbiniform shells, while derived clade Stomatella has depressed auriform shells [21]. However, there are no species with limpet-shaped shells in Stomatellinae. Stomatella snails having strongly flattened shells inhabit undersides of boulders, and their soft bodies are too large to draw them into the shells. These data suggest that the shell flattering in Stomatella has Fig 7. Diurnal changes of tidal level (wavy line) and observed number of R. eximia snails (solid columns). The habitat of the snail in reference to the tidal level is illustrated as a gray band. Roya snails were observed on wet rock surfaces at low tide, while they were found inside vacant barnacle shells on submerged rock surfaces at high tide. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Morphology of the shell and soft body of Fossarininae https://doi.org/10.1371/journal.pone.0197719.g007 Fig 7. Diurnal changes of tidal level (wavy line) and observed number of R. eximia snails (solid columns). The habitat of the snail in reference to the tidal level is illustrated as a gray band. Roya snails were observed on wet rock surfaces at low tide, while they were found inside vacant barnacle shells on submerged rock surfaces at high tide. Fig 7. Diurnal changes of tidal level (wavy line) and observed number of R. eximia snails (solid columns). The habitat of the snail in reference to the tidal level is illustrated as a gray band. Roya snails were observed on wet rock surfaces at low tide, while they were found inside vacant barnacle shells on submerged rock surfaces at high tide. https://doi.org/10.1371/journal.pone.0197719.g007 https://doi.org/10.1371/journal.pone.0197719.g007 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fig 8. Side and ventral views of living snails and shells of four Fossarininae species, and opercula of F picta and S. pulchella. F picta (a, e, i, m, q); S. pulchella (b, f, j, n, r); B. iridescens (c, g, k, o); R. eximia (d, h, l, p). Scale bar = 1 mm. https://doi.org/10.1371/journal.pone.0197719.g008 Fig 8. Side and ventral views of living snails and shells of four Fossarininae species, and opercula of F picta and S. pulchella. F picta (a, e, i, m, q); S. pulchella (b, f, j, n, r); B. iridescens (c, g, k, o); R. eximia (d, h, l, p). Scale bar = 1 mm. https://doi.org/10.1371/journal.pone.0197719.g008 https://doi.org/10.1371/journal.pone.0197719.g008 occurred as an adaptation to life beneath boulders, and that loss of coiling was not necessary in such protected interstitial habitats. The soft bodies of Fossarininae snails corresponded to the shell morphology. In F. picta, the body is coiled and its anterior is covered by black mantle (Fig 9A and 9E), while anterior parts of the bodies of the other species are whitish and scarcely protruded from the shells. F. picta has a short, coiled columellar muscle attaching to the columella (Fig 9I). In S. pulchella, the coiling of the soft body was greatly reduced, and the body is only slightly twisted (Fig 9B). The columellar muscle of S. pulchella was elongated, horseshoe-shaped and arranged in the poste- rior part of the body (Fig 9F). In B. iridescens and R. Morphology of the shell and soft body of Fossarininae eximia, the bodies were vertically con- tracted, and had completely lost coiling (Fig 9C and 9D), the visceral mass is retracted into the flattened body, and the long, horseshoe-shaped, zygomorphic columellar muscle was situated along the inner margin of the posterior part of the shell. (Fig 9G and 9H). This horseshoe- shaped muscle is suggested to function to appress their shells tightly to the hard substrates. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Radula morphology and diet of Fossarininae Irrespective of shell and soft body morphology differences among the four Fossarininae spe- cies, they have largely similar rhipidoglossate radulae. The central and lateral teeth of F. picta clearly differ from those of the other three species, whereas the marginal teeth were very simi- lar in all four species. The formula of F. picta radula is (30−40)−4−1−4−(30−40) (Fig 10A). The central tooth has no triangular edge and lies flat along the radula, with the cusp curled slightly upward. The teeth become much narrower in the basal region (Fig 10B). The lateral teeth on each side are hidden by the central tooth or the adjacent lateral tooth. The size and shape of the lateral teeth are similar to those of the central tooth, but the lateral teeth are slightly asym- metric with the outer side being wider than the inner side. The marginal teeth are oar-shaped, with 4−6 serrations on each side (Fig 10C). The formula of S. pulchella radula is (30−40)−5−1 −5−(30−40) (Fig 10D). The central tooth has a sharp triangular edge with 2−3 serrations on PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 9 / 13 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fig 9. Dorsal views and schematic drawings of soft bodies of four Fossarininae species. F. picta (a, e, i); S. pulchella (b, f); B. iridescens (c, g); R. eximia (d, h). Columellar muscles in schematic drawings were colored in yellow. cm columellar muscle, ct cephalic tentacle, ept epipodial tentacle, e eye, ft foot, g gill, h heart, ki kidney, nl neck lobe, ov ovary, sn snout, st stomach. Scale bar = 1 mm. https://doi.org/10.1371/journal.pone.0197719.g009 Fig 9. Dorsal views and schematic drawings of soft bodies of four Fossarininae species. F. picta (a, e, i); S. pulchella (b, f); B. iridescens (c, g); R. eximia (d, h). Columellar muscles in schematic drawings were colored in yellow. cm columellar muscle, ct cephalic tentacle, ept epipodial tentacle, e eye, ft foot, g gill, h heart, ki kidney, nl neck lobe, ov ovary, sn snout, st stomach. Scale bar = 1 mm. Fig 9. Dorsal views and schematic drawings of soft bodies of four Fossarininae species. F. picta (a, e, i); S. pulchella (b, f); B. iridescens (c, g); R. eximia (d, h). Columellar muscles in schematic drawings were colored in yellow. Radula morphology and diet of Fossarininae cm columellar muscle, ct cephalic tentacle, ept epipodial tentacle, e eye, ft foot, g gill, h heart, ki kidney, nl neck lobe, ov ovary, sn snout, st stomach. Scale bar = 1 mm. https://doi.org/10.1371/journal.pone.0197719.g009 https://doi.org/10.1371/journal.pone.0197719.g009 https://doi.org/10.1371/journal.pone.0197719.g009 each side (Fig 10E). The lateral teeth on each side are similar in size and shape to the central teeth, and bent toward the center of the radula. The marginal teeth are oar-shaped with serra- tions (Fig 10F). The formula of B. iridescens radula is (30−40)−5−1−5−(30−40) (Fig 10G). The central tooth has a shovel-shaped triangular edge with 4−6 serrations on each side (Fig 10H). The lateral teeth on each side are similar in size and shape to the central teeth, and are bent toward the center of the radula. The marginal teeth are oar-shaped and serrations are present on all teeth (Fig 10I). The formula of R. eximia radula is (30−40)−7−1−7−(30−40) (Fig 10J). The shapes and sizes of all teeth of R. eximia are very similar to those of B. iridescens, but the cutting edges of the teeth are slightly sharper (Fig 10K and 10L). As a result of diet examination, diatoms were the primary component of the gastric con- tents of all four Fossarininae species (Table 3). Fragments of red algae were also observed in small amounts in all snail species, while nematodes and sponge ossicles were rarely observed. The similarity of radulae and diet among Fossarininae species suggest that the differing shell morphologies were not influenced by diet or foraging habits. General discussion By superimposing the obtained information on morphology and ecology of Fossarininae snails into their cladogram, we can discuss the evolutionary trend accompanying shell-flattering (Fig PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 10 / 13 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats Fig 10. Views of the whole radulae (left column) and close-up views of the central and marginal teeth (middle and right columns) of four Fossarininae species. F. picta (a–c); S. pulchella (d–f); B. iridescens (g–i); R. eximia (j–l). Scale bar = 30 m. https://doi.org/10.1371/journal.pone.0197719.g010 Table 3. Organic matter seen in the gastric contents of each Fossarininae species. F. picta S. pulchella B. iridescens R. eximia Pennales diatom 158 19 49 135 red alga 5 2 3 2 Nematoda 0 0 0 1 ossicle of sponge 3 0 2 1 Total 163 21 52 138 https://doi.org/10.1371/journal.pone.0197719.t003 Fig 11. A cladogram of subfamily Fossarininae with the information acquired from this study. Fig 10. Views of the whole radulae (left column) and close-up views of the central and marginal teeth (middle and right columns) of four Fossarininae species. F. picta (a–c); S. pulchella (d–f); B. iridescens (g–i); R. eximia (j–l). Scale bar = 30 m. Fig 10. Views of the whole radulae (left column) and close-up views of the central and marginal teeth (middle and right columns) of four Fossarininae species. F. picta (a–c); S. pulchella (d–f); B. iridescens (g–i); R. eximia (j–l). Scale bar = 30 m. Fig 10. Views of the whole radulae (left column) and close-up views of the central and marginal teeth (middle and right columns) of four Fossarininae species. F. picta (a–c); S. pulchella (d–f); B. iridescens (g–i); R. eximia (j–l). Scale bar = 30 m. https://doi.org/10.1371/journal.pone.0197719.g010 Table 3. Organic matter seen in the gastric contents of each Fossarininae species. F. picta S. pulchella B. iridescens R. eximia Pennales diatom 158 19 49 135 red alga 5 2 3 2 Nematoda 0 0 0 1 ossicle of sponge 3 0 2 1 Total 163 21 52 138 https://doi.org/10.1371/journal.pone.0197719.t003 Fig 11. A cladogram of subfamily Fossarininae with the information acquired from this study. https://doi.org/10.1371/journal.pone.0197719.g011 Fig 10. Views of the whole radulae (left column) and close-up views of the central and marginal teeth (middle and right columns) of four Fossarininae species. F. picta (a–c); S. pulchella (d–f); B. iridescens (g–i); R. eximia (j–l) Scale bar = 30 m. Fig 10. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Supporting information S1 Table. Species found in quadrats and degree of coverage of each macroalgal species (n = 10). SW strong wave, GW gentle wave, CB calcareous branched, CE calcareous encrusting, B branched, E encrusting. (DOCX) Project administration: Luna Yamamori, Makoto Kato. Resources: Luna Yamamori. Supervision: Makoto Kato. Supervision: Makoto Kato. Visualization: Luna Yamamori, Makoto Kato. Writing – original draft: Luna Yamamori, Makoto Kato. Acknowledgments We are grateful to all the staff of the Seto Marine Biological Laboratory and Shirahama Aquar- ium for supporting our survey; and Y. Henmi and K. Kondo of Kyoto University for field assistance. General discussion Views of the whole radulae (left column) and close-up views of the central and marginal teeth (middle and right columns) of four Fossarininae species. F. picta (a–c); S. pulchella (d–f); B. iridescens (g–i); R. eximia (j–l). Scale bar = 30 m. https://doi.org/10.1371/journal.pone.0197719.g010 Table 3. Organic matter seen in the gastric contents of each Fossarininae species. F. picta S. pulchella B. iridescens R. eximia Pennales diatom 158 19 49 135 red alga 5 2 3 2 Nematoda 0 0 0 1 ossicle of sponge 3 0 2 1 Total 163 21 52 138 https://doi.org/10.1371/journal.pone.0197719.t003 Table 3. Organic matter seen in the gastric contents of each Fossarininae species. Fig 11. A cladogram of subfamily Fossarininae with the information acquired from this study. https://doi.org/10.1371/journal.pone.0197719.g011 Fig 11. A cladogram of subfamily Fossarininae with the information acquired from this study. Fig 11. A cladogram of subfamily Fossarininae with the information acquired from this study. https://doi.org/10.1371/journal.pone.0197719.g011 11 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats 11). The basal lineage of the subfamily Fossarininae, Fossarina, has a small coiled shell and uti- lizes narrow refugia in intertidal rock reefs. In S. pulchella, the coiling of the shell is reduced, aperture is enlarged, and the shell is flattened auriform. S. pulchella lives in crevices in exposed rock reefs, suggesting that their flat shell with large aperture is adaptive to both the narrow spaces and the wave-swept environment. In the clade comprising Broderipia and Roya, evolu- tion of a completely zygomorphic limpet-shaped shell has occurred, although selective pres- sures may differ between the two genera. In Broderipia, the extremely flattened limpet-shaped shell is presumed to be an adaptation to the narrow free space offered by the pit of its host sea urchin [7]. In Roya, the non-flat limpet-shaped shell with radial ribs is considered to be an adaptation to life moving between wave-swept rock surfaces and refugia of vacant barnacle shells. Our results show that the evolution of limpet-shaped shells occurs under various selec- tive pressures in coiled snail lineages. One intriguing topic for future research is determining the gene whose mutation has contributed to the evolution of limpet-shaped shells in multiple lineages and various marine environments. Author Contributions Conceptualization: Luna Yamamori, Makoto Kato. Formal analysis: Luna Yamamori. Funding acquisition: Luna Yamamori, Makoto Kato. Funding acquisition: Luna Yamamori, Makoto Kato. Investigation: Luna Yamamori. Methodology: Luna Yamamori, Makoto Kato. Methodology: Luna Yamamori, Makoto Kato. Project administration: Luna Yamamori, Makoto Kato. References 1. Eagar RMC. Shape and function of shell—Comparison of some living and fossil bivalve molluscs. Bio- logical Reviews of the Cambridge Philosophical Society. 1978; 53(2):169–210. https://doi.org/10.1111/ j.1469-185X.1978.tb01436.x PubMed PMID: WOS:A1978FC64800001. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 12 / 13 Morphological and ecological adaptation of limpet-shaped top shells to wave-swept rock reef habitats 2. Palmer AR. Fish predation and the evolution of gastropod shell sculpture–experimental and geological evidence. Evolution. 1979; 33(2):697–713. https://doi.org/10.1111/j.1558-5646.1979.tb04722.x PubMed PMID: WOS:A1979HE57800016. PMID: 28563927 3. Branch GM. Limpets: evolution and adaptation. The Mollusca. 1985; 10:187–220. 4. Vermeij GJ. The limpet form in gastropods: evolution, distribution, and implications for the comparative study of history. Biological Journal of the Linnean Society. 2017; 120(1):22–37. PubMed PMID: WOS:000400943400002. 5. Collin R. The utility of morphological characters in gastropod phylogenetics: an example from the Calyp- traeidae. Biological Journal of the Linnean Society. 2003; 78(4):541–93. https://doi.org/10.1046/j.0024- 4066.2002.00166.x PubMed PMID: WOS:000182544500010. 6. Williams ST, Donald KM, Spencer HG, Nakano T. Molecular systematics of the marine gastropod fami- lies Trochidae and Calliostomatidae (Mollusca: Superfamily Trochoidea). Molecular Phylogenetics and Evolution. 2010; 54(3):783–809. https://doi.org/10.1016/j.ympev.2009.11.008 PubMed PMID: WOS:000275176500010. PMID: 19919851 7. Yamamori L, Kato M. The macrobenthic community in intertidal sea urchin pits and an obligate inquilin- ism of a limpet-shaped trochid gastropod in the pits. Marine Biology. 2017; 164(3). https://doi.org/10. 1007/s00227-017-3081-5 PubMed PMID: WOS:000395180300024. 8. National Institute of Advanced Industrial Science and Technology. 2011 [cited 2018 Apr 22]. Available from: https://gbank.gsj.jp/seamless/index_en.html 9. Airoldi L. Roles of disturbance, sediment stress, and substratum retention on spatial dominance in algal turf. Ecology. 1998; 79(8):2759–70. https://doi.org/10.1890/0012-9658(1998)079[2759:rodssa]2.0. co;2. PubMed PMID: WOS:000077501000014. 10. Airoldi L, Cinelli F. Effects of sedimentation on subtidal macroalgal assemblages: An experimental study from a Mediterranean rocky shore. Journal of Experimental Marine Biology and Ecology. 1997; 215(2):269–88. https://doi.org/10.1016/s0022-0981(96)02770-0 PubMed PMID: WOS: A1997XJ16400007. 11. Akester RJ, Martel AL. Shell shape, dysodont tooth morphology, and hinge-ligament thickness in the bay mussel Mytilus trossulus correlate with wave exposure. Canadian Journal of Zoology-Revue Cana- dienne De Zoologie. 2000; 78(2):240–53. https://doi.org/10.1139/cjz-78-2-240 PubMed PMID: WOS:000085820200010. 12. Davies MS, Edwards M, Williams GA. Movement patterns of the limpet Cellana grata (Gould) observed over a continuous period through a changing tidal regime. Marine Biology. 2006; 149(4):775–87. https:// doi.org/10.1007/s00227-006-0258-8 PubMed PMID: WOS:000238683900007. 13. Iwasaki K. Factors affecting individual variation resting site fidelity in the Patellid limpet, Cellana tor- euma (Reeve). Ecological Research. 1992; 7(3):305–31. https://doi.org/10.1007/bf02347099 PubMed PMID: WOS:A1992KM64100011. 14. Denny M. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197719 August 22, 2018 References Biology and the mechanics of the wave-swept environment: Princeton University Press; 2014. 15. Branch GM. The biology of limpets: physical factors, energy flow, and ecological interactions. 1981. 16. Vermeij GJ. A natural history of shells: Princeton University Press; 1995. 17. Vermeij GJ. Morphological patterns in high-intertidal gastropods–adaptive strategies and their limita- tions. Marine Biology. 1973; 20(4):319–46. https://doi.org/10.1007/bf00354275 PubMed PMID: WOS: A1973Q477700006. 18. Alexander RR, Dietl GP. The fossil record of shell-breaking predation on marine bivalves and gastro- pods. Predator—Prey Interactions in the Fossil Record: Springer; 2003. p. 141–76. 19. Vermeij GJ. Biogeography and adaptation: patterns of marine life: Harvard University Press; 1978. 20. Checa AG, Jimenez-Jimenez AP. Constructional morphology, origin, and evolution of the gastropod operculum. Paleobiology. 1998; 24(1):109–32. PubMed PMID: WOS:000072055800008. 21. Uribe JE, Williams ST, Templado J, Buge B, Zardoya R. Phylogenetic relationships of Mediterranean and North-East Atlantic Cantharidinae and notes on Stomatellinae (Vetigastropoda: Trochidae). Molec- ular Phylogenetics and Evolution. 2017; 107:64–79. https://doi.org/10.1016/j.ympev.2016.10.009 PubMed PMID: WOS:000394200500008. PMID: 27746316 13 / 13
https://openalex.org/W4286698077	https://www.nature.com/articles/s41598-022-16597-2.pdf	English	null	Fractional quantum oscillator and disorder in the vibrational spectra	Scientific reports	2,022	cc-by	11,574	OPEN We study the role of disorder in the vibration spectra of molecules and atoms in solids. This disorder may be described phenomenologically by a fractional generalization of ordinary quantum-mechanical oscillator problem. To be specific, this is accomplished by the introduction of a so-called fractional Laplacian (Riesz fractional derivative) to the Scrödinger equation with three-dimensional (3D) quadratic potential. To solve the obtained 3D spectral problem, we pass to the momentum space, where the problem simplifies greatly as fractional Laplacian becomes simply kµ , k is a modulus of the momentum vector and µ is Lévy index, characterizing the degree of disorder. In this case, µ →0 corresponds to the strongest disorder, while µ →2 to the weakest so that the case µ = 2 corresponds to “ordinary” (i.e. that without fractional derivatives) 3D quantum harmonic oscillator. Combining analytical (variational) and numerical methods, we have shown that in the fractional (disordered) 3D oscillator problem, the famous orbital momentum degeneracy is lifted so that its energy starts to depend on orbital quantum number l. These features can have a strong impact on the physical properties of many solids, ranging from multiferroics to oxide heterostructures, which, in turn, are usable in modern microelectronic devices. Although disorder (especially strong like substance amorphization) in a solid is usually considered to be a trouble for its possible device applications, its constructive properties have become increasingly clear in recent years as they give an additional possibility to fine tune their physical properties. Specifically, by varying the type and concentration of different imperfections (like point or extended defects), we can customize the characteristics of such material to meet specific requirements, needed, for instance, in electronics. Disordered semiconductors and dielectrics like organometallic halide perovskites1,2 are widely used in photovoltaic cells, light-emitting devices, and nanolasers3,4. The above disorder influences phonon and electron spectra of a substance, leading to the distribution of the internal magnetic, electric and elastic fields. This is the case for so-called multiferro- ics, where ferroelectric and magnetic orders coexist5. It is well-known (see, e.g.6,7) that strong disorder is well described by Non-Gaussian probability distributions, having so-called long tails. In other words, these distri- butions decay at infinities (sometimes much) slower, that Gaussian one. The most prominent example here is so-called Lévy flights8–13, which are Markovian random processes whose probability density functions (pdf) are Lévy stable laws, characterized by the Lévy index 0 < µ ≤2 . www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2022) 12:12540 OPEN In the infinite space, it is profitable to define the corresponding pdf in terms of its characteristic function, i.e. Fourier transform. The pdf of a typical Lévy flight is usually determined by the fractional Fokker–Planck (FP) Eq. (8). In dimensionless units (diffusion coefficient and particle mass are set to unity) it reads (1) ∂tf (r, t) = −\|\|µ/2f (r, t) + ∇ ∇U(r)f (r, t) (1) where \|\|µ/2 is an n-dimensional fractional Laplacian (Riesz fractional derivative6,7) (2) −\|\|µ/2f (x) = An,µ f (u) −f (x) \|u −x\|µ+n dnu, (2) 1Institute of Physics, University of Opole, ul. Oleska 48, 45‑052 Opole, Poland. 2Department of Physics and Medical Engineering, Rzeszów University of Technology, al. Powstańców Warszawy 6, 35‑959 Rzeszów, Poland. 3Department of Mathematics, Universidad de Las Palmas de Gran Canaria, Campus de Tafira Baja, C.P. 35017 Las Palmas, Spain. *email: stef@uni.opole.pl \| https://doi.org/10.1038/s41598-022-16597-2 Scientific Reports \| (2022) 12:12540 www.nature.com/scientificreports/ An,µ = 2µŴ µ+n 2 πn/2\|Ŵ(−µ/2)\|, (3) which at µ = 2 yields the ordinary one6,7. Here Ŵ(x) is Ŵ-function14, ∇ is (also n- dimensional) gradient operator and U(r) is an external potential, in which the anomalous diffusion occurs15. The operator (Eq. 2) is spatially nonlocal, which is a source of memory effects in strongly disordered systems. f It had been shown by Laskin, that the substitution of the Gaussian measure by the Lévy one in the Feyn- man path integrals generates so-called fractional quantum mechanics16,17, where an ordinary Laplacian in the Schrödinger equation is substituted with fractional one (Eq. 2). In context of the above disordered solids, it is important to consider the influence of disorder onto the spectra of their collective atomic vibrations, which con- stitute well-known phonons in an ordered substance. As the latter vibrations are described by the quantum har- monic oscillator model, it is reasonable to consider the fractional analog of real 3D quantum oscillator problem.i g q p In the present paper we shall find the spectrum of 3D fractional oscillator for arbitrary index 0 < µ ≤2 . As this problem resides on the whole 3D space, we shall solve it using Fourier transformation to the momentum space. In latter space the problem becomes local as the Fourier image of the operator (Eq. 2) is simply kµ ( k ≡\|k\| , where k is the momentum vector), while the oscillator potential gives the ordinary Laplacian in the momentum space. OPEN In other words, the initial fractional Schrödinger equation gives ordinary one in k space. The only differ- ence is that the potential in this new equation will be kµ . This implies that to solve our problem in momentum space, we can use well developed numerical and variational techniques. The spectral problem for fractional quantum harmonic oscillator In the units, where ℏ= m = ω = 1 (m and ω are the mass and frequency of oscillating particle respectively), the spectral problem for 3D fractional oscillator reads The spectral problem for fractional quantum harmonic oscillator The spectral problem for fractional quantum harmonic oscillator In the units, where ℏ= m = ω = 1 (m and ω are the mass and frequency of oscillating particle respectively), the spectral problem for 3D fractional oscillator reads (4) −\|\|µ/2ψnlmµ(r) + (x2 + y2 + z2)ψnlmµ(r) = 2Enlµψnlmµ(r), (4) where the operator \|\|µ/2 is defined by the expression (2) and ψnlmµ(x) is the eigenfunction of a fractional quantum harmonic oscillator having the eigenenergy Enlµ for any specific µ value. In general 3D case, as it is typical for the quantum-mechanical problems in central force potentials, the eigenfunction ψ and eigenenergy E of the problem (4) depend on three quantum numbers n, l, m which are principal, orbital and magnetic quantum numbers respectively18,19.i p y As we discussed above, the most profitable method to solve this problem is to pass to the momentum space, which permits to reduce the integral equation (4) to the differential one. Namely, as it is well-known that the combinations x2ψ(x, y, z) , y2ψ(x, y, z) and z2ψ(x, y, z) (here we suppress the indices nlmµ for brevity) in momentum space render as second derivatives ∂2 ∂k2x ψ(kx, ky, kz) , ∂2 ∂k2y ψ(kx, ky, kz) and ∂2 ∂k2z ψ(kx, ky, kz) , the potential term in the equation (4) renders simply as an ordinary Laplacian k ≡∂2 ∂k2x + ∂2 ∂k2y + ∂2 ∂k2z in momentum space. At the same time, the fractional Laplacian in momentum space becomes simply kµ , where k ≡= k2x + k2y + k2z is a vector k modulus. Latter definition immediately implies that 0 ≤k < ∞ . This means that in momentum space, the equation (4) renders as (5) Hkψnlmµ(k) = Enlµψnlmµ(k), Hk = −k + kµ, (5) where Hk is the system Hamiltonian in momentum space. OPEN One note is in place here. Namely, the equation (5) shows that at µ < 2 , the famous orbital 3D oscillator degeneracy (see Ref.18 for details) is lifted so that the eigenenergy E starts to depend on the orbital quantum number l. At the same time, as in the fractional case of µ < 2 , the time-reversal symmetry remains (it is lifted, for instance, in the external magnetic field18), we still have degeneracy with respect to the quantum number m. Namely, the spectrum is invariant as m ↔−m . That is why, in equation (5), we put index m explicitly in the wave functions, which are different for different m, although the energy E does not depend on m. gy p As for any central potential the wave functions are invariant with respect to rotation around the origin18,19, this is also the case for our 3D fractional oscillator. This implies that the wave functions ψnlmµ(k) admit the separation of angular and radial (in k space) variables in the form (6) ψnlmµ(k) = Rnlmµ(k)Ylm(θ, ϕ), (6) where Ylm(θ, ϕ) = AlmP\|m\| l (cos θ)eimϕ are spherical harmonics14,18. Here P\|m\| l (cos θ) are associate Legendre poly- nomials of the first kind and Alm is the corresponding normalization coefficient, which is obtained from the following normalization condition14,18 (7) Y∗ lm(θ, ϕ)Ylm(θ, ϕ)d = 1, (7) where d = sin θdθdϕ. ϕ It is well-known from “ordinary” quantum mechanics18,19 that 3D quantum oscillator problem for µ = 2 admits the factorization of the wave function ψ(x, y, z) = ψx(x)ψy(y)ψz(z) (here we also suppress the lower indices for a moment), which permits to reduce the problem to the three equations for 1D oscillator, containing https://doi.org/10.1038/s41598-022-16597-2 Scientific Reports \| (2022) 12:12540 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Potential in the Schrödinger equation (9) at l = 0 (a) and l = 1 (b). Lévy indices µ are coded by colors (legend in (a)). Mind the different vertical scales in (a,b). Figure 1. Potential in the Schrödinger equation (9) at l = 0 (a) and l = 1 (b). Lévy indices µ are coded by colors (legend in (a)). Mind the different vertical scales in (a,b). three different energies, say Ex , Ey and Ez . In this case, the initial energy E = Ex + Ey + Ez . OPEN After problem being in such a way factorized, the eigenenergy can be derived in the well-known form18,19 three different energies, say Ex , Ey and Ez . In this case, the initial energy E = Ex + Ey + Ez . After problem being in such a way factorized, the eigenenergy can be derived in the well-known form18,19 (8) Eµ=2 = n + 3 2, (8) where n = nx + ny + nz with nx,y,z being the quantum numbers, corresponding to Ex,y,z respectively. Such fac- torization in the “ordinary” case µ = 2 reduces the problem of the 3D oscillator to the combinatorial problem with respect to its 1D “components”. In other words, the quantum numbers nlm of the resulting 3D problem comprise different combinations (obeying certain combinatorial rules, see Refs.18,19 for details) of the initial nx , ny and nz numbers. y It had been shown in19, that the possibility of such factorization at µ = 2 is based on additivity of both Laplacian operator (kinetic energy) and the potential ∼(x2 + y2 + z2) . At the same time, in r space (Eq.4), the fractional Laplacian (Eq. 2) is not additive. That is to say, it does not consist of a sum of the terms, containing separately x, y and z variables. This fact is “transferred” to k space, where the potential kµ ≡(k2 x + k2 y + k2 z)µ/2 is factorable for µ = 2 (ordinary case) only.hifh y y This signifies the difference between fractional µ < 2 and ordinary µ = 2 3D oscillator problems. This dif- ference implies that for our fractional case we should proceed directly in 3D case. This, however, does not give an essential complication as problem still reduces to 1D one. Namely, after the decomposition (Eq. 6), we obtain following fractional 1D Scrödinger equation for the radial part Rnlmµ(k) (9) d2R dk2 + 2 k dR dk + 2E −l(l + 1) k2 −kµ R = 0. (9) Here we also suppress the indices nlmµ in the functions R and energy E for a moment. In the quantum mechanics of the central force systems, it is also customary (see, e.g.18,19) to pass to the function Here we also suppress the indices nlmµ in the functions R and energy E for a moment. OPEN In the quantum mechanics of the central force systems, it is also customary (see, e.g.18,19) to pass to the function (10) χ(k) = kR(k) (10) for which the Eq. (9) assumes particularly simple form for which the Eq. (9) assumes particularly simple form (11) d2χ dk2 + 2E −l(l + 1) k2 −kµ χ = 0, (11) which resembles that for 1D system. This form will be used below to construct trial functions for variational method as well as for numerical calculations, where the form (Eq. 11) permits to avoid divergencies at k = 0 . We note here, that if for the states with l = 0 , the function R(k) has a maximum at k = 0 , the corresponding function χ is zero by obvious reason. As we mentioned above, the Eq. (11) can be readily solved numerically. On the other hand, even approximate analytical solution like variational one is much more desired as it can represent the useful orthogonal set, which can be further employed for the solutions of the problems, not obligatory dealing with fractional quantum mechanics. For instance, it could be well used to look for the solutions of the fractional FP equation (1) in the from of the expansion over the infinite sets of orthogonal functions. That is why we will solve the radial equation (11) variationally and confirm our results by direct numerical solution.hf 2h i The Eqs. (9), (11) represent Schrödinger equation with the effective 1D potential \|k\|µ + l(l + 1)/k2 . The plots of this potential at different µ and l are shown in Fig.1. It is seen from Fig.1a that for l = 0 the problem is identical Scientific Reports \| (2022) 12:12540 \| https://doi.org/10.1038/s41598-022-16597-2 www.nature.com/scientificreports/ to that for 1D fractional oscillator20, while for l = 0 (Fig.1b), the centrifugal force drives it outwards. The only difference with the case of 1D oscillator is that now the problem is defined on positive semi-axis 0 < k < ∞ as k is now the modulus of a momentum vector. As it is seen from Fig.1b, the centrifugal term in the potential l(l + 1)/k2 is dominant at k →0 , while at k →∞ the main contribution comes from kµ . This means that the whole spectrum of 3D fractional oscillator remains to be discrete similar to the “ordinary” case µ = 2 . Variational solution. Classification of the states of 3D fractional oscillator ll k h h l l f h 18 fi d h It is seen that for all 0 < µ ≤2 the asymptotics (Eq. 12) decays sufficiently fast for corresponding integrals to be convergent. Namely, in worst case of k = 0 ψ(k →∞) ∼e−k √ 2 , i.e. decays exponentially. Good localization of the wave functions in k space yields their localization in coordinate space by Riemann–Lebesgue lemma, see e.g.22. Our analysis show that at small k the functions R(k) have the same asymptotics R ∼kl (and accordingly χ ∼kl+1 ) as in ordinary case, corresponding to µ = 218. p g µ To classify the states of the 3D fractional oscillator according to the quantum numbers nlm, we notice that although the Eq. (9) resembles that for the ordinary 3D oscillator, it is not equivalent to it as we have the term kµ instead of k2 in the potential. The former term “spoils” the neat classification18,19 of the states of 3D quantum mechanical oscillator so that in the fractional case µ < 2 there is no obvious way to introduce the principal quantum number n. We recollect that for ordinary 3D oscillator n = 2nr + l (see, e.g.19), where nr is so-called radial quantum number, defining the number of nodes of the radial function for a given l. That is why to classify the states of the oscillator in our 3D fractional case we begin with general consideration of a quantum particle, moving in a central force field. Namely, to apply the oscillation theorem to the radial part χ(k) in Eq. (11), we arrange the eigenenergies corresponding to a given l in ascending order. It had been shown18,19, that the low- est eigenenergy for a given l corresponds to a nodeless function so that we assign nr = 0 to this state. The next (excited) state for the same l has one node, i.e. nr = 1 etc. This means that we may classify the states of our fractional oscillator by essentially two quantum numbers - nr and l. To be specific, for l = 0 (s - state; here we use the common notations of the states with the given l, see, e.g.18) we have the whole sequence nr = 0, 1, 2, ... ; the same is true for l = 1, 2, 3, .. . Variational solution. Classification of the states of 3D fractional oscillator ll k h h l l f h 18 fi d h Variational solution. Classification of the states of 3D fractional oscillator It is well-known that the variational principle of quantum mechanics18 permits to find the approximate ana- lytical expressions for the wave functions and eigenenergies of corresponding Schrödinger equations. This is especially true for the states of discrete spectrum, which are spatially localized. As usually, the “correct” (i.e. that which minimizes corresponding energy functional with minimal number of variational parameters) set of trial functions should be obtained on the class of functions, having the asymptotics, dictated by the corresponding differential equation.fi f q Note that for the above variational principle to work, it is sufficient even to use the trial functions obeying the correct boundary conditions rather than have the “prescribed” asymptotics. In our case, this means that the trial functions have to be zero at k →∞ . Such “simple” trial functions can be constructed on the base of the Gaussian one, inherent in an “ordinary” (i.e. that at µ = 2 ) 3D oscillator. In this case, the whole variational spectrum can be thought of in the form of “Hermite polynomials with unknown coefficients”, where the latter coefficients are to be found from the orthogonality conditions, see below. In the next section, we shall compare the relative errors of the variational approaches, based on “fractional” (i.e. with predefined asymptotics) and “Gaussian” trial functions. To construct the “fractional” trial functions, we should analyze the large k asymptotics of the χ(k) , given by the radial (in k space) Schrödinger equation (11). For that we observe that at large k we can neglect all the terms in the square brackets in Eq. (11) except kµ . This yields the equation for large k in the form d2χ(k)/dk2 = kµχ . The spatially decaying solution to this equation is proportional to k1/2Kν(u) , where ν = 1/(µ + 2) and u = 2 √ 2\|k\|1+µ/2/(µ + 2)21. Here Kν(x) is so-called MacDonald function with the following large x asymp- totics Kν(x →∞) ≈(π/(2x))1/2e−x14. Substitution of latter asymptotics into the above expression generates following wave function asymptotics at all admissible µ’s (12) χ(k →∞) ∼exp −2 √ 2 µ + 2k1+µ/2 . (12) Here we once more suppressed indices nlm. In the case of µ = 2 (ordinary quantum oscillator) the asymptotics (Eq. 12) reproduces well known result for “ordinary” 3D quantum oscillator18. OPEN Note that at small µ = 0.2 the potential begins to grow at very large k, while at µ = 0 the potential goes to 1 at k →∞ . The same situation at l = 0 gives constant value one for µ = 0 , see Fig. 1a. Our numerical analysis shows that in 3D case despite the contribution of centrifugal term, at µ = 0 , the whole oscillator spectrum collapses into one value, see below. This property is similar to that in 1D case20. Note that at l = 0 and µ < 1 the potential at k →0 the potential has nonanalytical behavior with infinite first derivative. At l = 0 this asymptotics is “killed” by the centrifugal term. Variational solution. Classification of the states of 3D fractional oscillator ll k h h l l f h 18 fi d h Introducing the principal quantum number n = nr + 1 , we obtain following explicit classification of the states of the fractional quantum oscillator: 1s ( nr = 0 , hence n = 1 , l = 0 ), 1p ( n = 1 , l = 1 ), 1d ( n = 1 , l = 2),... 2s, 2p, 2d,... etc. In this case, the commonly known degeneracy of the states 1d and 2s, 1f and 2p etc, inherent in “ordinary” 3D oscillator, is lifted so that all states with different nr and l have different energies. This classification will be used below to construct the trial functions. ghi To construct the above functions, we need the asymptotics at small and large k as well as the information bout the number of nodes, i.e. the numbers nr and l. Specifically, in terms of functions χ(k) , we have https://doi.org/10.1038/s41598-022-16597-2 Scientific Reports \| (2022) 12:12540 \| www.nature.com/scientificreports/ (13) χ1s = A1ske−a1sk1+µ/2, χ2s = A2sk(a1k + b1) × e−a2sk1+µ/2, χ3s = A3sk(a2k2 + b2k + c2) × e−a3sk1+µ/2, ... (13) (14) χ1p = A1pk2e−a1pk1+µ/2, χ2p = A2pk(a3k + b3) × e−a2pk1+µ/2, χ3p = A3pk(a4k2 + b4k + c4) × e−a3pk1+µ/2, ... (14) (15) χ1d = A1dk3e−a1dk1+µ/2, ... χ1d = A1dk3e−a1dk1+µ/2, ... (15) Here, Anl are normalization constants, found from the “1D-like” conditions Here, Anl are normalization constants, found from the “1D-like” conditions Here, Anl are normalization constants, found from the “1D-like” conditions (16) < χ2(k) >= 1, < ... >= ∞ 0 ...dk. (16) Also, anl are variational parameters, while the constants ai , bi and ci (here i = 1 −4 ) are found from the orthogo- nality conditions (17) (17) < χnlχn′l′ >= δnn′δll′, < χnlχn′l′ >= δnn′δll′, where δjj′ is Cronecker delta. We note that the trial functions χnl for different l (for instance χ1p and χ2d ) are orthogonal automatically because of orthogonality of spherical harmonics14,18,19. That’s why the integration in Eqs. (16) and (17) runs over k only. This implies, that only the functions with different n′s (and the same l) should be orthogonalized. Namely, in the particular case of the above trial functions we have explicitly (18) < χ1sχ2s > =< χ1sχ3s >=< χ2sχ3s >= 0, < χ1pχ2p >=< χ1pχ3p >=< χ2pχ3p >= 0. (18) The orthogonalization procedures Eqs. p g y Orthogonality condition < χ1sχ2s >= 0 yields Variational solution. Classification of the states of 3D fractional oscillator ll k h h l l f h 18 fi d h (17), (18) along with normalization of the functions χnl determine niquely the coefficients ai through variational parameters anl. W h li i l q yfi We have explicitly (19a) A1s = (2a1s) 3 µ+2 √µ + 2 2Ŵ 6 µ+2 , (19a) (19b) A2s = 1 √I2s , I2s = 2(2a2s)−10 µ+2 µ + 2 a2 1Ŵ 10 µ + 2 + + b2 1(2a2s) 4 µ+2 Ŵ 6 µ + 2 + + 2a1b1(2a2s) 2 µ+2 Ŵ 8 µ + 2 , (19b) (19c) A1p = (2a1p) 5 µ+2 √µ + 2 2Ŵ 10 µ+2 , (19c) (19d) A2p = 1 I2p , I2p = 2(2a2p)−14 µ+2 µ + 2 a2 3Ŵ 14 µ + 2 + + b2 3(2a2p) 4 µ+2 Ŵ 10 µ + 2 + + 2a3b3(2a2p) 2 µ+2 Ŵ 12 µ + 2 , ... (19d) Here Ŵ(x) is the Ŵ-function14. The other normalization coefficients can be calculated, but their explicit expres- sions become progressively more cumbersome. Here Ŵ(x) is the Ŵ-function14. The other normalization coefficients can be calculated, but their explicit expres- sions become progressively more cumbersome. g y Orthogonality condition < χ1sχ2s >= 0 yields https://doi.org/10.1038/s41598-022-16597-2 https://doi.org/10.1038/s41598-022-16597-2 https://doi.org/10.1038/s41598-022-16597-2 Scientific Reports \| (2022) 12:12540 \| www.nature.com/scientificreports/ (20) b1 = −a1, = Ŵ 8 µ+2 Ŵ 6 µ+2 (a1s + a2s)− 2 2+µ . (20) Substitution of the expression (20) into the normalization coefficient A2s Eq. (19b) generates following explicit expression for the function χ2s (21) χ2s = k(k −) √κ2s e−a2sk1+µ/2, (21) where is determined by the expression (20) and where is determined by the expression (20) and (22) κ2s = 2 µ + 2 (2a2s)−10 µ+2 Ŵ 10 µ + 2 −2(2a2s)− 8 µ+2 × Ŵ 8 µ + 2 + 2(2a2s)− 6 µ+2 Ŵ 6 µ + 2 . (22) Now, after variational determination of the parameter a1s (see below), the parameter (20) starts to depend on the single parameter a2s , which can also be determined variationally. Having determined the parameters a1s and a2s , we then use the orthogonality condition < χ1sχ3s >= 0 to express both c2 and b2 through a2 . After substitution of the above found a1s and a2s into the corresponding normalization coefficient, the function χ3s starts to depend on a3s only. Variational solution. Classification of the states of 3D fractional oscillator ll k h h l l f h 18 fi d h Such “chain rule” can be applied to determine uniquely all higher order functions, corresponding to l = 0 (s-state), l = 1 (p-state) and for any arbitrary n and l.hh The expressions (13)–(15) show the algorithm of construction of trial functions. This algorithm is essentially the same as that for “ordinary” case of µ = 2 . Namely, the arbitrary trial function χnl is the product of the fac- tors kl+1 (small k asymptotics), e−anlk1+µ/2 (large k asymptotics) and a polynomial of degree nr = n −1 with unknown coefficients. These coefficients, in turn, are determined from the orthogonality conditions (17) by the above “chain rule”. As usually, the variational solution of the Schrödinger equations should minimize the energies (23) Wnlµ = ψ∗ nlmµ(k) Hkψnlmµ(k)d3k, (23) where Hk is the Hamiltonian (5) and Wnlµ are the variational approximations of the eigenenergies Enlµ . Normally Wnlµ ≥Enlµ . As the radial parts Rnlmµ(k) of the wave functions (6) are well localized (see asymptotics (12)), the expression (23) could be rendered (by the integration by parts) to the more convenient form where Hk is the Hamiltonian (5) and Wnlµ are the variational approximations of the eigenenergies Enlµ . Normally Wnlµ ≥Enlµ . As the radial parts Rnlmµ(k) of the wave functions (6) are well localized (see asymptotics (12)), the expression (23) could be rendered (by the integration by parts) to the more convenient form (24) Wnlµ = 1 2 ∞ 0 kR′ nlmµ 2 + kµ+2 + l(l + 1) R2 nlmµ dk, (24) where R′ = dR/dk . After substitution of the trial functions (13), (14) and (15) into the integral (24), with respect to the above “chain rule”, we find the variational parameters anl from its minimization. Such a procedure can be done for all wave functions of higher excited states, giving the approximate spectrum of the operator (5). Note that the substitution of found a1s into W1sµ (24) gives the approximate value of the ground state energy for all µ , the same with, say, a2p gives the energy of the excited state W2pµ and so on for higher eigenenergies. Although the expressions for higher excited states become extremely cumbersome (see above), the variational method permits to obtain the approximate analytical expressions for the eigenvalues and eigenfunctions of the operator (Eq. 5) for all admissible 0 < µ ≤2. Variational solution. Classification of the states of 3D fractional oscillator ll k h h l l f h 18 fi d h q ) µ Substitution of the trial function χ1s into the integral (Eq. 24) with subsequent minimization over a1s yields (25) (a1s)min = √2µ 2 + µ, (25) which is similar to the expression for 1D fractional oscillator20. Further substitution of this value to the expres- sion (24) generates the approximate value of the ground state energy for arbitrary µ which is similar to the expression for 1D fractional oscillator20. Further substitution of this value to the expres- sion (24) generates the approximate value of the ground state energy for arbitrary µ (26) (W1sµ)min ≈E1sµ = 1 2 µ + 4 µ + 2 2 + µ 2√2µ 2µ 2+µ Ŵ 2 2+µ Ŵ 6 2+µ . (26) It is seen that (W1sµ)min gives correct value 3/2 of the ground state energy for µ = 2 , corresponding to the ordi- nary quantum oscillator with the spectrum En = n + 3/2 in our units. Below we shall see, that for the case µ = 0 all the spectrum shrinks into a single value E0 = 1/2 , which is also obtained correctly from the expression (26). The dependence (Eq. 26) will be plotted below and compared with the numerical solution.hh It is seen that (W1sµ)min gives correct value 3/2 of the ground state energy for µ = 2 , corresponding to the ordi- nary quantum oscillator with the spectrum En = n + 3/2 in our units. Below we shall see, that for the case µ = 0 all the spectrum shrinks into a single value E0 = 1/2 , which is also obtained correctly from the expression (26). The dependence (Eq. 26) will be plotted below and compared with the numerical solution.hh h p q p p The same procedure with χ1pµ gives that (a1pµ)min = (a1sµ)min , which is given by Ex. (25). The variational expression for the energy of the 1p state reads https://doi.org/10.1038/s41598-022-16597-2 Scientific Reports \| (2022) 12:12540 \| www.nature.com/scientificreports/ (27) (W1pµ)min ≈E1pµ = 3 2 8 + µ 2 + µ Ŵ 6 2+µ Ŵ 10 2+µ 2 + µ 2√2µ 2µ 2+µ . (27) The expression (27) shows that at µ = 2 we obtain the correct answer 5/2. We recollect here that the energy of “ordinary” 3D quantum oscillator is determined by the expression (8), where n = 2nr + l , see above. Numerical analysish 24) with its subsequent minimi- zation over σ generates following minimizing wave function width Substitution of Eq. 28 with respect to Eq. 29 into the expression for energy (Eq. 24) with its subsequent minimi- zation over σ generates following minimizing wave function width (30) σmin =  3 2 √π µŴ 3+µ 2   1 µ+2 . (30) Further substitution of the ground state wave function (28), with respect to Eqs. 29 and 30, into the energy functional (24), yields the Gaussian analog of the variational ground state energy (Eq. 26) Further substitution of the ground state wave function (28), with respect to Eqs. 29 and 30, into the energy functional (24), yields the Gaussian analog of the variational ground state energy (Eq. 26) (31) (WG1sµ)min = 1 √π Ŵ 3 + µ 2 σ µ min + 3 4(σmin)−2, (31) where σmin is determined by Eq. (30). At µ = 2 , the energy (WG1sµ)min also gives correct value 3/2 of the oscillator ground state energy. The same is valid for µ = 0 , where (WG1sµ=0)min = Ŵ(3/2)/√π = 1/2 . Here we also have a removable divergency of the type limµ→0(µ−µ/2) = limµ→0 exp(−(µ/2) ln µ) = 1 in the first term of Eq. (31). µ µi Having both “Gaussian” (31) and “fractional” (26) and (27) expressions for variational energy levels of our 3D oscillator, we are now in a position to compare them with corresponding numerical values. Such compari- son is reported in Fig. 2a,c. As is the case for the variational method, in both panels the variational curves lie higher than numerical ones as variational energy should be larger than its exact (in our case numerical) value18. It is seen that the agreement is a little worse for the 2s state, which has one node. For the nodeless states with n = 1 , the variational and numerical curves are indistinguishable in the scale of the plots. The quantitative error analysis for 1s state is reported in Fig. 2c. It shows that as both “Gaussian” and “Fractional” trial functions give exact results at µ = 0 and µ = 2 , the relative error should be maximal somewhere inside this interval. The inset in Fig. 2c shows that the maximal relative error occurs at µ ≈0.5 and does not exceed 0.6%. Latter error occurs for “Gaussian” trial function. Variational solution. Classification of the states of 3D fractional oscillator ll k h h l l f h 18 fi d h As for 1p state we have nr = 0 (no nodes) and l = 1 so that n = 1 and the energy E1p(µ = 2) = 1 + 3/2 = 5/2 . At the same time, at µ →0 we have removable divergence limµ→0(2µ−µ/2) = limµ→0 exp(−µ ln(2µ)) = 1 , while the rest of the expression (27) gives E1p,µ=0 = 6Ŵ(3)/Ŵ(5) = 1/2 , i.e. once more the correct answer. The dependence (27) will also be plotted below and compared with numerical results. We will see that the expressions (26) and (27) give very good approximate expressions for corresponding energies of a 3D fractional quantum oscilla- tor for all admissible µ . Within the suggested variational approach, the energy of any desired level Enlµ can be evaluated analytically, although the calculations for higher excited states become very cumbersome, see above. Numerical analysish y The easiest way to solve our problem numerically is to use the Eq. (11) for the function χ(k) , which has only one divergency (in the centrifugal term l(l + 1)/k2) ) at k = 0 . The numerical solution of the Eq. (11) should be augmented by the boundary conditions χ(0) = χ(∞) = 0 . As usually for boundary value problems, our Eq. (11) can be solved numerically by its reduction to the eigenproblem for the corresponding finite-dimensional matrix. In this case, the energies E are the eigenvalues of the above matrix and the eigenvectors of the latter comprise the wave functions of the problem in momentum space. The transition to the usual coordinate space is accomplished by 3D inverse Fourier transformation. We note that the satisfactory accuracy of the numerical solution is achieved for typical matrix dimensions 10000 × 10000, which makes the task quite computer intensive. As we can reproduce numerically the spectrum of the problem for arbitrary µ , we are now in a position to compare the results of variational and numerical treatments. We begin with comparison of different classes of trial functions (see above), namely aforementioned “fractional” (13)–(15) and “Gaussian”, which we are going to construct below. As the consideration of a ground (1s in our case) state wave functions give the lower limit of the relative errors, here we consider just this function for “Gaussian” case. In terms of χ functions (10), we have (28) χG1s = Ake−k2 2σ2 , (28) where “G” in lower index stands for “Gaussian”, A is normalization coefficient and σ is variational parameter. We note that at µ = 2 the function χG1s is equivalent to χ1s (see (13)) with a1s = 1/(2σ 2). The normalization of (28) with the help of condition (16) yields where “G” in lower index stands for “Gaussian”, A is normalization coefficient and σ is variational parameter. We note that at µ = 2 the function χG1s is equivalent to χ1s (see (13)) with a1s = 1/(2σ 2). The normalization of (28) with the help of condition (16) yields µ χ q χ The normalization of (28) with the help of condition (16) yields (29) A = 2π−1/4σ −3/2. (29) Substitution of Eq. 28 with respect to Eq. 29 into the expression for energy (Eq. Numerical analysish (d) Portrays the comparison between variational (both types of the functions, shown in the legend) and numerical 1s wave functions for the same µ values, as in (b). Insets report the relative errors for µ = 1.4 and 0.4 as the wave function for µ = 2 is exact. Black curves correspond to “Gaussian”—numerical and red ones to “fractional”—numerical relative errors as the functions of momentum k. Figures near curves in (b,d) correspond to Lévy indices µ. and numerical 1s wave functions become visible. Panel (b) of Fig. 2 shows that functions for different µ have different decay rates, which are dictated by the asymptotics (Eq. 12). For instance, the 2s function for µ = 0.4 at k = 5 is not yet achieved its final part (compare to that for µ = 1.4 ), which occurs at k ≈15 . For smaller µ , the spatial extension of the wave functions in k space increases drastically. The functions for µ →0 in k space are almost delocalized, which means that in coordinate space they are Dirac δ-function-like. This behavior is qualitatively similar to that of 1D fractional quantum oscillator20 as well as for 2D23,24 and 3D25 fractional hydrogenic problems. and numerical 1s wave functions become visible. Panel (b) of Fig. 2 shows that functions for different µ have different decay rates, which are dictated by the asymptotics (Eq. 12). For instance, the 2s function for µ = 0.4 at k = 5 is not yet achieved its final part (compare to that for µ = 1.4 ), which occurs at k ≈15 . For smaller µ , the spatial extension of the wave functions in k space increases drastically. The functions for µ →0 in k space are almost delocalized, which means that in coordinate space they are Dirac δ-function-like. This behavior is qualitatively similar to that of 1D fractional quantum oscillator20 as well as for 2D23,24 and 3D25 fractional hydrogenic problems.h The same tendency is seen in Fig. 2d. Here, the “Gaussian” trial function has stronger deviation from the numerical curve, while the “fractional” one goes closer to it. This difference is more visible at µ = 0.4 , which is close to the above value µ ≈0.5 , where the relative error in the energy is maximal. Numerical analysish This is because the latter function does not take into account the actual asymptotics (Eq. 12) of the wave functions.hf The selected numerical wave functions in k-space are reported in Fig. 2b,d for different values of µ . It can be checked that functions are normalized, i.e. they obey condition (16). Note that corresponding “fractional” wave functions (13) are almost indistinguishable from numerical ones in the scale of the plot in Fig. 2b. At the same time, in the Fig. 2d, we choose the vertical scale so that the (minute) differences between “fractional” (variational) Scientific Reports \| (2022) 12:12540 \| https://doi.org/10.1038/s41598-022-16597-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. (a) Reports the lowest state eigenenergies (indicated near the curves) as functions of Lévy index µ , calculated numerically (solid lines) and variationally (dashed lines, “fractional” functions (13)–(15)). (b) Displays wave functions of the lowest states, shown in the legend. The numerical and variational plots in (b) are similar in the scale of the plot. (c) The 1s state energies, calculated variationally (expressions (26) and (31)) as well as numerically. The curves are indistinguishable in the scale of the plot. Inset in (c) reports the “Gaussian” - numerical (black curve) and “fractional”—numerical (red curve) relative errors in the entire µ domain. (d) Portrays the comparison between variational (both types of the functions, shown in the legend) and numerical 1s wave functions for the same µ values, as in (b). Insets report the relative errors for µ = 1.4 and 0.4 as the wave function for µ = 2 is exact. Black curves correspond to “Gaussian”—numerical and red ones to “fractional”—numerical relative errors as the functions of momentum k. Figures near curves in (b,d) correspond to Lévy indices µ. Figure 2. (a) Reports the lowest state eigenenergies (indicated near the curves) as functions of Lévy index µ , calculated numerically (solid lines) and variationally (dashed lines, “fractional” functions (13)–(15)). (b) Displays wave functions of the lowest states, shown in the legend. The numerical and variational plots in (b) are similar in the scale of the plot. (c) The 1s state energies, calculated variationally (expressions (26) and (31)) as well as numerically. The curves are indistinguishable in the scale of the plot. Inset in (c) reports the “Gaussian” - numerical (black curve) and “fractional”—numerical (red curve) relative errors in the entire µ domain. Scientific Reports \| (2022) 12:12540 \| Numerical analysish That said, we can safely assert that (in the intermediate range of momenta k, where the error can be calculated reliably, see above) with the accuracy not higher than 3% the analytical expressions (13)–(15) approximate the corresponding numerical curves. One more observation is in place here. Namely, the eigenenergies in fractional case µ < 2 are smaller than those for “ordinary” case µ = 2 . This means that in the system, where the substitution of conventional Laplacian by the fractional one in Schrödinger equation is admissible (like in strongly disordered solids like amorphous ones, see, e.g.23 for discussion), it is energetically favorable for an oscillator to “become fractional”. g g y As we have discussed above, the wave functions in the coordinate space can be obtained by the inverse Fourier transform. They are reported in the Fig. 3 for 1s (panel (a)) and 2s (panel (b)) states. It is seen from the Figure, that the curves are qualitatively similar to those in the momentum space. The regularities here are similar to those for 1D case20, i.e. the slowest decay have the functions for µ →2 (“ordinary” quantum oscillator), while the fastest (like Dirac δ , see above) have the functions for µ →0 ( µ = 0.4 in Fig. 3), corresponding physically to the strongest disorder. This may be one more signature of the famous Anderson localization26 phenomenon. Numerical analysish As numerical and vari- ational curves are identical for µ = 2 (the corresponding trial functions are exact), the relative error is reported for µ = 1.4 and 0.4 only. The behavior of relative error reflects the well-known fact that different classes of trial functions can give a very good accuracy for the energy, while the approximation of the numerical wave function can be much worse. We note here that the values of relative error at both k →0 and k →∞ are not that reliable as in this case we are dealing with the difference of small (in the sense of machine precision) numbers, divided by those (small) numbers. That’s why we limit ourselves by k ≈3 as for k > 3 because of latter effect, the error grows to infinity. It is seen from the insets in Fig. 2d, that in the intermediate region of k values, the maximal relative error of the “Gaussian” trial function is around 10%, while that for “fractional” one is less than 1%. Our analysis shows that for the “fractional” trial functions with nodes (including 2s and higher) the average error is 2–3%, the larger error around 3% occurs for the higher excited states with many nodes. This shows that the Scientific Reports \| (2022) 12:12540 \| https://doi.org/10.1038/s41598-022-16597-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Wave functions of 1s (a) and 2s (b) states in the coordinate space. The values of µ are encoded by colors (legend in (a)). Figure 3. Wave functions of 1s (a) and 2s (b) states in the coordinate space. The values of µ are encoded by colors (legend in (a)). variational expressions like (13)–(15) (with respect, of course, to (19a)–(19d)) can be regarded as pretty good wave functions in k space. Such good variational approximation by the functions, having only one parameter may be because the energies are monotonous functions of µ . That said, we can safely assert that (in the intermediate range of momenta k, where the error can be calculated reliably, see above) with the accuracy not higher than 3% the analytical expressions (13)–(15) approximate the corresponding numerical curves. variational expressions like (13)–(15) (with respect, of course, to (19a)–(19d)) can be regarded as pretty good wave functions in k space. Such good variational approximation by the functions, having only one parameter may be because the energies are monotonous functions of µ . Possible experimental applications. Outlookh 33a–33c) with respect to numerically calculated R00 and R11 wave func- tions into the expression (32), generates the Lévy index dependence of oscillator strength components fi ≡fi,1s−1p ( i = x, y, z ), which is reported in Fig. 4. It is seen that the oscillator strength goes to zero in the case of strongest disorder, corresponding to µ = 0 , see Ref.23 for details. We observe (see above) that as µ →0 , both E1p and E1s go to the same value (as, actually, the whole spectrum) 1/2. This means that for µ = 0 , the denominator of the expression (32) becomes zero. Despite that, the whole expression (32) tends to zero because of much faster decay of dipole matrix elements (33a)–(33c) in this case. The origin of this stems from coordinate space, where the wave functions of all states look very much like Dirac δ-functions. This means that the probability of optical transitions is much higher in relatively ordered regions, where µ > 1 . This fact may be useful in the theoretical description of the experimental data either in polymers, where chains can vibrate, or in disordered solids. Namely, the developed formalism can be well applied to the calculations of the properties of real physical systems, where disorder (like lattice imperfections and/or impurities) influences phonon and electron spectra of a substance, leading to non-Gaussian distribution of the internal electric, magnetic and elastic fields. A compelling example is the properties of the above multiferroics5. The non-Gaussian statistics due to disorder and frustration plays an important role in these substances28–30 and it is challenging to apply the above formalism to explain their dis- order (in phonon and magnon subsystems) phenomenologically in terms of introduction fractional derivatives to the corresponding Schrödinger equations. The dynamics of such “disordered phonons and magnons” can be described in terms of the above FP equation. Here, essentially the same variational approach can be used as there exist a mapping between FP and Schrödinger equation in terms of so-called Lévy–Schrödinger semigroup31,32. Of course, direct numerical solutions are always possible. In this context, we also mention one more interesting physical problem regarding the onset of chaos33 in the systems (like semiconductors1,2 and trapped cold atomic gases34) with the joint effect of the Lorentz force, Zeeman splitting, and spin-orbit coupling35,36. This suggests one more generalization of our 3D fractional quan- tum-mechanical oscillator problem. Possible experimental applications. Outlookh The obtained spectrum of the fractional quantum 3D oscillator can be used for the calculation of arbitrary observable characteristic of real physical systems. As an illustration, here we consider the so-called oscillator strength or dipole matrix element (the matrix element of a dipole moment operator d = er ) for the transition between the energy levels with different orbital quantum numbers l and l′ = l ± 1 . The latter relation stems from the selection rule, which gives zero (because of angular integration) for any combination of orbital quantum numbers, other than the latter one. The oscillator strength is important for the evaluation of such experimentally observable characteristics like the intensities of electric dipole transitions, which are the dominant effects of the interaction of a charge carrier in solids (electrons or holes) with the external electromagnetic field. One of the most important matrix elements of this type defines a transition between the states with l = 1 and l = 0 . As here we are working in momentum space, the formula for the oscillator strength contains the matrix element a\|k\|b for the transition between states a and b. In our dimensionless units, the expression for the oscillator strength reads (see, e.g.19,27) (32) fab = −2\|a\|k\|b\|2 Ea −Eb , (32) where the transition occurs between states a (like 1p) and b (like 1s) with the eigenenergies Ea and Eb respectively. For the transition between the states 1p and 1s, the explicit expressions for matrix elements assume the form where the transition occurs between states a (like 1p) and b (like 1s) with the eigenenergies Ea and Eb respectively or the transition between the states 1p and 1s, the explicit expressions for matrix elements assume the form (33a) 1s\|kx\|1p = k sin θ cos ϕR00(k)R11(k)× × Y00Y1,±1(θ, ϕ)d3k, (33a) (33b) 1s\|ky\|1p = k sin θ sin ϕR00(k)R11(k)× × Y00Y1,±1(θ, ϕ)d3k, (33b) https://doi.org/10.1038/s41598-022-16597-2 https://doi.org/10.1038/s41598-022-16597-2 https://doi.org/10.1038/s41598-022-16597-2 Scientific Reports \| (2022) 12:12540 \| www.nature.com/scientificreports/ p / Figure 4. The components (shown near curves) of oscillator strength (dipole matrix element Eq. (32)) for transition between the states 1s and 1p as functions of Lévy index µ . Both functions are strongly decreasing as µ →0. Figure 4. The components (shown near curves) of oscillator strength (dipole matrix element Eq. (32)) for transition between the states 1s and 1p as functions of Lévy index µ . Both functions are strongly decreasing as µ →0. Possible experimental applications. Outlookh (33c) 1s\|kz\|1p = k cos θR00(k)R11(k)× × Y00Y10(θ, ϕ)d3k. (33c) where ...d3k = ∞ 0 ...k2dk π 0 sin θdθ 2π 0 dϕ. ...d3k = ∞ 0 ...k2dk π 0 sin θdθ 2π 0 dϕ. Substitution of the matrix elements (Eqs. 33a–33c) with respect to numerically calculated R00 and R11 wave func- tions into the expression (32), generates the Lévy index dependence of oscillator strength components fi ≡fi,1s−1p ( i = x, y, z ), which is reported in Fig. 4. It is seen that the oscillator strength goes to zero in the case of strongest disorder, corresponding to µ = 0 , see Ref.23 for details. We observe (see above) that as µ →0 , both E1p and E1s go to the same value (as, actually, the whole spectrum) 1/2. This means that for µ = 0 , the denominator of the expression (32) becomes zero. Despite that, the whole expression (32) tends to zero because of much faster decay of dipole matrix elements (33a)–(33c) in this case. The origin of this stems from coordinate space, where the wave functions of all states look very much like Dirac δ-functions. This means that the probability of optical transitions is much higher in relatively ordered regions, where µ > 1 . This fact may be useful in the theoretical description of the experimental data either in polymers, where chains can vibrate, or in disordered solids. Namely, the developed formalism can be well applied to the calculations of the properties of real physical systems, where disorder (like lattice imperfections and/or impurities) influences phonon and electron spectra of a substance, leading to non-Gaussian distribution of the internal electric, magnetic and elastic fields. A compelling example is the properties of the above multiferroics5. The non-Gaussian statistics due to disorder and frustration plays an important role in these substances28–30 and it is challenging to apply the above formalism to explain their dis- order (in phonon and magnon subsystems) phenomenologically in terms of introduction fractional derivatives to the corresponding Schrödinger equations. The dynamics of such “disordered phonons and magnons” can be described in terms of the above FP equation. Here, essentially the same variational approach can be used as there exist a mapping between FP and Schrödinger equation in terms of so-called Lévy–Schrödinger semigroup31,32. Of course, direct numerical solutions are always possible. Substitution of the matrix elements (Eqs. www.nature.com/scientificreports/ www.nature.com/scientificreports/ energies and wave functions of s-states (i.e. those for l = 0 ) are in a very good agreement with those obtained from exact solutions in terms of Airy functions. A quantitative comparison is out of the frames of the present work. Note also, that 1D and 2D problems for the fractional Schrödinger equation in a linear potential had been considered in the paper41. For 1D case this paper complements both our studies of 1D fractional oscillator20 for the entire µ domain and those in the paper40 for µ = 1 , where the exact solution in terms of Airy functions had been presented. The consideration of Cauchy distribution time evolution, made in41 is also complementary to our study42. y One more promising approach to non-relativistic quantum problems in central force potentials like rq , is to suggest an approximation to the spectrum of the problem for arbitrary q. This has been done in the paper43 for the case of ordinary (i.e. non-fractional) laplacians in the Schrödinger equation. Moreover, the author43 was instrumental to generalize the approximation both to the case of logarithmic potential, corresponding to (drq/dq)q=0 = ln r and negative q like hydrogenic problems with q = −1 . It would be interesting in future to generalize the approach43 to the case of fractional laplacians in corresponding Schrödinger equations.i Several one-dimensional problems (like fractional quantum harmonic oscillator and infinite quantum well as a limiting case of the potentials \|x\|p as p →∞ ) had been considered in Ref.44. There, the corresponding time- dependent Schrödinger equation had been solved by the matrix method. Different nonlocal operators, based on fractional Riesz derivatives had been discussed at length. Numerical and variational solutions had been presented. The variational solution of 1D fractional quantum oscillator problem had been reported in terms of Hermite and Laguerre polynomial type functions. This approach is complementary to ours in the sense that here we consid- ered the “Gaussian” trial functions for comparison. The approach of the paper44 is also complementary to our 1D case, considered in Ref.20. The consideration of the infinite potential well problem is complementary to our work45, where the same problem have been solved by the explicit matrix method. Note that the matrix method had been applied in our numerical solutions of 3D (present consideration) and 1D fractional oscillators problems. Methods Th d l The details of our theoretical methodology and those of working with fractional derivatives and fractional Lapla- cians, in particular, have been described in the sections “The spectral problem for fractional quantum harmonic oscillator” and “Variational solution. Classification of the states of 3D fractional oscillator”. The numerical solu- tions of boundary value problems for partial differential equation (11) have been conducted using the commercial Mathematica software package. www.nature.com/scientificreports/ I h di d h l bl f 3D f i l h i ill i h In summary, we have studied the spectral problem for a 3D fractional quantum harmonic oscillator with arbitrary Lévy index 0 < µ ≤2 . Our main supposition here is that Laskin’s construction of path integrals with Lévy measure is equivalent to “extraction” of probability density function from stochastic fractional Langevin equation and, in turn, to the assumptions made in seminal Anderson paper26. This permits us to assert that fractional Schrödinger equation accounts for disorder phenomenologically with Lévy index µ being the measure of the degree of disorder. The presence of potential in the system “tames” the initial Lévy distribution, making it decay faster than that in a corresponding free problem. In other words, here once more we have an interplay between the width of disorder distribution and system potential, which makes the square of the modulus of the corresponding wave function decay faster in space. This makes the problem of a fractional 3D oscillator resem- blant to Anderson localization26 in disordered systems. Data availabilityh y The datasets used and/or analysed during the current study available from the corresponding author on reason- able request. Received: 23 February 2022; Accepted: 12 July 2022 Possible experimental applications. Outlookh Namely, the spin-orbit interaction term can be added to the fractional Schrödinger equation(4). In this case, the solution will be more sophisticated as the wave function will be spinor now (see, e.g.37) although the problem can be solved in the momentum space similar to the present case. This problem turns out to be very important for amorphous semiconductors with atomic vibrations1,2,38, where chaos can adversely influence possible optoelectronic, spintronic, and/or photovoltaic devices functionality. h l bl b d d h f h ll f h l d h l Many physical problems can be reduced to that of a quantum harmonic oscillator. One of them is related to the theoretical studies of quark-antiquark bound states (so-called quarkonium, see, e.g. Ref.39), which had been a hot topic several decades ago. In the context of 1D fractional quantum oscillator, the problem had been introduced by Laskin17, where the 1D potentials of the form \|x\|γ , 1 < γ ≤2 had been considered instead of quadratic one. At the same time, as the consideration was phenomenological by its nature, the linear potential had also been applied for that problem39. As it is well-known18,19, such potential in the 1D Schrödinger equation admits exact solution in terms of Airy functions14. In our context, such problem arises in Eq. (11) for l = 0 and µ = 1 . This problem had been considered by us earlier40 in the context of Lévy flights confinement by linear potential. The https://doi.org/10.1038/s41598-022-16597-2 Scientific Reports \| (2022) 12:12540 \| References 1. Snaith, H. J. Perovskites: The emergence of a new era for low-cost, high-efficiency solar cells. J. Phys. Chem. Lett. 4, 3623 (2013). 2. Stranks, S. D. & Snaith, H. J. Metal-halideperovskites for photovoltaic and light-emitting devices. Nat. Nanotechnol. 10, 391 (2015). 3. Zhu, H. et al. Lead halide perovskite nanowire lasers with low lasing thresholds and high quality factors. Nat. 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https://openalex.org/W2914107647	https://trialsjournal.biomedcentral.com/track/pdf/10.1186/s13063-018-3151-0	English	null	Chinese herbal medicine bi min fang for allergic rhinitis: protocol for a double-blind, double-dummy, randomized controlled trial	Trials	2,019	cc-by	9,014	Abstract Background: People with allergic rhinitis (AR) often seek help from Chinese medicine due to dissatisfaction with conventional treatments. Lung-spleen qi deficiency syndrome (LSQDS) is the most common type of AR, and the Chinese herbal medicine formula bi min fang (BMF) is commonly prescribed for AR patients with LSQDS. However, direct evidence supporting its efficacy and safety is not available, and its potential mechanism of action remains unclear. Methods/design: This paper presents a double-blind, double-dummy, randomized controlled trial. After a 2-week run-in period, 80 AR patients with LSQDS will be recruited and randomly allocated to the BMF group or the control group in a 1:1 ratio. The patients in the BMF group will receive BMF and the placebo for levocetirizine Methods/design: This paper presents a double-blind, double-dummy, randomized controlled trial. After a 2-week run-in period, 80 AR patients with LSQDS will be recruited and randomly allocated to the BMF group or the control group in a 1:1 ratio. The patients in the BMF group will receive BMF and the placebo for levocetirizine hydrochloride orally, while the control group participants will receive levocetirizine hydrochloride and the placebo for BMF orally. All participants will receive 4 weeks of treatment and 12 weeks of follow-up. hydrochloride orally, while the control group participants will receive levocetirizine hydrochloride and the placebo for BMF orally. All participants will receive 4 weeks of treatment and 12 weeks of follow-up. The primary outcome is a change in the Total Nasal Symptom Score (TNSS). Secondary outcomes include changes in scores for the standard version of the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ(S)), and visual analog scale (VAS); changes in serum levels of the cytokines interleukin-4, interferon-γ, transforming growth factor β-1, and interleukin-17; and changes in the gut microbiota composition in the stool. The TNSS, RQLQ(S), and VAS will be recorded at the beginning of, middle of and after the treatment period and at the end of each month in the 3-month follow-up period. Blood and stool samples will be collected at baseline and the end of the treatment. The aforementioned four cytokines will be detected in the serum using enzyme-linked immunosorbent assays, and the stool gut microbiota will be detected using 16S ribosomal ribonucleic acid sequencing. Any side effects of the treatment will be recorded. Discussion: The results of this trial will provide consolidated evidence of the effect of BMF on AR and the potential mechanism by which BMF acts. STUDY PROTOCOL Chinese herbal medicine bi min fang for allergic rhinitis: protocol for a double-blind, double-dummy, randomized controlled trial Qiulan Luo1,2†, Shiqing Zhou3†, Xiaoshan Li4,5, Qubo Chen6, Wenmin Lin1,2, Liming Lu7, Hua Li1,2, Caifeng Chen1,2, Wenyong Chen1,2 and Yunying Li1,2* Chinese herbal medicine bi min fang for allergic rhinitis: protocol for a double-blind, double-dummy, randomized controlled trial Qiulan Luo1,2†, Shiqing Zhou3†, Xiaoshan Li4,5, Qubo Chen6, Wenmin Lin1,2, Liming Lu7, Hua Li1,2, Caifeng Chen1,2, Wenyong Chen1,2 and Yunying Li1,2* Luo et al. Trials (2019) 20:66 https://doi.org/10.1186/s13063-018-3151-0 Luo et al. Trials (2019) 20:66 https://doi.org/10.1186/s13063-018-3151-0 Open Access Abstract This study will be the first to explore the mechanism of action of Chinese herbal medicine on the gut microbiota in AR. Trial registration: Chinese Clinical Trial Registry, ChiCTR-IPR-17010970. Registered on 23 March 201 Keywords: Allergic rhinitis, Chinese herbal medicine, Randomized controlled trial, Cytokines, Gut m * Correspondence: docliyunying@gzucm.edu.cn † * Correspondence: docliyunying@gzucm.edu.cn †Qiulan Luo and Shiqing Zhou contributed equally to this work. 1Otorhinolaryngology Department, Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 111 Dade Road, Yuexiu District, Guangzhou 510120, Guangdong Province, China 2Otorhinolaryngology Department, Guangdong Provincial Hospital of Chinese Medicine, 111 Dade Road, Yuexiu District, Guangzhou 510120, Guangdong Province, China Full list of author information is available at the end of the article * Correspondence: docliyunying@gzucm.edu.cn †Qiulan Luo and Shiqing Zhou contributed equally to this work. 1Otorhinolaryngology Department, Second Affiliated Hospital of Guangzho University of Chinese Medicine, 111 Dade Road, Yuexiu District, Guangzhou 510120, Guangdong Province, China 2Otorhinolaryngology Department, Guangdong Provincial Hospital of Chinese Medicine, 111 Dade Road, Yuexiu District, Guangzhou 510120, Guangdong Province, China Full list of author information is available at the end of the article Background g p y p [ ] Chinese herbal medicine (CHM) is a well-tolerated choice for AR patients seeking complementary and al- ternative therapies to reduce AR symptoms [7]. The latest Chinese guidelines for the diagnosis and treat- ment of allergic rhinitis (2015, Tianjin) suggest that CHM can be applied as an auxiliary method of treat- ing AR [4]. CHM is an important type of comple- mentary alternative medicine, and several studies have focused on the use of CHM to treat AR. A double-blind randomized controlled trial (RCT) with a design incorporating repeated measures and three parallel groups showed that CHM is useful for ameli- orating symptoms, enhancing quality of life, and strengthening body constitution in patients with AR, and all participants in this study were subjected to syndrome differentiation [8]. Another two RCTs showed that CHM safely reduces the nasal symptoms of AR, although the participants in those studies were included without syndrome differentiation [9, 10]. One systematic review and meta-analysis of seven RCTs comparing oral CHM to a placebo showed that CHM was able to reduce total nasal symptom scores. However, details of the CHM formulas used in the studies included were not investigated, and the syn- dromes of the participants were not described. Fur- thermore, the sample sizes of the RCTs were small, and all the trials had certain methodological limita- tions. Thus, the authors could not draw a firm con- clusion regarding the effects of CHM on AR [11]. Overall, CHM appears to be a promising intervention Existing evidence supports the beneficial effects of two original TCM formulas on AR [8, 20–22]. Clinical stud- ies have shown that yu ping feng san can improve symp- toms [8, 21] and quality of life [8] and decrease the levels of interleukin-4 (IL-4) and IgE in AR patients [21]. Indeed, a glucosidic extract from yu ping feng san re- portedly exerts anti-inflammatory and immunoregula- tory effects by inducing activation of T helper cells and regulating other subsets of T lymphocytes [22]. Bu zhong yi qi tang was also found to reduce AR symptoms, with suppressive effects on the total serum level of IgE and IL-4-stimulated production of prostaglandin E2, leukotriene C4, and COX-2 mRNA expression in IL-4-stimulated polymorphonuclear neutrophils [20]. Thus, it is reasonable to hypothesize that BMF is effect- ive for the treatment of AR. Background for patients with AR, and more rigorous RCTs with large sample sizes are needed to further define its effectiveness. g Allergic rhinitis (AR) is a symptomatic nasal disorder caused by an immunoglobulin E (IgE)-mediated im- munological reaction to allergen exposure [1] and is a global health problem that affects people of all ages. The worldwide incidence of AR is 10–20% [2], and the preva- lence of self-reported AR in China is 11.1–19.1% [3, 4]. Classic symptoms of AR include rhinorrhea, nasal ob- struction, nasal itching, and sneezing [1], and the dis- order is classified as intermittent AR or persistent AR based on the duration of symptoms [1]. AR can lead to sleep cycle disorders, emotional imbalance, impaired ability to perform normal daily activities, severely de- creased quality of life, and significant economic burden [1, 5]. Moreover, AR also serves as a trigger for other diseases, such as bronchial asthma [1]. Current conven- tional management of AR primarily includes allergen avoidance, pharmacotherapy, immunotherapy, and pa- tient education [1, 6]. Second-generation H1 antihista- mines, nasal glucocorticosteroids and leukotriene antagonists are recommended as the first-line therapy [1, 2, 6]. However, due to unsatisfactory results with conventional treatment, AR patients are increasingly seeking complementary and alternative therapies [7]. Syndrome differentiation or pattern identification is the core treatment principle of traditional Chinese medi- cine (TCM), and an accurate treatment for an AR pa- tient must be prescribed according to their body constitution. Based on our clinical observations, a con- siderable number of AR sufferers are diagnosed with lung and spleen qi deficiency syndrome (LSQDS), and this observation is supported by the results of published studies [12–16]. Therefore, according to TCM theory, tonifying lung and spleen qi is the treatment principle for AR patients with LSQDS. Two classic CHM formulas, bu zhong yi qi tang and yu ping feng san, are commonly used for allergic diseases [17]. In our clinical practice, bi min fang (BMF), which is composed of modified bu zhong yi qi tang and yu ping feng san, is the most commonly used CHM formula for AR patients with LSQDS. The composition of BMF is based on a systematic review of classic reference books, articles obtained from the PubMed database [8, 17], the Chinese scientific literature, clinical guidelines [18], and our clinical experience [19]. © 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. Luo et al. Trials (2019) 20:66 Luo et al. Trials (2019) 20:66 Page 2 of 10 Page 2 of 10 Page 2 of 10 Study design and setting This is a double-blind, double-dummy RCT. In total, 80 participants will be recruited from the Guangdong Pro- vincial Hospital of Chinese Medicine (GDPHCM), Guangzhou, China. The flow chart and study period are shown in Figs. 1 and 2, respectively. After obtaining written informed consent, a 2-week run-in period will be implemented, and then eligible participants will be ran- domly assigned to a CHM group or a control group in a 1:1 ratio. Because anther study designed to identify dif- ferences in the gut microbiota between AR patients and healthy controls will utilize stool samples from the same group of AR participants, additional consent provisions for collection and use of these participants’ data and stool samples will be explained to the participants. Placeboes have been widely used in the control groups of previous clinical studies of AR treated with pharma- cotherapy and immunotherapy, with the placebo control treatment resulting in over 50% symptom relief [6]. Therefore, in this study, we selected Western medicine as the positive control instead of a placebo. Levocetiri- zine, a second-generation antihistamine that acts on per- ipheral histamine H1 receptors, has a strong anti-allergic effect without a sedative effect, and it is used for reliev- ing and preventing not only AR symptoms but also those of other allergic diseases. After careful consider- ation of the adverse effects and the desired effect, we chose levocetirizine as the intervention for the control group. To blind the participants and the researchers ad- equately, a placebo for the intervention given to the other group will be administered to both groups. We de- signed a double-blind, double-dummy RCT for AR Background Most CHM is administered orally, whereby formula- tions are ingested, transferred, digested, and absorbed through the gastrointestinal system. Accordingly, CHM may influence the gastrointestinal system, including the intestinal mucosa and gut microbiota, the latter of which is essential for health and closely linked to diseases [23]. For instance, several studies have demonstrated that low gut microbiota diversity is associated with a high risk of allergic diseases [24–28]. Other studies have reported that the characteristics of seasonal AR include a lower diversity of intestinal Bifidobacterium [29], with a reduc- tion of Bifidobacterium, Clostridium, and Bacteroides [30] and increased populations of Bacteroides fragilis [31]. A few systematic reviews and studies indicate that Luo et al. Trials (2019) 20:66 Luo et al. Trials (2019) 20:66 Page 3 of 10 patients with LSQDS. The objectives are to assess the ef- ficacy of BMF for AR patients with LSQDS and to ex- plore the mechanism of BMF in treating AR with respect to the immune response and gut microbiota. certain probiotics are beneficial for patients with AR [32–35]. We hypothesize that components of CHM, such as glycosides and oligosaccharides [36], may influ- ence the gut microbiota in the same manner as an oral probiotic by regulating local intestinal immunological conditions and thereby, achieving systematic immuno- modulation [37, 38]. Table 1 Western medicine diagnostic criteria for allergic rhinitis Table 1 Western medicine diagnostic criteria for allergic rhinitis Symptoms Two or more symptoms such as rhinorrhea, nasal obstruction, nasal itching, and sneezing, persisting for a cumulative period greater than 1 h per day. These symptoms may be accompanied by itchy and red eyes and tears. Signs The nasal mucosa is pale and edematous. Nasal secretions are watery. Allergen test At least one type of skin prick test or serum-specific IgE test is positive. Participants who cannot fully cooperate with the trial due to mental or behavioral disorders Symptoms Two or more symptoms such as rhinorrhea, nasal obstruction, nasal itching, and sneezing, persisting for a cumulative period greater than 1 h per day. These symptoms may be accompanied by itchy and red eyes and tears. Participants who frequently change their work place, resulting in difficulty with follow-up Participants who drive or work high above the ground Signs The nasal mucosa is pale and edematous. Nasal secretions are watery. Participants p Participants will be recruited via a local advertisement and doctor referrals from otorhinolaryngology clinics at Fig. 1 Trial flow chart Fig. 1 Trial flow chart Page 4 of 10 Luo et al. Trials (2019) 20:66 Fig. 2 Study period Age 18 to 65 years old, with no sex limitation Age 18 to 65 years old, with no sex limitation Has not taken hormones or antibiotics in the preceding 3 months and has not consumed yogurt or prebiotics for 1 month preceding the trial Participants provided informed written consent and volunteered to participate in the study. Participants can complete the case report forms (CRFs) independently, and blood and stool samples can be collected according to the instructions. GDPHCM. Information about the study, processing, and scheduling will be carefully explained before enrollment. Participants must meet the Western medicine diagnostic criteria for AR (Table 1) [4] and the TCM syndrome diag- nostic criteria for LSQDS (Table 2). Syndrome differenti- ation will be determined by two independent qualified TCM otolaryngologists. Participants can complete their case re- port forms (CRFs) independently, and blood and stool sam- ples can be collected according to the instructions. Has not taken hormones or antibiotics in the preceding 3 months and has not consumed yogurt or prebiotics for 1 month preceding the trial g Participants provided informed written consent and volunteered to participate in the study. Participants can complete the case report forms (CRFs) independently, and blood and stool samples can be collected according to the instructions. Exclusion criteria: Eligibility criteria Eligibility criteria Inclusion criteria: Patients with similar nasal diseases such as acute rhinitis, vasomotor rhinitis, autonomic nervous rhinitis, eosinophilia nonallergic rhinitis, and allergic sinusitis In accordance with the Western medicine diagnostic criteria for AR. Presence of persistent AR and meeting the criteria for lung and spleen qi deficiency in TCM Pregnant women. The trial will be suspended if a participant becomes pregnant Table 1 Western medicine diagnostic criteria for allergic rhinitis Outcome measurement The primary outcome is a change in the Total Nasal Symptom Score (TNSS) [40]. Secondary outcome mea- sures are as follows: (1) changes in the standard version of the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ(S)) score; (2) the frequency of AR episodes and their severity (visual analog scale, VAS); (3) changes in the serum levels of cytokines IL-4, interferon-γ (IFN-γ), transforming growth factor β-1 (TGF-β1), and interleukin-17 (IL-17), and changes in the gut micro- biota composition in stools before and after treatment. In addition, participants will complete a medicine diary and report any AEs related to the intervention drugs throughout the trial. This study is a double-blind, double-dummy RCT, and the medicine used for treatment and the placebo will be identical in appearance. In addition, the research team will be instructed not to communicate with the partici- pants regarding their possible treatment group allocation. Blinding The research team members, with the exception of the clinical research methodology personnel and eligible par- ticipants, will be blinded to the treatment allocation. The study code will not be revealed until the end of the study, unless there is a serious adverse event (AE). Table 2 Diagnostic criteria for TCM differentiation of lung and spleen qi deficiency syndrome The clinical symptoms are characteristically sudden and repeated. The main symptoms of AR are nasal itching, paroxysmal sneezing, watery rhinorrhea, and nasal congestion. These symptoms may be accompanied by itchiness of the eyes, pharynx, or soft palate. Other laboratory tests Immunological tests such as a skin prick test and serum IgE test are helpful in making a diagnosis. AR allergic rhinitis, TCM traditional Chinese medicine AR allergic rhinitis, TCM traditional Chinese medicine PROCPLAN process statements of the SAS statistics and analysis system (SAS Institute Inc., Cary, NC, USA) will be used to generate random numbers, which will then be enclosed in opaque envelopes. Attention will be paid to the concealment of the randomization. Eligible participants will be randomly assigned to either the BMF group or the control group. PROCPLAN process statements of the SAS statistics and analysis system (SAS Institute Inc., Cary, NC, USA) will be used to generate random numbers, which will then be enclosed in opaque envelopes. Attention will be paid to the concealment of the randomization. Eligible participants will be randomly assigned to either the BMF group or the control group. If a participant cannot take the medication for any particular reason, a record will be made, and the medi- cine will be conserved and returned to the researchers at the end of the study. Two follow-up telephone visits will be performed during the treatment period. Participants who complete treatment will be followed up three times over the course of 3 months (Fig. 2). No other treatment for AR will be allowed for any participant in either group during the study period. The rescue medication, deslora- tadine tablets (5 mg orally every night, Hainan Poly Pharmaceutical Co., Ltd., Chinese State Food and Drug Administration approval number: H20040972), will be provided for participants who may experience AR epi- sodes. Administration of the rescue medication will be recorded and statistically analyzed as an indicator of a curative effect. Treatment allocation will be concealed and held by one member of the research team. To ensure adequate concealment, the participants will be given sequential treatment cards by independent researchers. The partici- pants will receive an opaque envelope and allocated to one of the two groups according to the serial number and group name printed on their treatment card. Randomization and allocation concealment Allergen test At least one type of skin prick test or serum-specific IgE test is positive. Randomization will be performed by the Key Unit of Methodology in Clinical Research of GDPHCM. The Luo et al. Trials (2019) 20:66 Page 5 of 10 Luo et al. Trials (2019) 20:66 Table 2 Diagnostic criteria for TCM differentiation of lung and spleen qi deficiency syndrome History Participants may have a personal or family history of allergies. Clinical symptoms The clinical symptoms are characteristically sudden and repeated. The main symptoms of AR are nasal itching, paroxysmal sneezing, watery rhinorrhea, and nasal congestion. These symptoms may be accompanied by itchiness of the eyes, pharynx, or soft palate. Signs The nasal mucosa is pale gray, pale blue, or red. The nasal turbinate is congested, and the nasal cavity is filled with a watery discharge. All these signs are unapparent when in remission. A light pink tongue body with a thin white coating or teeth marks on the sides and a thin and weak pulse are also characteristic. Other laboratory tests Immunological tests such as a skin prick test and serum IgE test are helpful in making a diagnosis. AR allergic rhinitis, TCM traditional Chinese medicine The ingredients, dosage, and pharmacological action of BMF are presented in Table 3 [39]. The placebo for BMF compound granules will be composed of starch and an edible pigment and will be matched as closely as possible to the appearance and taste of BMF. The pla- cebo for the levocetirizine oral solution has the same ap- pearance and dosage as the levocetirizine oral solution but lacks the active ingredient. All interventions will be produced by the manufacturers following good manufac- turing practices. The CHM compound granules and CHM placebo will be produced by Jiangyin Tianjiang Pharmaceutical Co., Ltd. (Jiangyin City, Jiangsu Prov- ince, China, Good Manufacturing Practice certificate number: JS20140328, batch number: 1608359). Levoce- tirizine oral solution and its placebo will be produced by Chongqing Huapont Pharmaceutical Co., Ltd. (Chong- qing City, China, production license: CQ20130006, batch number: 20160801). All interventions meet the require- ments of the regulatory guidelines issued by the China Food and Drug Administration. Table 2 Diagnostic criteria for TCM differentiation of lung and spleen qi deficiency syndrome Interventions The participants in the CHM group will be orally given BMF compound granules (30 mg twice daily) and a pla- cebo for levocetirizine (10 ml/5 mg every night) for 4 weeks. The participants in the control group will be or- ally given levocetirizine (10 ml/5 mg every night) and a placebo for BMF compound granules (30 mg twice daily) for 4 weeks. The TNSS evaluates the following four nasal symp- toms: rhinorrhea, nasal obstruction, nasal itching, and sneezing. The symptoms are self-assessed and recorded Page 6 of 10 Luo et al. Trials (2019) 20:66 Luo et al. Trials (2019) 20:66 Table 3 Composition and action of bi min fang Ingredients Percentage (%) g/ sachet g/ day Action Radix astragali (huang qi) 17.6 7.5 15 TCM: 1 Tonifying the spleen and lung qi and increasing yang. 2. Strengthening the defense qi and securing the exterior. 3. Inducing diuresis and removing edema Pharmaceutical study: 1. Enhancing the immune effect. 2. Inducing diuresis Rhizoma atractylodis macrocephalae (bai zhu) 17.6 7.5 15 TCM: 1 Tonifying the spleen and replenishing qi. 2. Eliminating dampness and inducing diuresis. 3. Hidroschesis Pharmaceutical study: 1. Maintaining a strong constitution. 2. Enhancing immunologic function. 3. Inducing diuresis Radix saposhnikoviae (fang feng) 11.8 5 10 TCM: 1. Dispelling wind and releasing the exterior. 2. Dispelling dampness and relieving pain. 3. Stopping convulsions Pharmaceutical study: 1. Antimicrobial and anti-inflammatory effects. 2. Analgesic and anti- convulsive effects Radix paeoniae alba (bai shao) 11.8 5 10 TCM: 1. Nourishing blood and constraining yin. 2. Emolliating the liver and relieving pain Pharmaceutical study: 1. Analgesic effect. 2. Antimicrobial and anti-inflammatory effect. 3. Immunologic effect Herba ecliptae (mo han lian) 11.8 5 10 TCM: 1. Enriching the yin of the kidney and the liver. 2. Cooling the blood and stopping bleeding Pharmaceutical study: 1. Enhancing immunity. 2. Protecting the liver. 3. Antimutagenic activity Radix bupleuri (chai hu) 11.8 5 10 TCM: 1. Releasing the exterior and abating fever. 2. Soothing liver-qi stagnation. 3. Increasing yang qi Pharmaceutical study: 1. Inhibiting the central nervous system. 2. Anti-inflammatory and antimicrobial effects. 3. Enhancing immunologic function. 4. Protecting the liver Xanthii fructus (cang er zi) 11.8 5 10 TCM: 1. Dispersing wind and cold. 2. Dispelling dampness. 3. Relieving nasal congestion. 4. Relieving pain Pharmaceutical study: 1. Relieving nasal symptoms. 2. Antimicrobial activity. 3. Preventing coughing Radix et rhizoma glycyrrhizea (gan cao) 5.9 2.5 5 TCM: 1. Interventions Tonifying the spleen and replenishing qi. 2. Dispelling phlegm and suppressing coughing. 3. Relaxing tension and relieving pain. 4. Clearing heat and detoxifying. 5. Harmonizing medications Pharmaceutical study: 1. Protecting the digestive system. 2. Antiallergic and anti- inflammatory effects Table 3 Composition and action of bi min fang by subjects using a four-point scale (0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms, and 3 = se- vere symptoms), with low scores indicating less severe nasal symptoms [40]. The RQLQ(S) is a disease-specific questionnaire that can assess quality of life impairment in AR patients. This questionnaire consists of 28 ques- tions covering the following seven domains: activities, sleep, non-nose/eye symptoms, practical problems, nasal symptoms, eye symptoms and emotional problems [41]. The RQLQ(S) with standardized activities is an updated version of the original RQLQ [42]. It has been translated into different languages and has frequently been used in different countries. The VAS, which ranges from 0 (nasal symptoms not at all bothersome) to 10 (nasal symptoms extremely bothersome) was designed to assess the sever- ity of nasal symptom disturbance and has been validated for use in the quantitative evaluation of AR severity [43]. The TNSS, RQLQ(S), and frequency and severity of AR episodes will be evaluated at the beginning of, middle of and after the treatment period and at the end of each month during the 3-month follow-up period (Fig. 2). by subjects using a four-point scale (0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms, and 3 = se- vere symptoms), with low scores indicating less severe nasal symptoms [40]. The RQLQ(S) is a disease-specific questionnaire that can assess quality of life impairment in AR patients. This questionnaire consists of 28 ques- tions covering the following seven domains: activities, sleep, non-nose/eye symptoms, practical problems, nasal symptoms, eye symptoms and emotional problems [41]. The RQLQ(S) with standardized activities is an updated version of the original RQLQ [42]. It has been translated into different languages and has frequently been used in different countries. The VAS, which ranges from 0 (nasal symptoms not at all bothersome) to 10 (nasal symptoms extremely bothersome) was designed to assess the sever- ity of nasal symptom disturbance and has been validated for use in the quantitative evaluation of AR severity [43]. Interventions The TNSS, RQLQ(S), and frequency and severity of AR episodes will be evaluated at the beginning of, middle of and after the treatment period and at the end of each month during the 3-month follow-up period (Fig. 2). will be used to detect the serum levels of the cytokines IFN-γ, IL-4, TGF-β1, and IL-17 using enzyme-linked im- munosorbent assays (ELISAs). These cytokines are typ- ical inflammatory cytokines involved in immune disorders and are released by Th1, Th2, T-regulatory, and Th17 cells [44, 45]. To observe the influence of BMF on the composition of the gut microbiota, 16S ribosomal ribonucleic acid (16S rRNA) sequencing will be used to detect bacterial taxa present in stool samples. A Health and Diet Habits Questionnaire, sampling in- structions, a cardboard box, a sampling spoonand a fecal collection tube with cached liquid will be distributed to each participant before the stool samples need to be col- lected. All participants will be requested to complete the Health and Diet Habits Questionnaire, after which they will be asked to defecate after urinating. The stool will be collected in the cardboard box. The participants will use the sampling spoon to collect two spoonfuls of stool (approximately two grams) without having the sample contact the urine, sewage and fecal pool, and they will deposit the sample in the fecal collection tube. They will then tightly cover the tube and shake it until the sample has uniformly mixed with the cached liquid. The fecal Blood and stool samples will be collected before and after the treatment period (Fig. 2). The blood samples Page 7 of 10 Luo et al. Trials (2019) 20:66 Page 7 of 10 Luo et al. Trials (2019) 20:66 collection tube will keep the stool fresh for 48 h at room temperature. As some participants cannot defecate in a hospital, they will take the above materials home and send their stool samples to the researchers by S.F. Ex- press to arrive within 1 day. All blood samples will be sent to the Biological Resource Center of the GDPHCM within 2 h, and the stool samples will be sent within 24 h. All blood and stool samples will be stored at −80 °C. The 16S rRNA sequencing of the gut microbiota will be performed by BGI Shenzhen Co., Ltd. all the data have been cleaned. Interventions If a participant withdraws from the trial either during the treatment period or the follow-up phase, the reasons will be clarified and the rate of participant withdrawal or loss to follow-up will be analyzed statistically. The study will be monitored by the Scientific Research Department and Ethics Committee of GDPHCM, which is independent of both the investigators and the sponsor. Interim auditing will include off-site surveillance and the submission of a report about the progress of the study. Personal information will be collected from potential and enrolled participants by authorized researchers who will be trained at the beginning of the study. Personal in- formation will not be shared without the participants’ agreement. Quality assurance and data management To ensure strict adherence to the study protocol and fa- miliarity with the trial administration process, an inde- pendent steering committee will be formed by the principal investigator prior to the beginning of the study. This committee will be composed of one independent chairman (Dr. Wenyong Chen), Dr. Hua Li, Dr. Caifeng Chen, and two other independent members, including at least one patient and a public involvement representa- tive. The responsibilities of the steering committee will include approving any amendments to the main study protocol, monitoring the trial, and approving and com- menting on project deliverables. The entire research team will then be provided with a standard operating manual detailing the procedures and will be required to undergo training. Sample size calculation This study is mainly focused on determining the efficacy of BMF for AR patients with LSQDS. The primary out- come is the TNSS. With reference to data from a previ- ous study [46] and our clinical observations, the sample size was calculated using the noninferiority test for two means with software PASS 2011. We assumed that the TNSS of the treatment group will be 5.53 ± 2.79 and that of the control group will be 6.64 ± 3.06. To obtain a sig- nificance level of 5%, a power of 90%, and a noninferior- ity margin of 0.92, 74 participants are required. Allowing for an 8% dropout rate, 80 participants are needed, with 40 in each group. Criteria for stopping treatment A participant may stop treatment and withdraw from the research project for any reason at any time, and the reason for the withdrawal will be recorded in their CRF. The participants will be told that they have the right to withdraw from the trial and that they will be provided with standardized treatment if they withdraw. The criteria for stopping treatment and withdrawing from the research project are: All information from CRFs will be carefully recorded. All errors will need to be crossed out and corrected, after which the correction must be signed and dated by the participant or researcher. Patient withdrawals or missed follow-up visits will be recorded in the CRFs. An independent Data Monitoring Board will be established at the beginning of the study and will be composed of four fully independent members: a chairman, an oto- laryngologist, a biostatistician and a clinical pharmacol- ogy expert from the GDPHCM. The Data Monitoring Board will review and oversee all the source documents and CRFs. The essential information (consent informa- tion, enrollment, number and proportion of missed visits, patients lost to follow-up, and AEs) will be moni- tored and evaluated for completeness and accuracy. The participant has suffered an AE related to taking the drug, and the investigator believes it is not appropriate for them to continue taking the drug. The participant is diagnosed with asthma or other complications of AR during the study. The participant develops another severe disease that needs to be treated during the study. Poor compliance, such that the actual drug usage is less than 80% or more than 120% of the prescribed dose. Use of drugs proscribed for AR during the study. Data analysis Data will be entered using the double-entry method, and to decrease errors, the data will be checked regularly by the research assistants and overseen by the monitors. Research assistants will double check the data before logging them and will promptly notify the research team if any discrepancies are found. All modifications will be marked on the CRFs. The database will be locked after All data analyses will be conducted according to the intention-to-treat principle, and the analysis will be per- formed in a double-blind manner by qualified statisti- cians. The EpiData 3.1 software will be used to build the database, and missing data will be replaced with the mean or median of the item for the homogeneous Luo et al. Trials (2019) 20:66 Luo et al. Trials (2019) 20:66 Page 8 of 10 participant population according to complete data. Two similar participants with complete data will be subjected to repeated review, and a logic check will be performed to confirm that the data are correct before analysis. To the best of our knowledge, this is the first double-blind double-dummy RCT aimed at determining the efficacy of BMF in treating AR patients with LSQDS. This is also the first study to investigate changes in in- flammatory cytokines as well as changes in the gut microbiota with CHM-based therapy. The data will be analyzed using the Statistical Package for the Social Sciences version 17.0 (SPSS 17.0). Mea- sured data will be expressed as the mean, median, mini- mum, and maximum values. Comparisons between the two groups will be analyzed by analysis of variance and pairwise comparisons (univariate variance will be assessed with the rank-sum test). Count data will be expressed as the composition ratio and the rate. Between-group efficacy will be assessed using the χ2 test, and ranked data will be analyzed with the rank-sum test. A baseline comparison test will be performed at a sig- nificance level of α = 0.10, and an efficacy analysis will be performed at a significance level of α = 0.05. py There are limitations to this study. The first is a lack of validated outcome measures to evaluate the body constitution of AR patients with LSQDS. In Chinese medicine theory, the body constitution of a patient is dy- namic and may change after the BMF intervention. Abbreviations 16S rRNA: 16S ribosomal ribonucleic acid; AE: Adverse event; AR: Allergic rhinitis; BMF: Bi min fang; CHM: Chinese herbal medicine; CRF: Case report form; ELISA: Enzyme-linked immunosorbent assay; GDPHCM: Guangdong Provincial Hospital of Chinese Medicine; IFN-γ: Interferon-γ; IgE: Immunoglobulin E; IL-17: Interleukin-17; IL-4: Interleukin-4; LSQDS: Lung and spleen qi deficiency syndrome; RCT: Randomized controlled trial; RQLQ(S): Standard version of the Rhinoconjunctivitis Quality of Life Questionnaire; SPSS 17.0: Statistical Packages for the Social Sciences version 17.0; TCM: Traditional Chinese medicine; TGF-β1: Transforming growth factor β-1; TNSS: Total Nasal Symptom Score; VAS: Visual analog scale Trial status The first patient in the study was enrolled on 22 Decem- ber 2016. The trial has already enrolled participants: 76 AR patients have been recruited, and 40 patients had completed the trial by 7 November 2017. Data analysis The second limitation is that the sample size calculation was based on a previous study [46] and our clinical observa- tions; thus, the sample size of our study may have been different if more cases had been observed by a prior rigorous RCT. The last limitation is that this study ex- plores only the mechanism by which the BMF formula treats AR and does not address the mechanism at a component level. Discussion Although treatment for AR has been developed and standardized, the symptoms of some AR patients are still not well controlled with current pharmacological therap- ies [47, 48]. CHM, which may have an effect on AR, has gradually received increasing attention and has been adopted for the treatment of AR [8]. Syndrome differen- tiation or pattern identification is a specific analysis of individual body constitutions, symptoms, and signs based on TCM physiology and pathology and is per- formed before arriving at a diagnosis or drawing a con- clusion [49]. This step must be performed before prescribing CHM formulas because accurate syndrome differentiation or pattern identification ensures accurate therapeutic effects with fewer AEs [8]. BMF is composed of modified bu zhong yi qi tang and yu ping feng san, which are typically applied to treat allergic diseases [17]. Based on previous experience with the use of BMF to treat AR, this study combines syndrome differentiation and treatment with strict RCT design principles to as- sess the efficacy and safety of BMF for the treatment of AR. Regarding the mechanistic study of this formulation, this study aims to determine whether BMF can modulate cytokine levels and gut microbiota. Ethical approval Despite its limitations, we believe that this study has the potential to contribute to the development of an ef- fective intervention for AR patients with LSQDS. In the future, a multicenter RCT with a large sample of AR pa- tients and the implementation of multidimensional com- prehensive evaluations should be performed. A series of studies on BMF and its chemical components and the molecular mechanisms of actions in AR patients with LSQDS are needed and may provide consolidated evi- dence regarding the efficacy and safety of BMF for AR patients with LSQDS (Additional file 1). The study protocol was approved by the Ethics Commit- tee of GDPHCM (B2016–100-01), and it will be expli- citly explained to all participants that the trial involves two types of interventions, with a 2-week run-in period, 4 weeks of treatment and 12 weeks of follow-up. All par- ticipants will be given sufficient time to decide whether to sign the informed consent form. Written informed consent must be obtained from each participant before they are randomized to a group. Acknowledgements We would like to thank Professor Zhaoxiang Bian from Hong Kong Baptist University for his comments on this manuscript. Page 9 of 10 Luo et al. Trials (2019) 20:66 Page 9 of 10 Page 9 of 10 Luo et al. Trials (2019) 20:66 Competing interests h h d l h 13. Song WJ, Li X, Xie Q. Analysis of traditional Chinese medicine syndrome differentiation in 300 cases of allergic rhinitis. Chin Comm Doc. 2012;14(8): 219 [Article in Chinese]. 13. Song WJ, Li X, Xie Q. Analysis of traditional Chinese medicine syndrome differentiation in 300 cases of allergic rhinitis. Chin Comm Doc. 2012;14(8): 219 [Article in Chinese]. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 14. Yang S, Chen H, Lin Y, Chen Y. The exploration of disease pattern, zheng, for differentiation of allergic rhinitis in traditional Chinese medicine practice. Evid Based Complement Alternat Med. 2012. https://doi.org/10.1155/2012/ 521780. 14. Yang S, Chen H, Lin Y, Chen Y. The exploration of disease pattern, zheng, for differentiation of allergic rhinitis in traditional Chinese medicine practice. Evid Based Complement Alternat Med. 2012. https://doi.org/10.1155/2012/ 521780. Authors’ contributions 5. Meltzer EO, Bukstein DA. The economic impact of allergic rhinitis and current guidelines for treatment. Ann Allergy Asthma Immunol. 2011; 106(Suppl 2):12–6. 5. Meltzer EO, Bukstein DA. The economic impact of allergic rhinitis and current guidelines for treatment. Ann Allergy Asthma Immunol. 2011; 106(Suppl 2):12–6. QL, WC and YL contributed to the design of the study protocol. LL was involved in designing the statistical methods used in the study. XL, QC, WL, HL and CC participated in the project development and collected the outcome data. QL, SZ and WL prepared the initial draft of the manuscript. XL, SZ and LL will be involved in data management and the statistical analysis. YL is the project leader. All authors were responsible for drafting the manuscript and approved the final version. 6. Seidman MD, Gurgel RK, Lin SY, Schwartz SR, Baroody FM, Bonner JR, et al. Clinical Practice Guideline: Allergic Rhinitis. Otolaryngol Head Neck Surg. 2015;152(Suppl 1):1–43. 6. Seidman MD, Gurgel RK, Lin SY, Schwartz SR, Baroody FM, Bonner JR, et al. Clinical Practice Guideline: Allergic Rhinitis. Otolaryngol Head Neck Surg. 2015;152(Suppl 1):1–43. 7. Kern J, Bielory L. Complementary and alternative therapy (CAM) in the treatment of Allergic Rhinitis. Curr Allergy Asthma Rep. 2014;14(12):479. 7. Kern J, Bielory L. Complementary and alternative therapy (CAM) in the treatment of Allergic Rhinitis. Curr Allergy Asthma Rep. 2014;14(12):479. 8. Chan RY, Chien WT. The effects of two Chinese herbal medicinal formulae vs placebo controls for treatment of allergic rhinitis: a randomised controlled trial. Trials. 2014;15:216. 8. Chan RY, Chien WT. The effects of two Chinese herbal medicinal formulae vs placebo controls for treatment of allergic rhinitis: a randomised controlled trial. Trials. 2014;15:216. Availability of data and materials h l bl h Data sharing is not applicable to this article because no datasets were generated or analyzed during the study. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 15. Qi W. Distribution investigation of related pathogenic factors and traditional Chinese medicine clinical syndrome types of allergic rhinitis. Guid J Tradit Chin Med Pharm. 2013;10(31):125–8 [Article in Chinese]. 15. Qi W. Distribution investigation of related pathogenic factors and traditional Chinese medicine clinical syndrome types of allergic rhinitis. Guid J Tradit Chin Med Pharm. 2013;10(31):125–8 [Article in Chinese]. Funding h d This study is supported by the Special Scientific Research for Chinese Medicine of Guangdong Provincial Hospital of Chinese Medicine (Grant number: YN2015MS18) and National Natural Science Foundation of China (Grant number: 81603667), and the contact information is as follows: Scientific Research Department, Guangdong Provincial Hospital of Chinese Medicine; 111 Dade Road, Yuexiu District, Guangzhou, 510120, China; kyc30907@126.com. These funding agencies had no role in the development of the study design, data collection, or preparation of the manuscript for publication. The principal investigator of this study is Professor Yunying Li, a clinical doctor in the Otorhinolaryngology Department in Guangdong Provincial Hospital of Chinese Medicine. She has no conflicts of interest with regard to the two pharmaceutical companies mentioned in this protocol. References 1. 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https://openalex.org/W2988528514	https://europepmc.org/articles/pmc6888251?pdf=render	English	null	Exploring the Interface between Inflammatory and Therapeutic Glucocorticoid Induced Bone and Muscle Loss	International journal of molecular sciences	2,019	cc-by	15,449	Received: 30 October 2019; Accepted: 14 November 2019; Published: 16 November 2019 Abstract: Due to their potent immunomodulatory anti-inflammatory properties, synthetic glucocorticoids (GCs) are widely utilized in the treatment of chronic inflammatory disease. In this review, we examine our current understanding of how chronic inflammation and commonly used therapeutic GCs interact to regulate bone and muscle metabolism. Whilst both inflammation and therapeutic GCs directly promote systemic osteoporosis and muscle wasting, the mechanisms whereby they achieve this are distinct. Importantly, their interactions in vivo are greatly complicated secondary to the directly opposing actions of GCs on a wide array of pro-inflammatory signalling pathways that underpin catabolic and anti-anabolic metabolism. Several clinical studies have attempted to address the net effects of therapeutic glucocorticoids on inflammatory bone loss and muscle wasting using a range of approaches. These have yielded a wide array of results further complicated by the nature of inflammatory disease, underlying the disease management and regimen of GC therapy. Here, we report the latest findings related to these pathway interactions and explore the latest insights from murine models of disease aimed at modelling these processes and delineating the contribution of pre-receptor steroid metabolism. Understanding these processes remains paramount in the effective management of patients with chronic inflammatory disease. Keywords: glucocorticoid; muscle wasting; osteoporosis Exploring the Interface between Inﬂammatory and Therapeutic Glucocorticoid Induced Bone and Muscle Loss Justine M. Webster 1,2,†, Chloe G. Fenton 1,3,†, Ramon Langen 4 and Rowan S. Har Justine M. Webster 1,2,†, Chloe G. Fenton 1,3,†, Ramon Langen 4 and Rowan S. Hardy 1,3,5,* 1 Institute of Metabolism and Systems Research, University of Birmingham, Birmingham B15 2TT, UK; JMW801@student.bham.ac.uk (J.M.W.); CXF637@student.bham.ac.uk (C.G.F.) 2 Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham B15 2TT, UK 3 Institute of Inﬂammation and Ageing, University of Birmingham, Birmingham B15 2TT, UK 4 Department of Respiratory Medicine, NUTRIM School of Nutrition and Translational Research in Metabolism, Faculty of Health, Medicine and Life Sciences, Maastricht University, 6211 LK Maastricht, The Netherlands; r.langen@maastrichtuniversity.nl 5 MRC Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Birmingham, Birmingham B15 2TT, UK 5 MRC Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Birmingham, Birmingham B15 2TT, UK * Correspondence: r.hardy@bham.ac.uk; Tel.: +44-0121-414-3776 † These authors contributed equally. Int. J. Mol. Sci. 2019, 20, 5768; doi:10.3390/ijms20225768 International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Glucocorticoids and Therapeutic Glucocorticoid Excess Synthetic glucocorticoids (GCs), such as dexamethasone, prednisolone and hydrocortisone, are widely utilized in the treatment of chronic inﬂammatory diseases such as chronic obstructive pulmonary disease (COPD), inﬂammatory bowel disease (IBD) and rheumatoid arthritis (RA), with approximately 1% of the adult population in the U.K. and U.S. receiving this class of drugs. Their mechanisms of action are diverse, with GCs suppressing a range of pro-inﬂammatory pathways including p38-mitogen Int. J. Mol. Sci. 2019, 20, 5768; doi:10.3390/ijms20225768 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5768 2 of 22 activated protein kinases (p38-MAPK), nuclear factor kappa-light-chain-enhancer (NF-κB) and activator protein (AP-1), in addition to inducing pro-resolving factors such as glucocorticoid induced leucine zipper (GILZ) and annexin-1 [1–3]. These signiﬁcantly reduce leukocyte inﬁltration at sites of inﬂammation, suppress the production of pro-inﬂammatory cytokines and chemokines and support resolution of inﬂammation and tissue remodelling [4,5]. Despite the potent immune-modulatory anti-inﬂammatory actions of therapeutic GCs, their clinical application is limited due to severe systemic side eﬀects. These occur in up to 70% of patients and can include muscle wasting and GC induced osteoporosis (GIO) [6–10]. The actions of GCs on bone and muscle metabolism are well established, but themselves complicated in the backdrop of chronic inﬂammation by separate inﬂammatory driven muscle wasting and bone loss. The inﬂammatory pathways that mediate bone and muscle loss in chronic inﬂammation are in turn suppressed by the anti-inﬂammatory actions of GCs, further complicating the prediction of their outcome on the musculoskeletal system. Understanding the complex interactions between GC and inﬂammatory regulation of bone and muscle metabolism remains paramount in the eﬀective management of patients with chronic inﬂammatory disease. In this review, we explore how inﬂammatory drivers and therapeutic GCs interact to regulate bone and muscle metabolism and consider the role of local steroid metabolism in shaping these processes. 2. Glucocorticoid Signalling and Regulation of Inﬂammation Lipophilic GCs readily diffuse across cell membranes, signalling through the cytoplasmic GC receptor (GR) superfamily, encoded by the NR3C1 gene. Classically, GC signalling and GR transactivation occur through ligand binding of the GRα homodimer. In its unbound state, GRα forms a multi-protein complex with chaperone proteins such as heat shock proteins (HSPs), HSPp-70, HSP90 and FK506 binding protein 52 that block their nuclear localization signal (NLS) and prevent translocation to the nucleus from the cytoplasm [11]. Upon GC binding, the GRα undergoes a conformational change, allowing dissociation of chaperone proteins. Homo-dimerization and exposure of the NLS are required for nuclear translocation of the ligand bound GR, where it can influence gene expression [12] (Figure 1). This is an oversimplified view of GC signalling, as several studies utilizing the GRdim mouse (possessing a mutation preventing GR dimerization) reveal that the anti-inflammatory properties of therapeutic GCs are mediated by both homo-dimeric GRα complexes and monomeric GRα to facilitate transactivation or transrepression of pro-inflammatory genes [5,13–16]. Whilst the mechanisms that underpin GR signalling have been reviewed extensively elsewhere, several key pathways are prominent in mediating the anti-inflammatory actions of GCs [17]. These include the direct GRα homodimer transactivation of anti-inflammatory genes such as secretory leukocyte protease inhibitor (SLPI), MAKP-1, GILZ and tristetraprolin (TTP), which suppress the NF-kB and p38-MAPK inflammatory pathways, in addition to the inhibition of pro-inflammatory transcription factors via their tethering to the GC bound GR [18–21]. In particular, GCs act via the GR to suppress the NF-κB and p38-MAPK inflammatory pathways and AP-1 pro-inflammatory pathways, which regulate the transcription of various genes relating to inflammation such as tumour necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1β) and -6 [22]. Many of these inflammatory pathways considered are direct contributors to the process of inflammatory bone and muscle wasting and are themselves opposed by the actions of therapeutic GCs. This review will now consider how inflammation and GCs influence bone and muscle metabolism, both in isolation and in concert with one another. 3 of 22 Int. J. Mol. Sci. 2019, 20, 5768 Int. J. Mol. Sci. 2019, 20 . J. Mol. Sci. 2019, 20, 5768 3 o Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 22 Figure 1. Overview of glucocorticoid (GC) signalling pathways. The majority of glucocorticoids (GCs) in the circulation are bound by corticosteroid-binding globulin (CBG), which prevents diffusion across the membrane. 2. Glucocorticoid Signalling and Regulation of Inﬂammation Here, the GR can either dimerise to transactivate anti-inflammatory genes or signal as a monomer to inhibit pro- inflammatory transcription factors. Cortisol (CORT), nuclear factor of activated T-cells (NFAT), CCAAT-enhancer-binding proteins (or C/EBPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), p38 mitogen-activated protein kinases (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), mitogen-activated protein kinase-1 (MKP-1), activator protein 1 (AP-1), signal transducer and activator of transcription 5 (STAT5), and response element (RE). 3. Bone Metabolism B b li i i h l l d h h i b b Figure 1. Overview of glucocorticoid (GC) signalling pathways. The majority of glucocorticoids (GCs) in the circulation are bound by corticosteroid-binding globulin (CBG), which prevents diﬀusion across the membrane. However, free GCs can readily enter the cell, where they bind to the GR in the cytoplasm. This induces a conformational change in the glucocorticoid receptor (GR), which causes the dissociation of chaperone molecules, such as heat shock proteins (HSPs), to expose the nuclear localisation signal (NLS) and allow translocation of the GC/GR complex to the nucleus. Here, the GR can either dimerise to transactivate anti-inﬂammatory genes or signal as a monomer to inhibit pro-inﬂammatory transcription factors. Cortisol (CORT), nuclear factor of activated T-cells (NFAT), CCAAT-enhancer-binding proteins (or C/EBPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), p38 mitogen-activated protein kinases (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), mitogen-activated protein kinase-1 (MKP-1), activator protein 1 (AP-1), signal transducer and activator of transcription 5 (STAT5), and response element (RE). 2. Glucocorticoid Signalling and Regulation of Inﬂammation However, free GCs can readily enter the cell, where they bind to the GR in the cytoplasm. This induces a conformational change in the glucocorticoid receptor (GR), which causes the dissociation of chaperone molecules, such as heat shock proteins (HSPs), to expose the nuclear localisation signal (NLS) and allow translocation of the GC/GR complex to the nucleus. Here, the GR can either dimerise to transactivate anti-inflammatory genes or signal as a monomer to inhibit pro- inflammatory transcription factors. Cortisol (CORT), nuclear factor of activated T-cells (NFAT), CCAAT-enhancer-binding proteins (or C/EBPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), p38 mitogen-activated protein kinases (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), mitogen-activated protein kinase-1 (MKP-1), activator protein 1 (AP-1), signal transducer and activator of transcription 5 (STAT5), and response element (RE). 3. Bone Metabolism Bone metabolism is a tightly regulated process that ensures homeostasis between bone Figure 1. Overview of glucocorticoid (GC) signalling pathways. The majority of glucocorticoids (GCs) in the circulation are bound by corticosteroid-binding globulin (CBG), which prevents diﬀusion across the membrane. However, free GCs can readily enter the cell, where they bind to the GR in the cytoplasm. This induces a conformational change in the glucocorticoid receptor (GR), which causes the dissociation of chaperone molecules, such as heat shock proteins (HSPs), to expose the nuclear localisation signal (NLS) and allow translocation of the GC/GR complex to the nucleus. Here, the GR can either dimerise to transactivate anti-inﬂammatory genes or signal as a monomer to inhibit pro-inﬂammatory transcription factors. Cortisol (CORT), nuclear factor of activated T-cells (NFAT), CCAAT-enhancer-binding proteins (or C/EBPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), p38 mitogen-activated protein kinases (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), mitogen-activated protein kinase-1 (MKP-1), activator protein 1 (AP-1), signal transducer and activator of transcription 5 (STAT5), and response element (RE). Figure 1. Overview of glucocorticoid (GC) signalling pathways. The majority of glucocorticoids (GCs) in the circulation are bound by corticosteroid-binding globulin (CBG), which prevents diffusion across the membrane. However, free GCs can readily enter the cell, where they bind to the GR in the cytoplasm. This induces a conformational change in the glucocorticoid receptor (GR), which causes the dissociation of chaperone molecules, such as heat shock proteins (HSPs), to expose the nuclear localisation signal (NLS) and allow translocation of the GC/GR complex to the nucleus. 4. Regulation of Bone Metabolism by Inﬂammation In diseases such as RA, IBD and COPD, ongoing systemic inﬂammation results in inﬂammatory osteoporosis, with localized destruction of bone at sites of inﬂammation in diseases such as RA [43–47]. Systemic bone loss is characterized by a general decrease in bone mineral density (BMD) at the femoral neck, hip and spine in patients, resulting in increased fracture rates [47–50]. It is widely accepted that this inﬂammatory bone loss results from an imbalance in the bone remodelling cycle, shifting towards resorption and away from formation [51]. Studies exploring inﬂammatory bone loss are complicated by immobility in patients and the impact of concurrent anti-inﬂammatory drugs that can inﬂuence bone metabolism. However, signiﬁcant insights have been derived from in vitro studies and clinical studies. A prominent mechanism associated with a shift toward inﬂammatory bone loss is the interaction of the activated immune system with bone resorbing osteoclasts. Here, changes in the inﬂammatory cytokine proﬁles in patients with chronic inﬂammation result in increased levels of pro-osteoclastogenic mediators and a decrease in anti-osteoclastogenic mediators. Many of the pro-osteoclastogenic cytokines upregulated in chronic inﬂammation, including TNF-α, IL-1, IL-6, IL-8 and IL-17, mediate their actions via an upregulation of RANKL on ﬁbroblasts and osteoblasts, which in turn promotes osteoclastic bone resorption [52–55]. In particular, combinations of cytokines including TNF-α, IL-1 and IL-6, act synergistically to increase RANKL in inﬂammation [56]. Activated Th17 and B cells also upregulate RANKL expression promoting resorptive bone lesions in patients and in vitro in a RANKL dependent manner [57–59]. A recent study identiﬁed a novel cytokine induced in response to TNF-α in T cells, known as secreted osteoclastogenic factor of activated T cells (SOFAT), which has the ability to cause osteoclastogenesis in a RANKL independent manner and may have implications in bone loss induced by chronic inﬂammatory disease [60]. Of particular interest, TNF-α also has eﬀects on the bone forming ability of osteoblasts in inﬂammation. TNF-α treatment of osteoblasts’ precursors inhibits their diﬀerentiation by suppressing the DNA binding ability of RUNX2, leading to inhibition of alkaline phosphatase expression and matrix deposition [61]. The pro-apoptotic properties of TNF-α on osteoblasts has also been observed [62]. Similarly, IL-6 treatment of osteoblasts leads to reductions in alkaline phosphatase activity and in the expression of RUNX2 and osteocalcin, with mineralisation dramatically reduced in a dose dependent manner [63]. Bone met resorption and h t i 3. Bone Metabolism 2019, 20, 5768 and an increase in factors that induce osteoblast diﬀerentiation (such as WNT, TGFβ and insulin-like growth factor 1 (IGF-1)) promote the formation of osteoblast pre-cursors from mesenchymal derived progenitors [33–35]. This process is tightly regulated through the master transcriptional regulator runt-related transcription factor 2 (RUNX2), mediating the expression of osteoblast speciﬁc genes such as osteocalcin, osteopontin and bone sialoprotein [36,37]. As osteoblasts continue to mature, RANKL levels (which maintain osteoclasts) decrease, whilst osteoprotegerin (OPG) (the dummy receptor for RANKL that suppresses RANK signalling) increases. Together with increasing TGFβ signalling, the decrease in RANK/RANKL signalling leads to reduced osteoclast diﬀerentiation, activity and survival [38]. The transition toward the reversal phase is characterised by an increase in mature osteoblasts at the vacated osteoclast lacunae site of bone resorption. One key cell type that appears to facilitate this transition appears to be a unique cell population known as reversal cells, which cover the eroded bone surface. Here, one study has revealed that the disruption of these cells results in a loss of the initiation of bone resorption, highlighting their importance in this process [39]. Mature osteoblasts then secrete factors required for osteoid formation including organic matrix rich in type 1 collagen, osteocalcin and bone sialoprotein (BSP) [40]. This is then mineralized via the deposition of hydroxyapatite crystals, created by the ﬂux of calcium and phosphate ions within vesicles that are deposited as a mineralized nodule, in a process that has been shown to require the enzyme alkaline phosphatase to release phosphate ions [41,42]. Ultimately, bone formation ceases as osteoblasts undergo apoptosis or are incorporated into the osteocyte network. Bone met resorption and h t i 3. Bone Metabolism homeostasis, as well as allowing constant healthy remodelling to compensate for external loading stress and damage and requires the close interaction between osteocytes, bone lining cells, bone forming osteoblasts and bone resorbing osteoclasts [23]. Here, in quiescent bone, osteocytes produce factors such as transforming growth factor-β (TGF-β), sclerostin and dickkopf WNT signaling pathway inhibitor 1 (DKK-1), which inhibits osteoclast and osteoblast maturation and differentiation [24]. Signals such as bone matrix damage or immobilization result in osteocyte apoptosis, leading to a removal of the inhibitory signals and increases in factors that promote osteoclastogenesis, such as macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-β ligand (RANKL) [25–27]. Together, these promote osteoclast differentiation from hematopoietic precursors and increase receptor activator of nuclear factor kappa-β (RANK) pathway activation, driving multinuclear polykaryon formation and the formation of mature osteoclasts that express osteoclast specific genes including tartrate-resistant acid phosphatase (TRAP) and cathepsin K [28– Bone metabolism is a tightly regulated process that ensures homeostasis between bone resorption and bone formation. This process maintains a balance in calcium and phosphate mineral homeostasis, as well as allowing constant healthy remodelling to compensate for external loading stress and damage and requires the close interaction between osteocytes, bone lining cells, bone forming osteoblasts and bone resorbing osteoclasts [23]. Here, in quiescent bone, osteocytes produce factors such as transforming growth factor-β (TGF-β), sclerostin and dickkopf WNT signaling pathway inhibitor 1 (DKK-1), which inhibits osteoclast and osteoblast maturation and diﬀerentiation [24]. Signals such as bone matrix damage or immobilization result in osteocyte apoptosis, leading to a removal of the inhibitory signals and increases in factors that promote osteoclastogenesis, such as macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-β ligand (RANKL) [25–27]. Together, these promote osteoclast diﬀerentiation from hematopoietic precursors and increase receptor activator of nuclear factor kappa-β (RANK) pathway activation, driving multinuclear polykaryon formation and the formation of mature osteoclasts that express osteoclast speciﬁc genes including tartrate-resistant acid phosphatase (TRAP) and cathepsin K [28–30]. Mature osteoclasts form tight integrin junctions on mineralized bone matrix, forming an acidiﬁed resorption compartment that facilitates the degradation of the inorganic hydroxyapatite component of the bone [31,32]. The organic component of bone can then be degraded by lysosomal enzymes, such as cathepsin K. In parallel to this process, a reduction in factors that suppress osteoblast diﬀerentiation (such as sclerostin and DKK-1) 4 of 22 Int. J. Mol. Sci. 5. Eﬀects of Glucocorticoids on Bone Metabolism Whilst GCs are widely used in the treatment of chronic inﬂammation, they are themselves associated with an increased risk of fractures and osteoporosis at therapeutic doses resulting in GIO. GIO is the most common form of secondary osteoporosis with risk of fracture increasing dramatically within three to six months of starting GC therapy [65]. Interestingly, these changes are reversed rapidly upon cessation of GCs, indicating a rapid and acute nature of action at the cellular level. The mechanism that underpins this appears to be primarily mediated by a substantial inhibition of osteoblastic bone formation [66]. Under physiological conditions, GCs promote osteoblast maturation. However, at higher therapeutic doses, GCs downregulate WNT agonists and upregulate WNT inhibitors, which induce apoptosis and suppress osteoblast diﬀerentiating [67–69]. In one clinical study examining children receiving exogenous glucocorticoids, serum levels of the WNT signalling inhibitor DKK-1 were shown to be signiﬁcantly elevated, suggesting it may play a key role in reduced bone formation in GIO [70]. In studies using transgenic mice with osteoblast targeted disruption of glucocorticoid signalling, GC signalling via the GR was shown to mediate reduced bone formation through the suppression of osteoblast diﬀerentiation via the WNT pathway and through inducing osteoblast apoptosis, with animals with GR signaling disruption being protected from GC induced bone loss [67,71]. The impact of GCs on osteoclasts is less clear. Studies have reported that GC treatment results in a decrease in osteoclast number, but an increase in osteoclast longevity, potentially mediated via a GC induced increase in M-CSF production [66,72,73]. In addition, studies have shown conﬂicting results on the expression of osteoclastic genes in response to GCs. One study showed that dexamethasone treatment of murine calvarial bones resulted in increased mRNA levels of Rank and Rankl, leading to increased markers of osteoclast activation [74]. Other studies showed that OPG levels are suppressed or reported no change at all in RANKL and OPG levels [72,75,76]. Some insight comes from one study in children receiving exogenous GCs, where serum levels of RANKL were elevated and OPG suppressed [77]. In these patients, spontaneous osteoclastogenesis in vitro was apparent in monocytic cell precursors. Certainly, one study utilizing a murine model of therapeutic GC delivery revealed that the targeting of osteoclasts using bisphosphonates was an eﬀective strategy to prevent both cortical and trabecular bone loss [78]. 5. Eﬀects of Glucocorticoids on Bone Metabolism There is some evidence to indicate that the responsiveness of osteoclasts to GCs is highly dependent on the stage of cell diﬀerentiation, but these ﬁndings require further investigation [79]. The variation in GC dose, the method of administration and the models employed may explain the variation in the results reported to date, whilst their interactions with inﬂammatory mediators in patients with chronic inﬂammation should also be taken into account when investigating their bone related eﬀects. 4. Regulation of Bone Metabolism by Inﬂammation The prominent role of the inﬂammatory activation of osteoclastogenesis was derived from murine models using the TNF-tg mouse of chronic polyarthritis and inﬂammatory bone loss. 5 of 22 Int. J. Mol. Sci. 2019, 20, 5768 Here, blockade of both the TNF-α and the RANKL/RANK signalling pathways using anti-TNF therapy in combination with anti-osteoclastic (OPG) was able to prevent inﬂammatory bone erosions [64]. Bone repair was then augmented through the addition of the pro-osteoblastic hormone parathyroid hormone (PTH). These results highlight the importance of bot inﬂammatory activation of osteoclasts and suppression of osteoblasts in mediating systemic and localized bone loss in chronic inﬂammation. Consequently, these results indicate that repair of bone erosions requires a therapy that simultaneously controls inﬂammation while also impacting both osteoclastic bone resorption and osteoblastic bone formation to shift the balance in bone homeostasis and promote normal repair and recovery of bone. 6. Glucocorticoids, Inﬂammation and Bone Homeostasis Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 22 appears to be prominent in mediating their bone sparing effects in chronic inflammatory joint destruction, through the direct suppression of osteoclastogenesis and osteoclast activation [80] (Figure 2). In contrast, their potent suppression of anabolic bone formation by osteoblasts may synergize with the deleterious actions of inflammation on osteoblasts. Consequently, the net balance of GCs on bone metabolism in the context of chronic inflammation is less clear. Several clinical studies shed light on the balance between beneficial and detrimental actions of GCs on bone metabolism in chronic inflammation. These include a study reporting no differences in BMD loss in RA patients receiving therapeutic GCs in combination with traditional disease modifying anti-rheumatic drugs (DMARDS), relative to a matched control cohort [81]. Of particular interest were studies exploring whether GCs at lower therapeutic doses might promote positive anti-inflammatory actions without eliciting detrimental bone loss. These studies reported that low dose GC therapy in RA did not increase the risk of generalized osteoporosis at the spine and hip [82,83]. Another study found that patients receiving GCs in combination with anti-TNF therapy had a 2.5% increase in BMD at the femoral neck compared to a 0.7% decrease in BMD in those using anti-TNF alone, suggesting that GCs may increase bone metabolism in this context [84]. In contrast, two further studies found that GCs’ use was associated with decreased BMD in RA patients [43,85]. Similarly, in juvenile chronic arthritis (JCA), two studies found that GC treated patients had significantly less trabecular bone and higher risk of vertebral collapse than a matched control cohort [86,87]. These studies found strong links with the dose of steroid applied, but were further complicated by the application of GCs in the developing skeleton of younger patients, who may be more vulnerable to the anti-anabolic actions of GCs than adults. The conflicting nature of these results may stem from a variety of issues, including differences in disease pathophysiology, disease activity, duration and variations in the delivery and dose of GC therapy. In addition, concomitant use of alternative therapies such as anti-TNF treatments causes further complications, making it difficult to dissect the contribution of GCs to changes in bone metabolism in chronic inflammatory disease. Figure 2. Schematic representation of the effects of inflammation and glucocorticoids (GCs) on bone remodelling. 6. Glucocorticoids, Inﬂammation and Bone Homeostasis Glucocorticoids directly oppose a wide array of the pathways that drive inﬂammatory bone loss. Amongst these, their suppression of pro-inﬂammatory factors such as RANKL, TNF-α and IL-6 appears to be prominent in mediating their bone sparing eﬀects in chronic inﬂammatory joint destruction, through the direct suppression of osteoclastogenesis and osteoclast activation [80] (Figure 2). In contrast, their potent suppression of anabolic bone formation by osteoblasts may synergize with the deleterious Int. J. Mol. Sci. 2019, 20, 5768 6 of 22 actions of inﬂammation on osteoblasts. Consequently, the net balance of GCs on bone metabolism in the context of chronic inﬂammation is less clear. Several clinical studies shed light on the balance between beneﬁcial and detrimental actions of GCs on bone metabolism in chronic inﬂammation. These include a study reporting no diﬀerences in BMD loss in RA patients receiving therapeutic GCs in combination with traditional disease modifying anti-rheumatic drugs (DMARDS), relative to a matched control cohort [81]. Of particular interest were studies exploring whether GCs at lower therapeutic doses might promote positive anti-inﬂammatory actions without eliciting detrimental bone loss. These studies reported that low dose GC therapy in RA did not increase the risk of generalized osteoporosis at the spine and hip [82,83]. Another study found that patients receiving GCs in combination with anti-TNF therapy had a 2.5% increase in BMD at the femoral neck compared to a 0.7% decrease in BMD in those using anti-TNF alone, suggesting that GCs may increase bone metabolism in this context [84]. In contrast, two further studies found that GCs’ use was associated with decreased BMD in RA patients [43,85]. Similarly, in juvenile chronic arthritis (JCA), two studies found that GC treated patients had signiﬁcantly less trabecular bone and higher risk of vertebral collapse than a matched control cohort [86,87]. These studies found strong links with the dose of steroid applied, but were further complicated by the application of GCs in the developing skeleton of younger patients, who may be more vulnerable to the anti-anabolic actions of GCs than adults. The conﬂicting nature of these results may stem from a variety of issues, including diﬀerences in disease pathophysiology, disease activity, duration and variations in the delivery and dose of GC therapy. In addition, concomitant use of alternative therapies such as anti-TNF treatments causes further complications, making it diﬃcult to dissect the contribution of GCs to changes in bone metabolism in chronic inﬂammatory disease. Int. J. 6. Glucocorticoids, Inﬂammation and Bone Homeostasis During inflammation, elevated levels of pro-inflammatory cytokines, such as TNF-α and IL-6, inhibit the differentiation of bone forming osteoblasts from their precursors. These cytokines, along with other pro-inflammatory mediators including IL-1, IL-17 and IL-8, also upregulate the expression of receptor activator of nuclear factor kappa-Β ligand (RANKL), which binds to receptor activator of nuclear factor kappa-Β (RANK) on pre-osteoclasts and triggers their differentiation into mature bone resorbing osteoclasts. Overall, bone formation is decreased while bone resorption is increased, leading to a net loss of bone. Although GCs suppress inflammation via suppression of Figure 2. Schematic representation of the eﬀects of inﬂammation and glucocorticoids (GCs) on bone remodelling. During inﬂammation, elevated levels of pro-inﬂammatory cytokines, such as TNF-α and IL-6, inhibit the diﬀerentiation of bone forming osteoblasts from their precursors. These cytokines, along with other pro-inﬂammatory mediators including IL-1, IL-17 and IL-8, also upregulate the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which binds to receptor activator of nuclear factor kappa-B (RANK) on pre-osteoclasts and triggers their diﬀerentiation into mature bone resorbing osteoclasts. Overall, bone formation is decreased while bone resorption is increased, leading to a net loss of bone. Although GCs suppress inﬂammation via suppression of pro-inﬂammatory factors and induction of anti-inﬂammatory mediators, they can also independently drive bone loss by inhibiting diﬀerentiation and inducing apoptosis of osteoblasts whilst increasing osteoclast diﬀerentiation by stimulating expression of RANKL and decreasing its decoy receptor osteoprotegerin (OPG). Osteoblasts (OBs), p38 mitogen-activated protein kinases (p-38-MAPK), glucocorticoid-induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), mitogen-activated protein kinase-1 (MKP-1), activator protein 1 (AP-1), OC (osteoclast), canonical WNT signalling (WNT), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Figure 2. Schematic representation of the effects of inflammation and glucocorticoids (GCs) on bone remodelling. During inflammation, elevated levels of pro-inflammatory cytokines, such as TNF-α and IL-6, inhibit the differentiation of bone forming osteoblasts from their precursors. These cytokines, along with other pro-inflammatory mediators including IL-1, IL-17 and IL-8, also upregulate the expression of receptor activator of nuclear factor kappa-Β ligand (RANKL), which binds to receptor activator of nuclear factor kappa-Β (RANK) on pre-osteoclasts and triggers their differentiation into mature bone resorbing osteoclasts. Overall, bone formation is decreased while bone resorption is increased, leading to a net loss of bone. Although GCs suppress inflammation via suppression of Figure 2. Schematic representation of the eﬀects of inﬂammation and glucocorticoids (GCs) on bone remodelling. 7. Muscle Mass Related Metabolism Similar to bone, muscle metabolism is tightly regulated to ensure a balance between anabolic and catabolic processes governing muscle mass. Its regulation is critical not only to facilitate mechanical locomotion, but also as a key site for whole body energy metabolism and homeostasis [88]. Several critical anabolic and catabolic signalling pathways determine muscle protein synthesis, muscle proteolysis and myogenesis as cellular processes in control of muscle mass. IGF-1 has been identiﬁed as a critical factor mediating the regulation of anabolic and catabolic muscle homeostasis in adult myoﬁbers. Produced primarily in the liver, its binding to the IGF-1 receptor (IGF1R) in skeletal muscle allows recruitment of the insulin receptor substrate 1 (IRS-1) and activation of phosphatidylinositol-3-kinase (PI3K) and phosphorylation of protein kinase B (nnown as AKT), [89,90]. Together, these result in the activation of the mammalian target of rapamycin (mTOR) signalling pathway, which results in suppression of proteolysis and activation of muscle protein synthesis. mTOR activation suppresses proteolytic, forkhead box class O family member proteins (FOXOs) and glycogen synthase kinase-3 beta (GSK-3β) pathways [91,92]. The activation of mTOR signalling promotes muscle protein synthesis through the downstream phosphorylation and inactivation of eIF4E-binding protein 1 (4E-BP1) and activation of the ribosomal protein S6 kinase beta-1 (p70S6K) [93–95]. When active, 4E-BP1 operates by suppressing the eukaryotic translation initiation factors (elF), which are a central rate limiting step in the regulation of protein synthesis in muscle. Here, eIF4F (a complex of initiation factors, eIF4e, eIF4G and eIF4A), promotes the translation of mRNA coding for muscle proteins by facilitating the cap dependent binding of messenger RNA to the 40S ribosomal subunit [96]. The repressor protein 4E-BP1 is a powerful negative regulator of eIF4F mediated protein translation, whilst its phosphorylation causes its dissociation from eIF4E and enables mRNA translation of anabolic muscle proteins. A second key stage in the regulation of anabolic protein metabolism in muscle occurs through the regulation of phosphorylated p70S6K by mTOR, which facilitates ribosomal biogenesis and translation capacity required for muscle protein anabolism [97]. An additional modulator of skeletal muscle mass downstream of the IGF-1/AKT pathway is GSK-3β. This protein kinase is a negative regulator of the translation initiation factor eIF2B and is phosphorylated and inactivated by AKT, allowing initiation of mRNA translation [98–100]. Together, the activation of these pathways by IGF-1 or insulin promote protein synthesis in muscle, favouring increased muscle mass. 6. Glucocorticoids, Inﬂammation and Bone Homeostasis During inﬂammation, elevated levels of pro-inﬂammatory cytokines, such as TNF-α and IL-6, inhibit the diﬀerentiation of bone forming osteoblasts from their precursors. These cytokines, along with other pro-inﬂammatory mediators including IL-1, IL-17 and IL-8, also upregulate the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which binds to receptor activator of nuclear factor kappa-B (RANK) on pre-osteoclasts and triggers their diﬀerentiation into mature bone resorbing osteoclasts. Overall, bone formation is decreased while bone resorption is increased, leading to a net loss of bone. Although GCs suppress inﬂammation via suppression of pro-inﬂammatory factors and induction of anti-inﬂammatory mediators, they can also independently drive bone loss by inhibiting diﬀerentiation and inducing apoptosis of osteoblasts whilst increasing osteoclast diﬀerentiation by stimulating expression of RANKL and decreasing its decoy receptor osteoprotegerin (OPG). Osteoblasts (OBs), p38 mitogen-activated protein kinases (p-38-MAPK), glucocorticoid-induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), mitogen-activated protein kinase-1 (MKP-1), activator protein 1 (AP-1), OC (osteoclast), canonical WNT signalling (WNT), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Int. J. Mol. Sci. 2019, 20, 5768 7 of 22 7. Muscle Mass Related Metabolism The regulation of muscle catabolism shares many of these pathways and involves their inverse activation state. Proteolysis of skeletal muscle proteins through their targeted degradation by the ubiquitin-proteasome system (UPS) and autophagy pathways is under stringent control of the PI3K/AKT and mTOR signalling pathways [101]. Here, a reduction in anabolic factors such as IGF-1 or an increase in negative regulators such as myostatin, TGFβ or FGF results in a decrease of the PI3K/AKT and mTOR signalling. As AKT and mTOR kinase activity is responsible for inhibitory phosphorylation of the FOXOs, including FOXO1, FOXO3 and FOXO4 [102,103], the lack thereof allows their nuclear translocation. FOXO transcription factors bind to promoter and enhancer regions of target genes such as the E3 ligases, Atrogin-1 and muscle RING-Finger protein-1 (MURF-1) and the autophagy-related genes LC3 and Bnip3 [104–107]. In addition to FOXO, increased GSK-3β secondary to reduced IGF-1/AKT signalling has also been implicated in upregulating Atrogin-1 and MURF-1 [108]. The E3 ligases are the largest family of ubiquitination factors targeting muscle proteins for degradation by the UPS [109,110] and can be highly upregulated in catabolic conditions. These include the muscle speciﬁc F-box protein Atrogin-1 encoded by the FBXO32 gene and MURF-1 encoded by the TRIM63 gene [111,112]. Their expression is elevated in a plethora of skeletal muscle atrophy models, including immobilisation, denervation, cancer, starvation and diabetes [111–113]. Atrogin-1 has been shown to ubiquitinate desmin and vimentin, muscle proteins essential to sarcomere Z-disk architecture [114]. In addition, Atrogin-1 stimulates the degradation of transcription factor EIF3F, Int. J. Mol. Sci. 2019, 20, 5768 8 of 22 leading to impaired muscle protein synthesis [115]. This E3 ligase has also been shown to play a pivotal role in repressing myogenesis through the ubiquitination of myoblast determination protein 1 (MYOD) [116]. MURF-1 encodes a protein containing a RING ﬁnger domain, which is responsible for its ubiquitin-ligase activity [112,117]. MURF-1 ubiquitinates and catalyses the degradation of contractile proteins and thick ﬁlaments, such as myosin and troponin I, with the sparing of thin ﬁlaments such as actin [118,119]. Besides a role in the UPS, increased FOXO activation also upregulates protein degradation and clearance through the autophagy pathways [106]. In muscle, this appears to be mediated through the direct upregulation of autophagy genes such as LC3, BNIP3 and ATG through the FOXO pathway during muscular atrophy [120–122]. Postnatal myogenesis is an anabolic process important to the maintenance of muscle mass and integrity. 7. Muscle Mass Related Metabolism Insulin-like growth factor 1 (IGF-1) has been shown to be a positive driver of myogenesis, whilst ﬁbroblast growth factor (FGF), transforming growth factor β (TGF- β) and myostatin are potent inhibitors [123–126]. In addition, various secreted WNT signalling factors positively inﬂuence myogenesis. These are regulated by an array of stimuli, including exercise, nerve innervation and dietary protein intake, and are mediated through various gene regulatory networks including the T-box family, tbx6, ripply1 and mesp-ba in mesenchymal stem cell populations [127–129]. Ultimately, this drives the expression of myogenic regulatory factors (MRFs) such as myogenic diﬀerentiation 1 (MYOD), myogenic factor 5 (MYF5) and myogenin (MYOG), this process being in mesenchymal derived muscle progenitor cells called satellite cells [130–133]. Although some redundancy exists in their cellular function, MYF5 is mostly implicated in mediating the proliferation of satellite cells and MYOD in their diﬀerentiation into myoblasts, whilst downstream factors, including myogenin, initiate further diﬀerentiation of mature myocytes followed by the fusion and formation of mature myotubes [130–132] or mostly relevant for adult muscle, fusion with myoﬁbers. y y y Below, we will describe how inﬂammation and glucocorticoids impact these regulatory processes of muscle mass metabolism, driving a shift towards anti-anabolic and catabolic protein metabolism, resulting in muscle wasting. 9. Eﬀects of Glucocorticoids on Muscle Metabolism Extended exposure to therapeutic GCs results in the rapid onset of a GC induced muscle atrophy, characterised by a decrease in myogenesis and protein synthesis and an increase in proteolysis and atrophy of muscle ﬁbres [9,154–157]. This leads to a signiﬁcant decrease in muscle ﬁbre size, with a greater degree of wasting apparent in fast-twitch or type II muscle ﬁbres [158]. The shift towards greater catabolic loss of protein and decreased anabolic synthesis in muscle is elicited by GCs through a number of pathways, including a decrease in IGF-1 signalling and an increase in negative regulators of the mTOR pathways such as myostatin and the protein regulated in development and DNA damage response 1 (REDD1) [157,159]. Similarly, as with inﬂammatory pathway activation, GCs also activate the UPS and autophagy secondary to upregulation of the FOXO1 pathway [80,154,160]. In particular, the marked increase in degradation of contractile skeletal muscle proteins through the UPS system is believed to be central in GC induced muscle wasting in vivo. This is supported by several studies demonstrating the downregulation of the PI3K/AKT/mTOR signalling pathways and the upregulation of the E3 ligases Atrogin-1 and MURF-1 in response to GCs [112]. Several studies have also demonstrated a signiﬁcant increase in 4E-BP1 and suppression of p70S6K in GC induced muscle atrophy, demonstrating a role for reduced protein synthesis and regeneration [157,159]. Of interest, the restoration of IGF-1 signalling can rescue GC induced myopathy in mice, demonstrating a crucial role for this growth factor in the process of GC-induced myopathy [9,154–156]. Glucocorticoid mediated muscle wasting has also been shown to be rescued through the in vivo deletion of myostatin, indicating that the negative regulation of the IGF-1 pathway may also be a crucial step in this process [161–163]. 8. Eﬀects of Inﬂammation of Muscle Metabolism Inflammation is a well established driver of muscle wasting in preclinical models and strongly relates to poor prognostic outcome and increased morbidity and mortality in patients with chronic inflammatory diseases [134]. Pro-inflammatory cytokines such as TNFβα, IL-1β and IL-6, which are elevated in chronic inflammation, are themselves reported to drive proteolysis and autophagy and suppress myogenesis and protein synthesis in muscle [135–138]. Of these, TNF-α, at the apex of the inflammatory cytokine cascade in many chronic inflammatory diseases, is critical in regulating inflammatory muscle wasting. Here, its activation of the NF-kB and p-38 MAPK pathways directly induces muscle wasting through the increased expression of the E3 ligases, atrogin-1 and MURF-1 and activation of the UPS system [139–143]. In models of chronic inflammation, TNF-α also downregulates circulating levels of IGF-1 and the downstream PI3K/AKT/mTOR signalling pathways, whilst upregulating the catabolic FOXO pathway to suppress protein synthesis and myogenesis in muscle [141,142,144,145]. Another factor implicated in inflammatory muscle wasting is myostatin. This is also reported to be increased in chronic inflammation, where it positively correlates with markers of disease severity. Elevated myostatin downregulates PI3K/AKT/mTOR signalling, promoting muscle atrophy [146,147]. Of interest, several studies have reported elevated endogenous GC levels as being central mediators of inflammatory muscle wasting. Here, the inflammatory activation of the hypothalamic/pituitary/adrenal (HPA) axis in response results in an elevation of circulating cortisol to mediate muscle wasting [148–151]. Of note, the blockade of endogenous GC production or muscle GR signalling could reverse muscle wasting in some experimental Int. J. Mol. Sci. 2019, 20, 5768 9 of 22 models [152,153]. This indicates that in addition to a direct impact of inflammation on intra-cellular muscle mass regulatory processes, activation of the HPA axis as an evolutionarily conserved response to suppress systemic inflammation can result in GC driven muscle wasting as an indirect effect of inflammation on skeletal muscle. 10. Interaction between Inﬂammation and Glucocorticoids in Muscle As with bone, many of the central inﬂammatory pathways that induce muscle wasting, including the NF-kB and p38-MAPK pathways, are themselves suppressed by GC signalling, suggesting that therapeutic application may protect against the process of inﬂammatory muscle wasting. However, other elements of inﬂammatory muscle wasting such as the suppression of IGF-1 and induction of myostatin and FOXO1 pathway activation are common components in both inﬂammatory and GC induced muscle wasting (Figure 3). Understanding how these interact in vivo remains paramount in our understanding of how therapeutic GCs should be applied in the setting of chronic inﬂammatory disease. Some insights arise from clinical studies exploring these processes in patients with inﬂammatory disease receiving GCs. Of note, in inﬂammatory myopathies arising directly from muscle inﬂammation, such as with polymyositis and dermatomyositis, GCs are eﬀective in controlling inﬂammation and protecting against inﬂammatory muscle wasting and associated weakness [164]. Similarly, therapeutic GCs are eﬀective in preventing muscle wasting in patients with Duchenne muscular dystrophy (DMD), where progressive muscle necrosis mediates loss of muscle [165–169]. However, in other inﬂammatory diseases, where muscle wasting occurs secondarily to inﬂammation at a non-muscle site, the application of therapeutic GCs is strongly associated with rapid loss of muscle mass [170,171]. These ﬁndings may suggest that GCs can oppose the process of inﬂammatory muscle wasting when the active inﬂammation is conﬁned to the muscle, but promote muscle wasting when used to manage other systemic chronic inﬂammatory diseases such as RA. As with GC induced osteoporosis in patients with chronic inﬂammatory disease, the interpretation of these ﬁndings in relation to muscle wasting is complicated by disease severity and duration and by concurrent DMARD therapies. 10 of 22 f 22 Int. J. Mol. Sci. 2019, 20, 5768 Int. J. Mol. Sci. 2019, 20, x F Figure 3. Schematic representation of signalling pathways involved in both inflammatory driven and GC induced muscle wasting and their interactions. Inflammatory cytokines such as TNF-α and IL-1 inhibit mammalian target of rapamycin (mTOR) signalling, dampening muscle protein synthesis, whilst simultaneously inducing transcription of E3 ligases, leading to muscle proteolysis. Glucocorticoids (GCs) inhibit inflammatory signalling, including nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB) signalling, and therefore decrease inflammatory driven muscle wasting. Despite this, GCs also drive muscle wasting through several pathways themselves, including suppression of the IGF-1/AKT/mTOR signalling cascade, leading to decreased protein synthesis and increased FOXO1 transcription. 10. Interaction between Inﬂammation and Glucocorticoids in Muscle GR activation and dimerization induce the transcription of myostatin (MSTN), FOXO1 and other E3 ligases, leading to increased proteolysis and diminished protein synthesis. Forkhead box protein O1 (FOXO1), mammalian target of rapamycin (mTOR), insulin like growth factor (IGF-1), (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), glucocorticoid receptor (GR), myostatin (MSTN), and IGF-1 receptor (IGF-1 R). Figure 3. Schematic representation of signalling pathways involved in both inﬂammatory driven and GC induced muscle wasting and their interactions. Inﬂammatory cytokines such as TNF-α and IL-1 inhibit mammalian target of rapamycin (mTOR) signalling, dampening muscle protein synthesis, whilst simultaneously inducing transcription of E3 ligases, leading to muscle proteolysis. Glucocorticoids (GCs) inhibit inﬂammatory signalling, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling, and therefore decrease inﬂammatory driven muscle wasting. Despite this, GCs also drive muscle wasting through several pathways themselves, including suppression of the IGF-1/AKT/mTOR signalling cascade, leading to decreased protein synthesis and increased FOXO1 transcription. GR activation and dimerization induce the transcription of myostatin (MSTN), FOXO1 and other E3 ligases, leading to increased proteolysis and diminished protein synthesis. Forkhead box protein O1 (FOXO1), mammalian target of rapamycin (mTOR), insulin like growth factor (IGF-1), (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), glucocorticoid receptor (GR), myostatin (MSTN), and IGF-1 receptor (IGF-1 R). Figure 3. Schematic representation of signalling pathways involved in both inflammatory driven and GC induced muscle wasting and their interactions. Inflammatory cytokines such as TNF-α and IL-1 inhibit mammalian target of rapamycin (mTOR) signalling, dampening muscle protein synthesis, whilst simultaneously inducing transcription of E3 ligases, leading to muscle proteolysis. Glucocorticoids (GCs) inhibit inflammatory signalling, including nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB) signalling, and therefore decrease inflammatory driven muscle wasting. Despite this, GCs also drive muscle wasting through several pathways themselves, including suppression of the IGF-1/AKT/mTOR signalling cascade, leading to decreased protein synthesis and increased FOXO1 transcription. GR activation and dimerization induce the transcription of myostatin (MSTN), FOXO1 and other E3 ligases, leading to increased proteolysis and diminished protein synthesis. 11 Insights from Murine Models of Chronic Inflammation Receiving Therapeutic Insights from Murine Models of Chronic Inflammation Receiving Therapeutic Glucocorticoids g g p Glucocorticoids Additional insight has come from murine models of polyarthritis receiving therapeutic GCs. These are able to circumvent issues related to differences in disease activity between patients and complications arising from the various alternative anti-inflammatory drugs used to manage disease in patients. In one such study, we examined the role of the GC corticosterone, delivered as a monotherapy in the TNF-tg murine model of polyarthritis, on net bone and muscle metabolism [80]. This revealed that therapeutic doses of GCs, whilst effective at suppressing disease activity, also potently suppressed inflammatory osteoporosis and juxta articular bone loss. This confirmed that their capacity to suppress inflammatory pathways that mediate inflammatory bone loss outweighed their deleterious effects on bone metabolism. These bone sparing effects of GCs were mediated through the suppression of pro-inflammatory osteoclasts’ activation, both systemically and at sites of inflammation. However, whilst these treatments protected from inflammatory bone loss, we still observed a suppression of anabolic bone formation in all mice receiving GCs, suggesting that long term administration may still ultimately result in GIO. Additional insight has come from murine models of polyarthritis receiving therapeutic GCs. These are able to circumvent issues related to diﬀerences in disease activity between patients and complications arising from the various alternative anti-inﬂammatory drugs used to manage disease in patients. In one such study, we examined the role of the GC corticosterone, delivered as a monotherapy in the TNF-tg murine model of polyarthritis, on net bone and muscle metabolism [80]. This revealed that therapeutic doses of GCs, whilst eﬀective at suppressing disease activity, also potently suppressed inﬂammatory osteoporosis and juxta articular bone loss. This conﬁrmed that their capacity to suppress inﬂammatory pathways that mediate inﬂammatory bone loss outweighed their deleterious eﬀects on bone metabolism. These bone sparing eﬀects of GCs were mediated through the suppression of pro-inﬂammatory osteoclasts’ activation, both systemically and at sites of inﬂammation. However, whilst these treatments protected from inﬂammatory bone loss, we still observed a suppression of anabolic bone formation in all mice receiving GCs, suggesting that long term administration may still ultimately result in GIO. Unlike bone, therapeutic GCs markedly exacerbated muscle wasting in mice with chronic inflammation. This was characterized by a marked activation of the catabolic FOXO1 and UPS Unlikebone, therapeuticGCsmarkedlyexacerbatedmusclewastinginmicewithchronicinflammation. This was characterized by a marked activation of the catabolic FOXO1 and UPS pathways [80]. 10. Interaction between Inﬂammation and Glucocorticoids in Muscle Forkhead box protein O1 (FOXO1), mammalian target of rapamycin (mTOR), insulin like growth factor (IGF-1), (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), glucocorticoid receptor (GR), myostatin (MSTN), and IGF-1 receptor (IGF-1 R). Figure 3. Schematic representation of signalling pathways involved in both inﬂammatory driven and GC induced muscle wasting and their interactions. Inﬂammatory cytokines such as TNF-α and IL-1 inhibit mammalian target of rapamycin (mTOR) signalling, dampening muscle protein synthesis, whilst simultaneously inducing transcription of E3 ligases, leading to muscle proteolysis. Glucocorticoids (GCs) inhibit inﬂammatory signalling, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling, and therefore decrease inﬂammatory driven muscle wasting. Despite this, GCs also drive muscle wasting through several pathways themselves, including suppression of the IGF-1/AKT/mTOR signalling cascade, leading to decreased protein synthesis and increased FOXO1 transcription. GR activation and dimerization induce the transcription of myostatin (MSTN), FOXO1 and other E3 ligases, leading to increased proteolysis and diminished protein synthesis. Forkhead box protein O1 (FOXO1), mammalian target of rapamycin (mTOR), insulin like growth factor (IGF-1), (p-38-MAPK), glucocorticoid induced leucine zipper (GILZ), secretory leukocyte protease inhibitor (SLPI), tristetraprolin (TTP), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), glucocorticoid receptor (GR), myostatin (MSTN), and IGF-1 receptor (IGF-1 R). 11 Insights from Murine Models of Chronic Inflammation Receiving Therapeutic Insights from Murine Models of Chronic Inflammation Receiving Therapeutic Glucocorticoids Similar findings had been reported in rats, where dexamethasone exacerbated inflammatory muscle wasting in models of sepsis [172]. These data indicate that the beneficial effects of inflammatory suppression by GCs in muscle were not sufficient to outweigh their deleterious actions on muscle metabolism. Whilst further Int. J. Mol. Sci. 2019, 20, 5768 11 of 22 11 of 22 work is required to better elucidate the actions of therapeutic GCs in the context of chronic inflammation, these data shed light on the potential strengths and weaknesses of their application in muscle and bone. In particular, they indicate that the management of side effects in muscle may need to be prioritised over those in bone, in patients with chronic inflammatory diseases receiving therapeutic GCs. 12. Pre-Receptor Regulation of Therapeutic GC Action to Protect against Side Eﬀects The pre-receptor metabolism of GCs by the 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes is recognised as a critical step in mediating GC signalling in many peripheral tissues. These are the 11β-HSD type 1 (11β-HSD1) and type 2 (11β-HSD2). 11β-HSD1 is expressed in many tissues, including the liver, bone, muscle and fat, where it converts inactive endogenous and therapeutic GCs (such as corticosterone and prednisone) to their active counterparts (such as cortisol and prednisolone), leading to a local accumulation and concentration of active GCs [173–175] (Figure 4). In contrast, 11β-HSD2 solely inactivates endogenous and therapeutic GCs within the kidney, providing circulating inactive GC substrate for the peripheral 11β-HSD1 enzyme and supporting renal clearance of GCs [175]. Several key studies have demonstrated a critical role for the pre-receptor activation of GCs by 11β-HSD1 in mediating the deleterious actions of therapeutic GCs in muscle and bone [176,177]. Here, animals with transgenic deletion of 11β-HSD1 are resistant to both exogenous GC induced muscle wasting and osteoporosis. This raises the exciting possibility that therapeutic 11β-HSD1 inhibitors, widely explored in the management of metabolic disease, may prevent bone loss and muscle wasting in patients with chronic inﬂammatory diseases receiving GCs [178,179]. Further studies lend strength to this concept, showing that 11β-HSD1 is potently upregulated within muscle cells and osteoblasts, where it is potently upregulated by circulating inﬂammatory cytokines such as TNF-α and IL-1β [174,180,181]. Despite this, caution should be applied in this context, given that systemic deletion of 11β-HSD1 can exacerbate disease activity in murine models of inﬂammation, secondary to a reduction in reactivation of endogenous GC at sites of inﬂammation [182,183]. Consequently, further studies are required to delineate the potential beneﬁts and risks of 11β-HSD1 inhibition in chronic inﬂammatory disease. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 12 of 22 Figure 4. Pre-receptor metabolism of GCs by 11β-HSD1. 11β-hydroxysteroid dehydrogenase (11β- HSD) type 1 is a bidirectional enzyme that predominantly reduces inactive GCs to their active Figure 4. Pre-receptor metabolism of GCs by 11β-HSD1. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 is a bidirectional enzyme that predominantly reduces inactive GCs to their active counterparts in Figure 4. Pre-receptor metabolism of GCs by 11β-HSD1. 11β-hydroxysteroid dehydrogenase (11β- HSD) type 1 is a bidirectional enzyme that predominantly reduces inactive GCs to their active counterparts in an NADPH dependent manner, whilst 11β-HSD type 2 is an NAD+ dependent unidirectional enzyme that converts active GCs to their inactive counterparts. References 1. Clayton, S.A.; Jones, S.W.; Kurowska-Stolarska, M.; Clark, A.R. The role of microRNAs in glucocorticoid action. J. Biol. Chem. 2018, 293, 1865–1874. [CrossRef] [PubMed] 1. Clayton, S.A.; Jones, S.W.; Kurowska-Stolarska, M.; Clark, A.R. 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Pre-receptor metabolism of GCs by 11β-HSD1. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 is a bidirectional enzyme that predominantly reduces inactive GCs to their active counterparts in an NADPH dependent manner, whilst 11β-HSD type 2 is an NAD+ dependent unidirectional enzyme that converts active GCs to their inactive counterparts. Int. J. Mol. Sci. 2019, 20, 5768 12 of 22 12 of 22 13. Conclusions Both chronic inﬂammation and therapeutic GCs are potent drivers for systemic bone and muscle wasting resulting from an imbalance between anabolic and catabolic homeostasis. Whilst therapeutic GCs oppose many of the inﬂammatory pathways that drive bone and muscle wasting, they share common pathways that promote anti-anabolic and catabolic metabolism of bone and muscle and can drive or exacerbate these deleterious processes in chronic inﬂammatory disease. 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https://openalex.org/W4225657794	https://www.researchsquare.com/article/rs-1076968/latest.pdf	English	null	Ethnobotanical study of cowpea (Vigna unguiculata (L.) Walp.) in Senegal	Journal of ethnobiology and ethnomedicine	2,022	cc-by	10,725	Ethnobotanical Study of Cowpea (Vigna Unguiculata (L.) Walp.) in Senegal Senegal Awa Sarr (  awa18.sarr@ucad.edu.sn ) Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Amy Bodian Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Mame Codou Gueye Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Badara Gueye International Institute of Tropical Agriculture (IITA), (BP) Ibadan Ghislain Kanfani Centre National de Recherches Agronomiques de Bambey Cyril Diatta Centre National de Recherches Agronomiques de Bambey Ali Lardia Bougma Université Joseph KI-ZERBO, Ouagadougou 03 Elisabeth A.M.C. Diop Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Ndiaga Cissé Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Diaga Diouf Université Cheikh Anta Diop (UCAD) Christian Leclerc UMR AGAP Institut, Université de Montpellier, CIRAD, INRAE, Institut Agro Research Article Keywords: Cowpea, ethnobotanical, local names, farming systems, Senegal Posted Date: November 16th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1076968/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Awa Sarr (  awa18.sarr@ucad.edu.sn ) Ethnobotanical Study of Cowpea (Vigna Unguiculata (L.) Walp.) in Senegal Ethnobotanical Study of Cowpea (Vigna Unguiculata (L.) Walp.) in Senegal Awa Sarr (  awa18.sarr@ucad.edu.sn ) Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Amy Bodian Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Mame Codou Gueye Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Badara Gueye International Institute of Tropical Agriculture (IITA), (BP) Ibadan Ghislain Kanfani Centre National de Recherches Agronomiques de Bambey Cyril Diatta Centre National de Recherches Agronomiques de Bambey Ali Lardia Bougma Université Joseph KI-ZERBO, Ouagadougou 03 Elisabeth A.M.C. Diop Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Ndiaga Cissé Centre d’Etude Régional pour l’Amélioration de l’Adaptation à la Sécheresse (CERAAS), Institut Sénégalais de Recherches Agricoles (ISRA) Diaga Diouf Université Cheikh Anta Diop (UCAD) Christian Leclerc UMR AGAP Institut, Université de Montpellier, CIRAD, INRAE, Institut Agro Research Article Keywords: Cowpea, ethnobotanical, local names, farming systems, Senegal Posted Date: November 16th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1076968/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 Journal of Ethnobiology and Ethnomedicine on February 5th, 2022. See published version at https://doi.org/10.1186/s13002-022-00506-y. Introduction Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important leguminous plant grown in tropical savannah zones in Africa [1]. Its cultivation makes a major contribution to food security for people living in the most marginal areas. Its seeds are rich in lysine and tryptophan, which are a valuable source of plant protein [2]. In addition, cowpea is an essential source of vitamins and minerals, which help to prevent birth defects [3,4]. Its capacity to fix atmospheric nitrogen improves soil fertility and helps to reduce the need of chemical fertiliser [5,6]. Cowpea is one of the legumes most often grown in association with cereals in rural areas. Several studies conducted in sub-Saharan Africa have shown that pulses, like cowpea, have a positive effect on cereal yield [7–10]. Young leaves and immature pods are eaten as a vegetable and the haulms are used as livestock fodder [11,12]. Formerly considered as a subsistence crop, it is now grown as cash crop and has a major socio-economic impact on Sahelian countries, particularly Senegal, where annual production is over that 180 000 tonnes in 2019 [13]. Despite the fact that its social and economic value has been demonstrated, our knowledge of the diversity of the varietal forms grown in family farming systems remains limited. Historically, local early flowering cowpea varieties were introduced from Nigeria for floodplain cropping in the Senegal River Valley, in the north of the country. In contrast, some late flowering varieties were introduced from Mali and grown in association with millet in more humid regions in Senegal [14]. These varieties spread to the rest of the country as a result of trade and migration. Today, the main cowpea production area is the centre and central north of the country [15]. Despite the key role of cowpea in Senegalese farming systems, little is known about the local management of cowpea. The ethnobotanical classification of cowpea diversity is essential for improving the conservation (in situ or ex situ) and valorisation of this legume. It is particularly relevant for breeding programmes, which require the availability of a wide genetic diversity [16]. In this respect, local cowpea varieties constitute a heritage of major importance. The surveys and/or collections that allowed us to identify cowpea varieties in the past focused on a limited number of regions. Cowpea collections were established between 1953 and 2003 in Senegal [14,17,18]. Research Article Keywords: Cowpea, ethnobotanical, local names, farming systems, Senegal DOI: https://doi.org/10.21203/rs.3.rs-1076968/v1 ork is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International Licen Version of Record: A version of this preprint was published at Journal of Ethnobiology and Ethnomedicine on February 5th, 2022. See the published version at https://doi.org/10.1186/s13002-022-00506-y. Page 1/25 Page 1/25 Abstract Cowpea (Vigna unguiculata) plays a key role in family farming systems in Senegal. It makes an essential contribution to economic, nutritional and food security. Although it is crucial, little is known about how farmers classify the diversity of local varieties or about the social practices associated with them. The aim of this study is to characterize the farming practices associated with growing cowpea in Senegal. Surveys were conducted involving 335 rural farmers living in 37 villages, spread across seven regions that produce cowpea. An average of ten farmers were randomly selected in each village. The results reveal that cowpea is a key feature of cropping systems in the studied area. Our findings highlight the high diversity of local cowpea varieties with 59 local names inventoried. In 75% of cases, the name refers to the seed’s morphology or color. Cowpea production is more diverse in Diourbel and Louga and less diverse in the south. More than half the farmers (57%) acquired their cowpea seeds (early, semi-early and late varieties) outside their village, either from markets, seed suppliers or NGOs. This new understanding of farmers’ expertise in the management of cowpea and its local variability will help to valorise local diversity in breeding programmes. Introduction However, these accessions have been partially lost, hence the need for a new assessment. Based on new collections and specific more exhaustive surveys, this study aims to characterize the farming practices associated with growing cowpea in Senegal for the first time. In particular, it aims to: (i) identify the role that cowpea has in the cropping system, by describing the range of species that it is associated with; (ii) survey and characterize its diversity in reference to the local nomenclature and the length of the varieties’ growth cycle; and (iii) identify the farmers’ seed supply. Study areas and sampling strategy The surveys were conducted between September 2015 and March 2016 in the main cowpea producing regions in Senegal (Louga, Thiès, Fatick, Diourbel, Sédhiou and Saint-Louis). The Kédougou region was also surveyed in order to identify the characteristics of the cowpea varieties grown there. The villages surveyed were chosen in consultation with agents from the Regional Rural Development Division’s services (DRDR) to facilitate access to villages that grow cowpea. To optimise the coverage of the main cowpea producing zones, three departments were visited in each region (Figure 1). The sampling strategy also aimed to provide the best representation of the diversity of ethnic groups that grow cowpea, based on the assumption that farming practices may vary from one group to another [19]. Thus, we Page 2/25 Page 2/25 selected average sized villages in different communes, located at least 15 km apart and 10 km from the national road and the market. The survey was organized in 37 villages, with from 4 to 6 villages per region (Additional file 1). selected average sized villages in different communes, located at least 15 km apart and 10 km from the national road and the market. The survey was organized in 37 villages, with from 4 to 6 villages per region (Additional file 1). Surveys on cropping diversity The focus groups, which brought together about ten farmers in the public square or at the village chief’s home, made it possible to check whether the full range of crop diversity in the village had been identified during the individual interviews. The spelling for the local names of the cowpea varieties was harmonized and the synonyms were identified to ensure that only one term of reference was kept and translated into english. Collecting the cowpea accessions After each individual interview, all the cowpea accessions grown by the farmer were collected. An accession corresponds to the name of a variety grown by a farmer. In fact, after recording the names of varieties grown by a farmer, a visit to the field and/or storage area was conducted to collect samples. Ideally, pod samples were taken in the field. Otherwise, seeds were sampled in the granary. The accessions collected were put in envelopes, labelled and kept before being transported to the "Centre d'Etudes Régional pour l'Amélioration à l'Adaptation à la Sécheresse" (CERAAS) in Senegal for conservation. The villages’ geographic coordinates were recorded on a tablet with the aid of the software Sygic Surveys on cropping diversity In each village surveyed, participatory research methods and tools were applied to find out about cowpea management and the varietal diversity used by farmers during the winter season in 2015 [20]. The floodplain cultivation system in the Saint-Louis Region was also considered (October 2015 – February 2016). The survey was conducted with the help of informal and semi-structured interviews, in addition to focus group discussions. The informal interviews were initially conducted with the village chief to find out about the site’s socio-cultural and demographic characteristics. The semi-structured interviews were conducted with the help of a questionnaire. This type of interview involves a discussion between the surveyor and the interviewee, which allows for reminders and interactions [21]. The semi-structured interviews made it possible to identify the range of species associated with cowpea (Additional file 2) describe the varietal diversity of cowpea using the local names, determine seed origin and the cowpea accession cycles (Additional file 3) (accessions were collected at the same time). A free listing method was used [22] to identify the diversity of species grown with cowpea, as well as the diversity of specific cowpea morphotypes or local varieties. Here, the term “variety” corresponds to local names to designate local morphotypes. Their importance is evaluated in relation with their frequency. Free listing is a technique that is widely used in ethnobotanical studies. It involves asking farmers to list all the known varieties for a given species [23]. This technique is used to explore and delimit a field of knowledge. It was used to classify the species and varieties of cowpea grown by farmers during the 2015 winter season. As Henley Henley (1969) and Borgatti (1999) pointed out, "the order in which elements are listed by individual respondents is not arbitrary”. A first list of species (or varieties) is proposed by farmers with no hesitation. After a pause, a second complementary list is proposed, following by a third one, and so on. The aim is to interpret these different series, by taking into account the order in which the species (or varieties) are listed by each interviewee. The hypothesis is that the most important species (or varieties) tend to be mentioned first. Data analysis The age, ethnic group and profession were used to characterize the farmers interviewed. The frequency, the average salience and Smith’s index for each species and variety were calculated with the R AnthroTools package [24]. The frequency with which an element was cited (species and varieties) reflects its importance and its perceptual distinctive character. Salience is determined by order of citation; an element is more important when cited at the beginning of the list [25]. Smith’s index is a weighted average of the reverse order for each element. A correspondence analysis was conducted between these species and the different regions of the study. The number of cowpea morphotypes that farmers identified and named was used to estimate the varietal richness [26]. To further our understanding of cowpea cultivation, we described the practices associated with each morphotype that was identified, in particular, how seeds were obtained (place of origin of seeds) and the cropping method (single or multiple cropping). A more detailed analysis of the Page 3/25 Page 3/25 vernacular names used by farmers made it possible to describe the naming process and identify the main categories of cowpea names. The farmers’ responses regarding the sowing and harvest dates allowed to propose a classification system according to the phenology of the cowpea varieties. The association between the variety types and the regions was checked using a chi-square test. The maps showing the village locations and the spatial distribution of the accessions were compiled using the software R (version 3.6.0 for Windows). The software packages Stats and FactoMinR were used for exploratory statistical analyses and to test the hypothesis. The farmers’ responses regarding the sowing and harvest dates allowed to propose a classification system according to the phenology of the cowpea varieties. The association between the variety types and the regions was checked using a chi-square test. The maps showing the village locations and the spatial distribution of the accessions were compiled using the software R (version 3.6.0 for Windows). The software packages Stats and FactoMinR were used for exploratory statistical analyses and to test the hypothesis. Table 2: Characteristics of the farmers interviewed in each region he average age of interviewees was 48 years, with no significant difference between the regions, ex Socio-cultural and demographic characteristics of the interviewees The panel of interviewees comprised 156 women and 179 men, for a total of 335 people. In the different regions, on average about ten farmers were randomly selected per village – except in the Kédougou Region, where it was only possible to interview four farmers per village (Table 1). Table 1: Number of villages and farmers surveyed per region Table 1: Number of villages and farmers surveyed per region The average age of interviewees was 48 years, with no significant difference between the regions, except in the Sédhiou region, where the average age was lower than elsewhere (37 years). Among those interviewed, 50.8% spoke Wolof, which is the language mainly spoken in the regions of Thiès, Louga, Diourbel and Saint-Louis. The Serer, which represented 17.9% of the interviewees, are found in the Fatick, Thiès and Diourbel regions. Lastly, the Toucouleur (10.5%) and Moors (3.3%) occupy the Louga and Saint-Louis regions, while the Mandinka, Jola, Bainuk, Bedick and Manjak live in the Fatick, Kédougou and Sédhiou regions (Table 2). age age was lower than elsewhere (37 years). Among those interviewed, 50.8% spoke Wolof, which of interviewees was 48 years, with no significant difference between the regions, except in the Sédh Table 1: Number of villages and farmers surveyed per region Regions Number of villages Average number of farmers Total number of farmers Diourbel 6 9.8 59 Fatick 5 9.6 48 Kédougou 5 3.6 18 Louga 6 9.7 58 Saint-Louis 6 10.3 62 Sédhiou 5 10.4 52 Thiès 4 9.5 38 Total 37 9 335 The average age of interviewees was 48 years, with no significant difference between the regions, except in the Sédhiou region, where the average age was lower than elsewhere (37 years). Among those interviewed, 50.8% spoke Wolof, which is the language mainly spoken in the regions of Thiès, Louga, Diourbel and Saint-Louis. The Serer, which represented 17.9% of the interviewees, are found in the Fatick, Thiès and Diourbel regions. Lastly, the Toucouleur (10.5%) and Moors (3.3%) occupy the Louga and Saint-Louis regions, while the Mandinka, Jola, Bainuk, Bedick and Manjak live in the Fatick, Kédougou and Sédhiou regions (Table 2). Page 4/25 Page 4/25 Page 4/25 Regions Regions Total % Variables Modalities Th Lg Dl Fk Sd Kg SL Age <25 2 1 0 0 2 1 3 9 2.68 25-50 20 30 19 17 43 8 25 162 48.36 50-75 15 26 36 28 7 9 32 153 45.67 ≥75 1 1 4 2 0 0 2 10 2.98 NA 0 0 0 1 0 0 0 1 0.3 Total 38 58 59 48 52 18 62 335 100 Wolof 22 35 40 9 31 0 33 170 50,75 Ethnic group Mandinka 0 0 0 3 7 10 0 20 5.97 Moor 0 3 0 0 0 0 8 11 3.28 Fulani 0 10 4 2 0 6 1 23 6.86 Serer 15 1 13 31 0 0 0 60 17.91 Toucouleur 1 9 2 3 0 0 20 35 10.45 Other 0 0 0 0 14 2 0 16 4.77 Total 38 58 59 48 52 18 62 335 100 Th=Thiès; Lg=Louga; Dl=Diourbel; Fk=Fatick; Sd=Sédhiou; Kg=Kédougou; SL=Saint-Louis; NA=data not provided Th=Thiès; Lg=Louga; Dl=Diourbel; Fk=Fatick; Sd=Sédhiou; Kg=Kédougou; SL=Saint-Louis; NA=data not provided Cowpea cropping systems Twenty-four (24) different species grown with cowpea were identified in the seven regions studied. The most frequently cited species grown with cowpea were groundnut and millet, which on average are grown, respectively, by 85% and 71% of the farmers interviewed (Table 3). However, the proportion of farmers that grow groundnut or millet varies depending on the region. While 98% of farmers grow groundnut in Diourbel, the figure is only 43% in Saint-Louis. This variation is also observed for millet, which is common in Diourbel and Louga, but more unusual in Saint-Louis. Other crops are far less common than these two species, such as guinea sorrel, maize, watermelon, rice and sorghum. Their distribution also varies depending on the region. The least common species grown with cowpea (only 0.3% of farmers interviewed) are calabash (Kédougou), cucumber and melon (Saint-Louis), turnip (Thiès) and Bambara groundnut (Diourbel). Table 3: Different species grown and their percentage in each region Page 5/25 Page 5/25 Page 5/25 Species Latin name Diourbel Fatick Kédougou Louga Saint-Louis Sédhiou Thiès Total Bambara nut Vigna subterranea 1.7 0 0 0 0 0 0 0.3 Calabash Lagenaria siceraria 0 0 5.6 0 0 0 0 0.3 Cassava Manihot esculenta 0 0 27.8 0 8.1 13.5 0 5.1 Chili Capsicum annuum 0 0 0 1.7 4.8 3.8 0 1.8 Cotton Gossypium spp. 0 0 33.3 0 0 0 0 1.8 Cowpea Vigna unguiculata 100 95.8 94.4 100 98.4 100 100 98.8 Cucumber Cucumis sativus 0 0 0 0 1.6 0 0 0.3 Eggplant Solanum melongena 1.7 0 33.3 0 3.2 0 5.3 3.3 Fonio Digitaria exilis 0 0 33.3 0 0 0 0 1.8 Groundnut Arachis hypogaea 98.3 95.8 100 94.8 43.5 84.6 94.7 84.8 Maize Zea mays 13.6 29.2 94.4 5.2 22.6 55.8 2.6 25.7 Melon Cucumis melo 0 0 0 0 1.6 0 0 0.3 Okra Abelmoschus esculentus 5.1 0 66.7 0 6.5 0 2.6 6 Onion Allium cepa 0 0 5.6 0 22.6 0 5.3 5.1 Pearl millet Pennisetum glaucum 100 79.2 44.4 87.9 24.2 69.2 84.2 71.3 Pumpkin Cucurbita spp. Cowpea cropping systems 5.1 0 33.3 0 6.5 0 0 3.9 Red sorrel Hibiscus sabdariffa 47.5 35.4 38.9 32.8 35.5 0 21.1 30.1 Rice Oryza glaberrima 0 16.7 77.8 0 12.9 23.1 0 12.5 Sesame Sesamum indicum 0 0 0 0 0 28.8 0 4.5 Sorghum Sorghum bicolor 18.6 12.5 55.6 17.2 1.6 0 2.6 11.6 Sweet potato Ipomoea batatas 0 0 0 0 12.9 7.7 0 3.6 Tomato Solanum lycopersicum 0 0 5.6 0 6.5 0 0 1.5 Turnip Brassica rapa 0 0 0 0 0 0 2.6 0.3 Watermelon Citrullus lanatus 3.4 8.3 0 20.7 62.9 5.8 10.5 19.1 Other 0 0 0 0 3.2 0 0 0.6 The correspondence analysis shows that the regions of Thiès, Sédhiou, Louga, Fatick and Diourbel have similar cropping profiles: red sorrel, sesame and sorghum, in addition to cowpea, groundnut and pearl millet. The Saint-Louis region differs, with watermelon (grown by 62.9 % of interviewees) and onion (22.6%), melon, cucumber and tomato, whereas the Kédougou region is characterized by fonio, pearl millet and cotton, rarely grown elsewhere (Figure 2). The three main species (cowpea, groundnut and pearl millet) are not randomly distributed between the regions. However, the disparity only concerns Saint-Louis, where quite a high proportion of farmers grow cowpea compared to what was expected randomly (residual > 3), although this proportion is low for millet (residual > 2.5, X-squared = 26.949, df = 12, p-value = 0.008). The number of species cultivated is structured according to the regions (Figure 3) and varies between three and nine species per farmer in the Thiès and Kédougou regions, respectively. On average, more than 4 species are grown per farmer in the regions of Kédougou, Saint- Louis and Sédhiou, whereas the number is between 3.5 and 4 per farmer in the regions of Louga, Diourbel and Fatick. Thiès is the region where the average number of species per farmer is the lowest (equal to 3.5) (Table 4). Table 5: Frequency, mean citation rank, Smith’s index, Sutrop index and B.score for species grown with cowpea Cowpea cropping systems Table 4: Number of species grown per region and the average ratio per farmer Table 4: Number of species grown per region and the average ratio per farmer Page 6/25 Page 6/25 Regions Number of species/farmers Number of farmers Average number of species/farmer Diourbel 233 59 3.95 Fatick 182 48 3.79 Kédougou 155 18 8.61 Louga 210 58 3.62 Saint-Louis 254 62 4.10 Sédhiou 208 52 4.00 Thiès 132 38 3.47 Using the free listing method, we established the frequency, Smith’s S index and average salience for each of the species. Groundnut and millet were the species cited the most often with cowpea. The Smith’s index was higher for these three species, with 0.682 for groundnut, 0.612 for cowpea and 0.559 for millet (Table 5). Cowpea is the third most important species in the zones visited, with a citation rank of 2.5, after groundnut (1.9) and millet (2). As expected [22], the citation rank obtained in the species free list is correlated with species frequency in a non-linear way (Figure 4). Table 5: Frequency, mean citation rank, Smith’s index, Sutrop index and B.score for species grown with cowpea Page 7/25 Cited Items N Frequency Mean rank Smith’s index Sutrop index B. score Cowpea 331 0.988 2.532 0.6122 0.3902 0.7209 Groundnut 290 0.866 1.872 0.6825 0.4624 0.7384 Pearl millet 245 0.731 2.012 0.5578 0.3635 0.6122 Red sorrel 102 0.304 4.039 0.1087 0.0754 0.1743 Maize 89 0.266 3.292 0.1487 0.0807 0.1902 Watermelon 59 0.176 3.153 0.0893 0.0559 0.1148 Rice 46 0.137 3.565 0.0788 0.0385 0.0998 Sorghum 42 0.125 4.024 0.0557 0.0312 0.0804 Okra 22 0.066 5.909 0.0223 0.0111 0.0388 Onion 20 0.06 4.6 0.0265 0.013 0.0382 Cassava 18 0.054 5.111 0.0243 0.0105 0.035 Sesame 16 0.048 4.438 0.0189 0.0108 0.0286 Pumpkin 14 0.042 4.571 0.018 0.0091 0.0255 Cotton 13 0.039 5.846 0.0176 0.0066 0.0253 Sweet potato 12 0.036 3.083 0.0248 0.0116 0.0278 Aubergine 11 0.033 6.091 0.0127 0.0054 0.0198 Fonio 8 0.024 6 0.0099 0.004 0.0145 Tomato 6 0.018 6 0.0055 0.003 0.0092 Pepper 6 0.018 5.833 0.0044 0.0031 0.0084 Other 3 0.009 4.333 0.0035 0.0021 0.0041 Melon 2 0.006 4.5 0.0036 0.0013 0.0032 Calabash 1 0.003 5 0.0019 6.00E-04 9.00E-04 Pea 1 0.003 7 0.0014 4.00E-04 6.00E-04 Cucumber 1 0.003 6 0.0013 5.00E-04 6.00E-04 B groundnut 1 0.003 5 6.00E-04 6.00E-04 0 Salad 1 0.003 6 5.00E-04 5.00E-04 0 Turnip 1 0.003 8 4.00E-04 4.00E-04 0 Collection and local nomenclature of cowpea varieties During the survey, 702 cowpea accessions were collected in Thiès (84), Louga (155), Diourbel (158), Fatick (85), Saint-Louis (122), During the survey, 702 cowpea accessions were collected in Thiès (84), Louga (155), Diourbel (158), Fatick (85), Saint-Louis (122), Kédougou (19) and Sédhiou (79) [27]. One to seven accessions were collected per farmer, with an average of two accessions per farmer These accessions were identified under 59 different local names. The informal interviews with farmers showed that, irrespective of their ethnic group, farmers translated "niébé", the French word for cowpea, into the local language to name the species Vigna unguiculata var unguiculata. In this way, the terms "Niébé" or "Seupe" are used by the Wolof and Halpulaar (Fula and Toucouleur), "Sosso" is used by the Mandinka, "Niao" by the Serer, "Deulleugane" by the Moors and "Oufithion" by the Manjak. A wide range of reasons is used by the farmers to identify their cowpea varieties. Indeed, 75% of names make reference to morphology (seed color and size or vegetative cycle), 14% are named after a person (the person who brought the variety to the village, a woman’s Page 8/25 Page 8/25 name if the variety is productive, etc.), 1% refer to the geographic origin (the zone they came from). Lastly, 9% have names that refer to a specific event (details not provided here) or are arbitrary (Table 6). Table 6: Percentage of name categories for cowpea Region Morphology % Person’s name % Zone of origin % Other % Total % Color Vegetative Cycle Color/Size Thiès 61 5 1 30 3 100 Louga 46 3 6 21 2 22 Diourbel 73 4 16 7 Fatick 62 9 22 7 Sédhiou 96 3 1 Kédougou 95 5 Saint-Louis 47 12 4 10 5 22 % Average 68.57 3.28 3.43 14.28 1 9.43 100 Most of the time, the names of varieties are composed of a generic name for cowpea in the local language plus a second term, which either refers to simple morphological characteristics (seed color), people’s names or zone of origin. Among the Mandinka, for example, cowpea is known by the generic name “Sosso”. In order to identify red cowpea, farmers add the suffix “wouléroung” (red) to the name “Sosso” . In all the regions visited, the names generally referred to morphology, particularly seed color (for example, “niebe bou wekh” or white cowpea). Collection and local nomenclature of cowpea varieties Sometimes seed size is added (for example, “niebe bou wekh bou didji” or white cowpea with large seeds). Some cowpea names are associated with the seeds’ geographic origin (Fouta cowpea) or a person (Baye Ngagne, Mame Fama, Marame Penda). In Senegal, the GOANA agriculture programme, launched in 2008 by the former President of the Republic, Abdoulaye Wade, coincided with the introduction of a cowpea variety that is now called after the programme. The Goana variety is sometimes called "pea" (because the shape of the seed is quite round or full) or "nenou naat", which means "guinea fowl’s egg", in reference to the marks on the seed’s integument (Table 7). After standardising the spelling and identifying the synonyms, 36 names of varieties were kept. Irrespective of the ethnic group, the cowpea varieties called white cowpea (26% of all the varieties in the collection), red cowpea (25%), black cowpea (15%) and Baye Ngagne (9%) are the most commonly grown in Senegal. Collection and local nomenclature of cowpea varieties Cowpea production is more diversified in the regions of Diourbel and Louga, followed by Thiès, Saint-Louis and Sédhiou, respectively (Figure 5). The average number of cowpea varieties per farmer varies between 1 (Kédougou) and 3 (Diourbel and Louga) (Table 8). The Diourbel and Louga regions are also where there is greater linguistic diversity among interviewed farmers. Therefore, the possible link between cowpea diversity and the farmers’ cultural diversity cannot be ruled out. Table 8: Number of varieties per farmer for each region Regions No. farmers No. var/region No. var/farmer Diourbel 59 158 2.678 Fatick 48 85 1.771 Kédougou 18 19 1.056 Louga 58 155 2.672 Saint-Louis 62 122 1.968 Sédhiou 52 79 1.519 Thiès 38 84 2.211 Cropping systems and acquiring seeds Cropping systems The majority of the farmers interviewed grow cowpea as a single crop (65%). This method of cultivating cowpea is far more frequent in four regions in Senegal, namely, Louga, Diourbel, Kédougou and Saint-Louis. Groundnut is the species most commonly associated with cowpea. This association was described for 28% of the farmers surveyed, especially in the regions of Thiès, Fatick, Sédhiou and Diourbel. Cowpea is also associated with maize (3%) in the Saint-Louis region, millet (0.3%) in the Kédougou region and market gardening (0.85%) Regions No. farmers No. var/region No. var/farmer Diourbel 59 158 2.678 Fatick 48 85 1.771 Kédougou 18 19 1.056 Louga 58 155 2.672 Saint-Louis 62 122 1.968 Sédhiou 52 79 1.519 Thiès 38 84 2.211 Regions No. farmers No. var/region No. var/farmer Diourbel 59 158 2.678 Fatick 48 85 1.771 Kédougou 18 19 1.056 Louga 58 155 2.672 Saint-Louis 62 122 1.968 Sédhiou 52 79 1.519 Thiès 38 84 2.211 Regions No. farmers No. var/region No. var/farmer Collection and local nomenclature of cowpea varieties Table 7: Local names, English translation and historical references Page 9/25 Page 9/25 Historical references Historical references Names g Baye Ngagne Baye Ngagne or black cowpea A person’s name Delleugane Labial White cowpea The color of the seed’s integument Delleugane Leukhmare Black cowpea The color of the seed’s integument Fithionouny oufithial White cowpea The color of the seed’s integument Gouana Goana Refer to the agricultural programme GOANA Hectare Hectare The seed’s pleasing appearance Mame Fama Mame Fama A person’s name Marame Penda Marame Penda A person’s name Melakh Melakh = Flash The variety’s early maturing cycle Mosse kham Taste to know The taste Ndao counda Ndao counda A person’s name Ndiaga aw Ndiaga aw A person’s name Ndiaye wekh White Ndiaye The color of the seed’s integument Ndieussiw Ndieussiw The capacity to produce fodder Nenou Naat Guinea fowl’s egg The color of the seed’s integument, which has brown speckles Niao balne Black cowpea The color of the seed’s integument Niao ndane White cowpea The color of the seed’s integument Niebe bou wekh White cowpea The color of the seed’s integument Niebe baledjo Black cowpea The color of the seed’s integument Niebe bodedjo Red cowpea The color of the seed’s integument Niebe bodedjo- baledjo Black-white cowpea The color of the seed’s integument Niebe bou khonk Red cowpea The color of the seed’s integument Niebe bou khonk bou didji Red cowpea with big seeds The seed’s size and color Niebe bou khonk bou sew Red cowpea with small seeds The seed’s size and color Niebe bou nioul Black cowpea The color of the seed’s integument Niebe bou wekh White cowpea The color of the seed’s integument Niebe bou wekh bou didj White cowpea with big seeds The seed’s size and color Niebe bou wekh bou sew White cowpea with small seeds The seed’s size and color Niebe danedjo White cowpea The color of the seed’s integument Niebe fouta Fouta cowpea Originally from Fouta and mainly used for floodplain cultivation Niebe Kell Kell cowpea Niebe Koudioule Niebe Mame Diarra Mame Diarra cowpea A person’s name Niebe poude Greyish cowpea The seed’s faded color Niebe poury Greyish cowpea The seed’s faded color Oufithion otopeul Black cowpea The color of the seed’s integument Oufithion oudjankfan Red cowpea The color of the seed’s integument Pakau Pakau Saneba sosso White cowpea The color of the seed’s integument Seupe bou khonk Red cowpea The color of the seed’s integument Seupe bou wekh White cowpea The color of the seed’s integument Sosso fima Sosso fing Black cowpea The color of the seed’s integument Sosso Khoyo Sosso koyma White cowpea The color of the seed’s integument Sosso meunie White cowpea The color of the seed’s integument Sosso meunie maynama Late white cowpea The seed’s size and color Sosso missew Sosso resse mesengo Sosso wouleroung Pale red cowpea The color of the seed’s integument Tachet Spotted The color of the seed’s integument, which is brown spotted Tamate awo First wives’ tomato The seed’s red color means that less tomato paste is used to prepare rice-based dishes Walette Early The seed’s early maturity Walette bou nioul Black Early The seed’s early maturity and color Walette bou wekh White Early The seed’s early maturity and color Yacine Yacine Yakhoul tamate That wastes no tomatoes The seed’s red color means that less tomato paste is used to prepare dishes The zone in the north and centre of the groundnut producing area has the greatest diversity (Louga and Diourbel), whereas Kédougou has the fewest varieties. Cropping systems The majority of the farmers interviewed grow cowpea as a single crop (65%). This method of cultivating cowpea is far more frequent in four regions in Senegal, namely, Louga, Diourbel, Kédougou and Saint-Louis. Groundnut is the species most commonly associated with cowpea. This association was described for 28% of the farmers surveyed, especially in the regions of Thiès, Fatick, Sédhiou and Diourbel. Cowpea is also associated with maize (3%) in the Saint-Louis region, millet (0.3%) in the Kédougou region and market gardening (0.85%) in the regions of Louga, Saint-Louis and Kédougou (Table 9). Table 9: Cowpea crop associations according to region Page 13/25 Page 13/25 Methods Regions Total Percentage% Th Lg Dl Fk Sd Kg SL Associated with groundnut 42 6 35 49 55 1 9 197 28 Associated with market gardening 0 3 0 0 0 1 2 6 0.85 Associated with maize 0 0 0 0 0 2 20 22 3 Associated with millet 0 0 0 0 0 2 0 2 0.3 Single crop 35 145 117 32 24 13 91 457 65 Single crop associated with groundnut 7 1 6 4 0 0 0 18 2.6 Total 84 155 158 85 79 19 122 702 100 Methods Regions Th=Thiès; Lg=Louga; Dl=Diourbel; Fk=Fatick; Sd=Sédhiou; Kg=Kédougou; SL=Saint-Louis Th=Thiès; Lg=Louga; Dl=Diourbel; Fk=Fatick; Sd=Sédhiou; Kg=Kédougou; SL=Saint-Louis In the regions of Thiès, Fatick, Diourbel, Kédougou and Sédhiou cowpea is grown in the winter season. In general, sowing is in June and July (53.42%) and harvesting is in September and October (93.44%). In the Louga region and part of the Saint-Louis and Diourbel regions, sowing is in August and September (42.60%) and harvesting is in November. Floodplain cultivation of cowpea is only found in the Saint- Louis region (3.99%). For this type of production, sowing is between November and January and harvesting is between February and March. There are three groups of cowpea varieties grown in Senegal that can be distinguished according to their development cycle: early (number of days <70), semi-early (between 70 and 90 days) or late (number of days ≥90). The early varieties represent 81.34% of the varieties grown. They are found in all regions, except Kédougou. Semi-early varieties (3.84%) are grown in Louga and Diourbel. Lastly, late varieties (14.67%) are generally grown in the regions of Kédougou, Thiès and Saint-Louis (Table 10). Cropping systems Table 10: Cropping calendar and cycle of accessions in each region Page 14/25 Features Conditions Regions Total % Chi-Square Th Lg Dl Fk Sd Kg SL Value df P- Value Sowing date June-July 83 34 103 81 63 11 0 375 53.42 455.5a 12 <0.001 Aug-Sept 1 121 55 4 16 8 94 299 42.60 Nov-Jan 0 0 0 0 0 0 28 28 3.99 Total 84 155 158 85 79 19 122 702 100.00 Harvest date Sept-Oct 84 155 158 84 79 10 86 656 93.44 302.6a 18 <0.001 Nov 0 0 0 0 0 8 8 16 2.28 Feb-March 0 0 0 0 0 1 28 29 4.13 DNR 0 0 0 1 0 0 0 1 0.14 Total 84 155 158 85 79 19 122 702 100.00 Cycle <70 45 133 148 81 79 3 82 571 81.34 251.955a 18 <0.001 70-90 0 16 7 2 0 1 1 27 3.84 ≥90 39 6 3 1 0 15 39 103 14.67 DNR 0 0 0 1 0 0 0 1 0.14 Total 84 155 158 85 79 19 122 702 100.00 Features Conditions Regions The p. value of the chi2 test for the sowing dates, harvest dates and length of cycle is below 0.001. The hypothesis of the independence between these variables and the regions has been rejected as a result. Th=Thiès; Lg=Louga; Dl=Diourbel; Fk=Fatick; Sd=Sédhiou; Kg=Kédougou; SL=Saint-Louis; MS=sowing date; MR=harvest date; June-July=June and July; Aug-Sept=August and September; Nov- Jan=November to January; Sept-Oct=September and October; Feb-March=February and March Discussion Drawing on the new collections and the recent surveys, which were more exhaustive than earlier surveys, the aim of this study was to characterize the farming practices associated with growing cowpea in Senegal. It focused particularly on the range of species grown in association with cowpea. The richness and variability of cowpea varieties were established in reference to the farmers’ nomenclature. We also identified where farmers obtained their seeds. Total h=Thiès; Lg=Louga; Dl=Diourbel; Fk=Fatick; Sd=Sédhiou; Kg=Kédougou; SL=Saint-Louis Nature Conditions Conditions Nature Regions How seeds are acquired Most of the interviewees (57%) stated that they obtained their first cowpea seeds at markets or from seed suppliers, NGOs, cooperatives or farmer organizations outside the village. Forty-two percent (42%) obtained them from relatives or neighbours in the village. How seeds are acquired varies depending on the region (Table 11). Eighty-one percent (81%) of interviewees stated that they acquired their first seeds in the last two decades, compared to only 11%, who obtained their seeds more than 25 years ago. More than 6% of interviewees cannot remember the year when they acquired their seeds. The majority (68%) of seeds from the last season were home-grown (Table 11). Table 11: Where seeds were acquired Page 15/25 Page 15/25 Nature Conditions Regions Total % Th Lg Dl Fk Sd Kg SL Place where first acquired Outside the village 43 95 68 60 48 0 85 399 57 Village 41 57 86 25 31 18 37 295 42 DNR 0 0 0 0 0 1 0 1 1 Total 84 155 158 85 79 19 122 702 100 Year when first acquired ˂25 66 122 106 73 79 13 115 574 81 ˃25 4 13 42 9 0 6 6 80 11.4 DNR 14 20 10 3 0 0 0 47 6.7 Total 84 155 158 85 79 19 122 702 100 Home-produced N 27 52 35 21 16 5 62 218 31 O 57 103 123 64 63 14 59 483 68 DNR 0 0 0 0 0 0 1 1 0.14 Total 84 155 158 85 79 19 122 702 100 Th=Thiès; Lg=Louga; Dl=Diourbel; Fk=Fatick; Sd=Sédhiou; Kg=Kédougou; SL=Saint-Louis Cowpea’s area of distribution and varietal richness This study helped confirm the area of distribution of cowpea production in Senegal. In fact, in the regions of Diourbel, Louga, Thiès and Saint-Louis, collecting several varieties from one farmer is common; whereas, in the Sédhiou and Kédougou regions, cowpea is less common and, on average, there is seldom more than one variety per farmer. The cultivation of this legume is more diversified in Diourbel and Louga. This reveals the importance and richness of the species in the central north and north, the main cowpea growing areas in Senegal [31]. The department of Louga, which is in the centre of this region, appears to be the preferred zone for growing cowpea: 21% of cultivated land is used to grow this species [32]. The analysis of diversity based on the local names for cowpea allowed us to identify 6 appellations for the cowpea species. On a varietal level, 59 different names were identified. Varieties whose seeds have the same morphological features may have different names depending on the ethnic group. These names essentially refer to seed color, size or people’s names. Thus, the farmer manages diversity by recognising perceptible characteristics, especially morphological features [33]. By studying the classification processes, we were able to determine the biological diversity of cowpea, as perceived by farmers. The diversity of the local names is an indicator of the plant’s importance in a geographic environment [34]. In Senegal, the fact that the local names that designate cowpea vary depending on locality or ethnic group was reported a long time ago [35]. This observation suggests that there is a close link between farmers’ cultural diversity and varietal diversity. In addition, this important diversity could be explained by the fact that Senegal belongs to the second zone of cowpea domestication [36]. A high level of diversity was also mentioned for fonio, with 52 local names [37], and maize, with 81 local names [38]. In this study, seed color is the most distinctive element and the most often used by farmers for naming varieties. This naming process can cause confusion between traditional and improved varieties because the latter’s names are sometimes constructed in the same way. For example, the improved variety, Yacine, is called “Niebe bou khonk" in Wolof, which means "red cowpea". In Burkina Faso, names are constructed using eye color (in over 35% of cases) and seed size (almost 45%) [39]. Diversity of species grown with cowpea In all the zones surveyed, cowpea producers also grow groundnut and millet. In Senegalese farming systems, these three species are complementary. Along with sorghum, cassava, watermelon and red sorrel, they are the main cash crops grown in the centre and north of the groundnut growing area, which is ideal for growing cowpea. Our findings on the regional distribution of species diversity are similar to those obtained when the FAO conducted inventories of the agricultural species in rural areas [28], in which Eastern Senegal and the Casamance appeared to be priority areas for plant breeding resources and crop biodiversity. This can be explained by the abundant rain in these zones, the diversified traditional farming practices, the ethnic diversity and, lastly, the proximity of the region to neighbouring countries, which favours exchanges. Although the Sédhiou region has as much rain as South-East Senegal (Kédougou), it has less species diversity. The Saint-Louis region is still diversified in terms of cultivated species, despite its rainfall deficit. This region’s Page 16/25 Page 16/25 geographical position offers favourable climatic conditions for farming. The potential in terms of irrigable land, estimated at 172 800 ha, and the abundance of water [29] no doubt contribute to this diversity as well. geographical position offers favourable climatic conditions for farming. The potential in terms of irrigable land, estimated at 172 800 ha, and the abundance of water [29] no doubt contribute to this diversity as well. Cropping system The majority of farmers surveyed grow cowpea as a single crop. This cropping system is found in the regions of Louga, Diourbel and Kédougou. In the groundnut growing area, which includes the regions of Diourbel and Louga, there has been a rainfall deficit for decades. However, cowpea is adapted to these conditions. More and more land is being used to grow cowpea. Between 2012-2017, cowpea was grown on 165 452 ha, on average. This increased to 257 219 ha in 2019 [30]. In these zones, where the harvest is destined for sale, cowpea is grown in huge fields. In contrast, in other regions, cowpea is considered as a subsistence crop and is associated with other crops, such as groundnut, maize, millet or even market gardening. Polyculture is practiced by farmers who do not have large areas of cultivable land. This association with other crops is used as a strategy to reduce the risks of production loss due to climatic hazards. In the regions of Thiès and Louga, young people grow cowpea, which could help reduce immigration. In fact, in this part of the country, the legume is grown as a cash crop on large areas of land. In the Sédhiou region, young people also grow cowpea, although it is often neglected in favour of other crops. This could be explained by the fact that varieties from other crops are better adapted to the groundnut producing zone, such as the Sédhiou region. In Sédhiou, cowpea is traditionally valorized by women. In the regions of Diourbel, Fatick and Saint-Louis, cowpea is grown by aged farmers, who probably know more about traditional accessions and their cropping practices. Cowpea’s area of distribution and varietal richness According to Ouedraogo et al., color and texture are only used for less than 10%. However, our findings, which are in line with the studies by Dabat et al. (2012) in Burkina Faso, show that white varieties appear to be more valued because the majority of seeds used by farmers are white. Page 17/25 Cowpea is mainly grown during the winter season in all the zones surveyed, except the Saint-Louis region, where cowpea is also grown on the floodplain. Three groups (early, intermediary and late) were identified according to the varieties’ development cycle. According to Kouakou et al. (2007), on a local level, cowpea diversity is generally due to its phenological adaptability to environmental constraints. The abundance of early maturing accessions may be due to the adoption of improved varieties that are early. Late varieties are no longer grown in the main cowpea producing areas because rainfall has been irregular or insufficient for four decades. This may also explain the high number of early varieties. The earliest varieties were collected in Sédhiou, which has the longest rainy season. However, in this region, very small areas were cultivated for home-consumption. The variability of rainfall in the different regions could explain the phenological diversity observed. In fact, more late accessions are grown in the Kédougou region, where rainfall is higher, and in the Saint- Louis region, where floodplain cropping plays an important role. These types of varieties are valuable because they are dual purpose and Page 17/25 Page 17/25 can be used as seed and fodder. In fact, under favourable conditions, they produce a large amount of seeds and fodder [3]. The late varieties found in the regions of Thiès, Kédougou and Saint-Louis could constitute an important pool for local and traditional varieties. In the cereal growing region of Thiès (where 47.2% of land is cultivated with maturing cowpea) [40], late maturing cowpea has a positive effect on cereal yields in the crop rotation because it produces huge quantities of biomass [41]. can be used as seed and fodder. In fact, under favourable conditions, they produce a large amount of seeds and fodder [3]. The late varieties found in the regions of Thiès, Kédougou and Saint-Louis could constitute an important pool for local and traditional varieties. Cowpea’s area of distribution and varietal richness In the cereal growing region of Thiès (where 47.2% of land is cultivated with maturing cowpea) [40], late maturing cowpea has a positive effect on cereal yields in the crop rotation because it produces huge quantities of biomass [41]. Acknowledgements We thank our interviewees warmly for their time and goodwill, Kodjo Mawuena Gbedevi, Ndeye Yacine Gueye and Mamadou Fall for their help on the surveys, the staff of “Direction Régional du Développement Rural (DRDR)” who guided and assisted us during the surveys and collection of the plant materials, and the three anonymous reviewers for their valuable comments. Conclusion Identifying the nomenclature for the local cowpea varieties and their seed management system is essential for optimising local diversity. This study revealed the considerable diversity of local names. This diversity is an indicator of the importance of cowpea in Senegalese farming systems. The names primarily refer to the seed morphology or color, a feature that facilitates identification. The named diversity of cowpea is greater in regions where the crop systems are less diversified. In the studied area, more than half the cowpea seeds grown by farmers are obtained from markets, NGOs, agricultural services and projects and then farmers produce and conserve their own seeds. Cowpea is generally grown as a single crop or associated with groundnut or maize. The length of the growing cycle is rarely used by farmers to identify their varieties. However, we classified varieties in terms of development cycles because of the difference observed between sowing and harvesting dates. This study made it possible to characterize the diversity of cowpea grown in Senegal. Undoubtedly, the diversity of farming practices and cowpea cropping systems is closely linked to the diversity of the biological types grown in the country and vice versa. Undoubtedly, the diversity of farming practices and cowpea cropping systems is closely linked to the diversity of the biological types grown in the country and vice versa. The seed supply In the last two decades, most of the seeds in the farmers’ possession were purchased at the market or obtained from agricultural services, NGOs, farmers’ organisations and cooperatives. These types of structure are common to several villages. Consequently, the same variety can be found in different villages or regions, even if it has different names. Thus, the pleasing appearance of seeds of one cowpea variety can encourage people to buy it at a market, even if they are unaware of its germination performance and agricultural value. Many of the people surveyed obtained their first seeds in the village, either through donations or by trading with relatives, friends or neighbours. Similarly, married women obtain their first seeds from their husband or parents-in-law, along with plots of land, after leaving their place of birth to go to their husband’s place of residence. Thus, women rarely take seeds from their home or continue to obtain seeds from their relatives, especially if they live in different villages. The majority of seeds from the season preceding this study were home-grown. In fact, farmers keep a share of their previous harvest for seed. Consequently, farmers only purchase or obtain seeds at the market or from relatives or neighbours the year after a poor harvest or a food shortage. Authors’ Contributions Awa Sarr, Amy Bodian and Mame Codou Gueye carried out the surveys and collected the plant material; Awa Sarr, Christian Leclerc, Ghislain Kanfani and Cyril Diatta analyzed the data; Awa Sarr, Christian Leclerc and Amy Bodian drafted the paper; Awa Sarr, Amy BODIAN, Mame Codou Gueye, Badara Gueye, Ghislain Kanfani, Cyril Diatta, Ali Lardia Bougma, Elisabeth A.M.C. Diop, Ndiaga Cissé, Diaga Diouf, Christian Leclerc edited and provided critical review of the manuscript. All authors read and approved the final manuscript. Funding Funding d like to thank the West African Agricultural Productivity Program (WAAPP) for the financial suppor he authors would like to thank the West African Agricultural Productivity Program (WAAPP) for the Availability of data and materials Availability of data and materials Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. References Ehlers JD, Hall AE. Cowpea (Vigna unguiculata L. Walp.). Field Crops Research. 1997;53:187–204. 2. Pungulani LL, Millner JP, Williams WM, Banda M. Improvement of leaf wilting scoring system in cowpea ('Vigna unguiculata’(L) Walp.): From qualitative scale to quantitative index. Australian Journal of Crop Science. 2013;7:1262. 3. Diouf D. Recent advances in cowpea [Vigna unguiculata (L.) Walp.] omics research for genetic improvement. African Journal of Biotechnology. 2011;10:2803–19. 4. Hall AE, Cisse N, Thiaw S, Elawad HOA, Ehlers JD, Ismail AM, et al. 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New York: D Jarvis, C Padoch, and D Cooper; 2007 [cited 2021 Jan 11]. p. 34–76. 26. Sadiki M, Jarvis DI, Rijal D, Bajracharya J, Hue N, Camacho-Villa T, et al. Variety names: An entry point to crop genetic diversity and distribution in agroecosystems? Managing Biodiversity in Agricultural Ecosystems [Internet]. D Jarvis, C Padoch, and D Cooper. New York: D Jarvis, C Padoch, and D Cooper; 2007 [cited 2021 Jan 11]. p. 34–76. 27. Sarr A. References Genetic diversity in cowpea [Vigna unguiculata (L.) Walp.] as revealed by RAPD markers. Genetic Resources and Crop Evolution. 2004;51:539–50. 36. Ba FS, Pasquet RS, Gepts P. Genetic diversity in cowpea [Vigna unguiculata (L.) Walp.] as revealed by RAPD markers. Genetic Resources and Crop Evolution. 2004;51:539–50. 37. Diop BM, Gueye MC, Agbangba CE, Cisse N, Deu M, Diack O, et al. Fonio (Digitaria exilis (Kippist) Stapf): A Socially Embedded Cereal for Food and Nutrition Security in Senegal. Ethnobiology Letters. 2018;9:150. 37. Diop BM, Gueye MC, Agbangba CE, Cisse N, Deu M, Diack O, et al. Fonio (Digitaria exilis (Kippist) Stapf): A Socially Embedded Cereal for Food and Nutrition Security in Senegal. Ethnobiology Letters. 2018;9:150. 38. N’da HA, Akanvou L, Zoro AIB. Prospection, Collecte, Nomenclature Paysanne Et Caractérisation Des Variables Qualitatives Des Variétés Locales De Maïs (Zea Mays L.) Cultivées En Côte d’Ivoire. European Scientific Journal. 2016;12:298–315. 38. N’da HA, Akanvou L, Zoro AIB. Prospection, Collecte, Nomenclature Paysanne Et Caractérisation Des Variables Qualitatives Des Variétés Locales De Maïs (Zea Mays L.) Cultivées En Côte d’Ivoire. European Scientific Journal. 2016;12:298–315. 39. Ouedraogo J, Sawadago M, Tignegre J-B, Drabo I, Balma D. Caractérisation agro-morphologique et moléculaire de cultivars locaux de niébé (Vigna unguiculata) du Burkina Faso. Cameroon Journal of Experimental Biology [Internet]. 2010 Jul 21 [cited 2019 Jul 22]; Available from: http://www.ajol.info/index.php/cajeb/article/view/56878 39. Ouedraogo J, Sawadago M, Tignegre J-B, Drabo I, Balma D. Caractérisation agro-morphologique et moléculaire de cultivars locaux de niébé (Vigna unguiculata) du Burkina Faso. Cameroon Journal of Experimental Biology [Internet]. 2010 Jul 21 [cited 2019 Jul 22]; Available from: http://www.ajol.info/index.php/cajeb/article/view/56878 40. Dabat M-H, Lahmar R, Guissou R. La culture du niébé au Burkina Faso : une voie d’adaptation de la petite agriculture à son environnement ? Autrepart. 2012;N° 62:95–114. 40. Dabat M-H, Lahmar R, Guissou R. La culture du niébé au Burkina Faso : une voie d’adaptation de la petite agriculture à son environnement ? Autrepart. 2012;N° 62:95–114. 41. ANSD/SRSD Thiès. Situation économique et sociale régionale 2013 [Internet]. http://www.ansd.sn/ressources/ses/SES-Thies- 2013.pdf. 2013 [cited 2019 Nov 27]. 41. ANSD/SRSD Thiès. Situation économique et sociale régionale 2013 [Internet]. http://www.ansd.sn/ressources/ses/SES-Thies- 2013.pdf. 2013 [cited 2019 Nov 27]. 42. Affokpon A, Djénontin PAJ, Zoffoun GA, Allagbé MC, Akondé TP, Aïhou K, et al. Effets des variétés de niébé à buts multiples comme précédent cultural sur le rendement du maïs cultivé sur terres de barre dégradées au Sud-Bénin. Bulletin de recherche agronomique du Bénin. References Caractérisation de la diversité du niébé (Vigna unguiculata) cultivé et des sauvages apparentés du Sénégal [Thèse de doctorat unique]. [Dakar]: Cheikh Anta Diop; 2020. 27. Sarr A. Caractérisation de la diversité du niébé (Vigna unguiculata) cultivé et des sauvages apparentés du Sénégal [Thèse de doctorat unique]. [Dakar]: Cheikh Anta Diop; 2020. 28. FAO-WIEWS. État des ressources phytogénétiques pour l’alimentation et l’agriculture dans le monde [Internet]. Sénégal: ISRA; 2008. Report No.: 2. Available from: http://www.fao.org/pgrfa-gpa-archive/sen/docs/senegal2.pdf 28. FAO-WIEWS. État des ressources phytogénétiques pour l’alimentation et l’agriculture dans le monde [Internet]. Sénégal: ISRA; 2008. Report No.: 2. Available from: http://www.fao.org/pgrfa-gpa-archive/sen/docs/senegal2.pdf 29. ANSD/SRSD Saint-Louis. Situation économique et sociale régionale 2015 [Internet]. AGENCE NATIONALE DE LA STATISTIQUE ET DE LA DEMOGRAPHIE. 2015 [cited 2019 Nov 27]. Available from: http://www.ansd.sn/ressources/ses/chapitres/8-agriculture- sl2013.pdf 29. ANSD/SRSD Saint-Louis. Situation économique et sociale régionale 2015 [Internet]. AGENCE NATIONALE DE LA STATISTIQUE ET DE LA DEMOGRAPHIE. 2015 [cited 2019 Nov 27]. Available from: http://www.ansd.sn/ressources/ses/chapitres/8-agriculture- sl2013.pdf 30. ANSD. 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Available from: http://www.ansd.sn/ressources/ses/chapitres/10-agriculture-Louga2013.pdf 32. ANSD/SRSD Louga. 10-agriculture-Louga2013.pdf [Internet]. http://www.ansd.sn/ressources/ses/SES-Louga-2013.pdf. 2013 [cited 2019 Oct 3]. Available from: http://www.ansd.sn/ressources/ses/chapitres/10-agriculture-Louga2013.pdf 33. Boster JS. Selection for perceptual distinctiveness: Evidence from Aguaruna cultivars ofManihot esculenta. Economic Botany. 1985;39:310–25. 34. Blench RM. Vernacular names for African millets and other minor cereals and their significance for agricultural history. Archaeological and Anthropological Sciences. 2016;8:1–8. 34. Blench RM. Vernacular names for African millets and other minor cereals and their significance for agricultural history. Archaeological and Anthropological Sciences. 2016;8:1–8. 35. Badiane FA, Gowda BS, Cissé N, Diouf D, Sadio O, Timko MP. Genetic relationship of cowpea (Vigna unguiculata) varieties from Senegal based on SSR markers. Genetics and Molecular Research. 2012;11:292–304. 35. Badiane FA, Gowda BS, Cissé N, Diouf D, Sadio O, Timko MP. Genetic relationship of cowpea (Vigna unguiculata) varieties from Senegal based on SSR markers. Genetics and Molecular Research. 2012;11:292–304. 36. Ba FS, Pasquet RS, Gepts P. References Numéro spécial Fertilité du maïs – Janvier 2013. Bénin; 2013;58–68. 42. Affokpon A, Djénontin PAJ, Zoffoun GA, Allagbé MC, Akondé TP, Aïhou K, et al. Effets des variétés de niébé à buts multiples comme précédent cultural sur le rendement du maïs cultivé sur terres de barre dégradées au Sud-Bénin. Bulletin de recherche agronomique du Bénin. Numéro spécial Fertilité du maïs – Janvier 2013. Bénin; 2013;58–68. Page 20/25 Page 20/25 Figures Figure 1 Location of villages surveyed. The size of the dots is proportional to the number of people surveyed Figure 5 Average number of cowpea varieties by region Figure 1 Figure 1 Location of villages surveyed. The size of the dots is proportional to the number of people surveyed Location of villages surveyed. The size of the dots is proportional to the number of people surveyed Location of villages surveyed. The size of the dots is proportional to the number of people surveyed Page 21/25 Page 21/25 Figure 2 Factor map of correspondence analysis. Association of species as a function of regions surveyed Figure 2 Factor map of correspondence analysis. Association of species as a function of regions surveyed Factor map of correspondence analysis. Association of species as a function of regions surveyed Page 22/25 Figure 3 Average number of species grown per region Figure 3 Figure 3 Average number of species grown per region Average number of species grown per region Page 23/25 Page 23/25 Figure 4 Average citation rank as a function of frequency of citation Figure 4 Average citation rank as a function of frequency of citation Page 24/25 Figure 5 Figure 5 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile1.csv Additionalfile2.csv Additionalfile3.csv Page 25/25
https://openalex.org/W4242381075	https://figshare.com/articles/thesis/Triply_Coupled_Bending-Bending-Torsion_Vibrational_Analysis_of_Rotating_Beams_Using_Fem_and_Dynamic_Finite_Element_Formulation/14651571/1/files/28133295.pdf	English	null	Triply Coupled Bending-Bending-Torsion Vibrational Analysis of Rotating Beams Using Fem and Dynamic Finite Element Formulation	null	2,021	cc-by	31,011	By PROPERTY OF fTÆRSQ[)l UiWVSnSlTY UBRARV PROPERTY OF fTÆRSQ[)l UiWVSnSlTY UBRARV UMI Number: EC53468 UMI UMI Microform EC53468 Copyright2009 by ProQuest LLC All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 UMI INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Author’s Declaration: I hereby declare that I am the sole author of this thesis. I authorize Ryerson University to lend this thesis to other institutions or individuals for the purpose of scholarly research. I further authorize Ryerson University to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. 11 ABSTRACT This research presents the numerical analysis of the triply coupled flap-wise, cord-wise and torsional vibrations of flexible rotating blades. Euler-Bemoulli bending and St. Venant torsion beam theories are considered to derive the governing differential equations of motion. Based on Finite Element Methodology (FEM), the cubic “Hermite” shape functions are implemented where the solution of the equations results in a linear eigenproblem. Then, the Dynamic (frequency dependent) Trigonometric Shape Functions (DTSF’s) for beam’s uncoupled displacements are derived. The application of the Dynamic Finite Element (DFE) approach to the solution of the governing equations is then presented. The DFE formulation, based on the weighted residual method and the DTSF’s, results in a nonlinear eigenproblem representing eigenvalues and eigenmodes of the system. The applicability of the DFE method is then demonstrated by illustrative examples, where a Wittrick-Williams root counting technique is used to find the system’s natural fi'equencies. The DFE approach, an intermediate method between FEM and “Exact” formulation, is characterized by higher convergence rates, and can be advantageously used when multiple natural frequencies and/or higher modes of beam-like structures are to be evaluated. IV Borrower’s page Ryerson University requires the signatures of all persons using or photocopying this thesis. Please sign below, and give address and date. I ll Dedicated to my life.... Dedicated to my life.... My wife, Famaz VI VI Acknowledgements I wish to express my gratitude to my supervisor Dr. Seyed M. Hashemi for his significant guidance and valuable advice during my graduate studies at Ryerson University. Dr. Hashemi has helped me to have the right direction in my research and to get a better understanding about numerical analysis of mechanical systems in general and vibration analysis in particular. I also would like to acknowledge the scholarship awarded by Ryerson University and the support provided by Natural Science and Engineering Research Council of Canada (NSERC). 4.5.2.1. Non-rotating Beam ............................................................................................. 35 4.5.2.2. Axially Loaded Beam ....................................................................................... 38 4.5.2.3. Non-rotating Pre-T\visted Beam .................................................................... 41 4.5.3. Coupled Bending-Bending Vibrations ....................................................................... 42 4.5.4. Coupled Bending-Bending-Torsion Vibrations ......................................................... 49 4.5.4.1. Vibrations of Non-rotating Uncoupled Beam .................................................. 49 4.5.4.2. Vibrations of Non-rotating Coupled Beam ..................................................... 50 4.5.4.3. Vibrations of Rotating Triply Coupled Beam ................................................. 52 g 4.5.3. Coupled Bending-Bending Vibrations ....................................................................... 42 4.5.4. Coupled Bending-Bending-Torsion Vibrations ......................................................... 49 4.5.4.1. Vibrations of Non-rotating Uncoupled Beam .................................................. 49 4.5.4.2. Vibrations of Non-rotating Coupled Beam ..................................................... 50 4.5.4.3. Vibrations of Rotating Triply Coupled Beam ................................................. 52 CHAPTER 5: DYNAMIC FINITE ELEMENT METHOD ...................................................... 56 5.1. Introduction ............................................................................................................................... 56 5.2. Frequency Dependent Approximation Functions ................................................................... 56 5.3. Derivation of System Eigenproblem ........................................................................................ 62 5.4. Refined Dynamic Finite Element (RDFE) ............................................................................... 65 5.5. Numerical results ................................................................................................................... 67 5.5.1. Uncoupled Bending Vibrations of Beam ................................................................... 68 5.5.2. Coupled Bending-Torsion Vibrations .......................................................................... 70 5.5.2.1. Non-rotating Beam ............................................................................................. 70 5.5.2.2. Axially Loaded Coupled Beam ....................................................................... 75 5.5.3. Coupled Bending-Bending Vibrations ....................................................................... 78 5.5.3.1. Axially Loaded Beam ....................................................................................... 79 5.5.3.2. Coupled Bending-Bending Rotating Beam ..................................................... 81 5.5.3.3. Rotating Beam with Variable Cross Section .................................................... 86 5.5.4. Triply Coupled Bending-Bending-Torsion Vibrations ............................................. 87 5.5.4.1. Uncoupled Beam Vibrations .......................................................................... 87 5.5.4.2. Coupled Vibrations of Non-rotating Beam ........................................................ 88 5.5.4.3. Vibrations of Rotating Triply Coupled Beam ................................................. 93 CHAPTER 6: CONCLUSION ...................................................................................................... 99 6.1. Concluding Remarks ............................................................................................................ 99 6.2. Comparison Between DFE and FEM ................................................................................... 101 5.3. Future Work ............................................................................................................................ 103 APPENDIX A: ANALYTICAL SOLUTION FOR BEAMS UNCOUPLED VIBRATIONS ... 104 A. 1. Pure Bending Vibrations of a Clamed-Free Beam ............................................................... 104 A.2. Torsional Vibrations of a Cantilever Beam ......................................................................... 107 APPENDIX B: COMPUTER PROGRAM, ALGORITHMS AND FUNCTIONS ................. 110 B.l. FEM Program and Algorithm ................................................................................................. 110 B.2. DFE Program and Algorithm ................................................................................................. 113 REFERENCES ............................................................................................................................................. 118 VIll Table of Contents: Author’s Declaration ................................................................................................................................. ii Borrower’s Page ....................................................................................................................................... iii Abstract ...................................................................................................................................................... iv Acknowledgements .................................................................................................................................. v Dedication .................................................................................................................................................. vi Tables of Contents ................................................................................................................................... vii List of Tables ............................................................................................................................................. ix List of Illustrations ................................................................................................................................... xi Nomenclature ............................................................................................................................................. xiii CHAPTER 1: INTRODUCTION ...................................................................................................... 1 1.1. Introduction .............................................................................................................................. 1 1.2. Objective ................................................................................................................................. 4 1.3. Thesis Organization ............................................................................................................... 5 CHAPTER 2: EQUATIONS OF MOTION GOVERNING COUPLED VIBRATIONS OF ... 7 ROTATING BEAMS 2.1. Introduction .............................................................................................................................. 7 2.2. Model ...................................................................................................................................... 7 2.3. Assumptions .......................................................................................................................... 8 2.4. Governing Differential Equations of Motion ......................................................................... 8 2.5. Boundary Conditions ............................................................................................................... 10 2.6. Equations of Motion for Dually Coupled Vibrations .............................................................. 11 2.6.1. Coupled Bending-Bending Vibrations ......................................................................... 11 2.6.2. Coupled Bending-Torsion Vibrations ............................................................................ 12 CHAPTER 3: SOLUTION OF EIGENPROBLEMS ................................................................... 14 3.1. Introduction .............................................................................................................................. 14 3.2. Wittrick-Williams Method ...................................................................................................... 15 3.2.1. Theory ............................................................................................................................. 15 3.2.2. Application to the Beam Vibrations ............................................................................ 17 3.3. Solution of the Eigenproblem ................................................................................................ 20 CHAPTER 4: FINITE ELEMENT METHOD .............................................................................. 22 4.1. Introduction ............................................................................................................................... 22 4.2. Galerkin Method ..................................................................................................................... 22 4.3. Approximation Functions ....................................................................................................... 26 4.4. Derivation of System Eigenproblem ....................................................................................... 28 4.5. Numerical results ..................................................................................................................... 32 4.5.1. Pure Bending Vibrations of Uncoupled Beam ............................................................. 32 4.5.2. Coupled Bending-Torsion Vibrations ............................................................................ 34 Author’s Declaration ................................................................................................................................. ii Borrower’s Page ....................................................................................................................................... iii Abstract ...................................................................................................................................................... iv Acknowledgements .................................................................................................................................. v Dedication .................................................................................................................................................. vi Tables of Contents ................................................................................................................................... vii List of Tables ............................................................................................................................................. ix List of Illustrations ................................................................................................................................... xi Nomenclature ............................................................................................................................................. xiii List of Illustrations ................................................................................................................................... xi Nomenclature ............................................................................................................................................. xiii CHAPTER 1: INTRODUCTION ...................................................................................................... 1 1.1. Introduction .............................................................................................................................. 1 1.2. Objective ................................................................................................................................. 4 1.3. Thesis Organization ............................................................................................................... 5 CHAPTER 2: EQUATIONS OF MOTION GOVERNING COUPLED VIBRATIONS OF ... 7 ROTATING BEAMS 2.1. Introduction .............................................................................................................................. 7 2.2. Model ...................................................................................................................................... 7 2.3. Assumptions .......................................................................................................................... 8 2.4. Governing Differential Equations of Motion ......................................................................... 8 2.5. Boundary Conditions ............................................................................................................... 10 2.6. Equations of Motion for Dually Coupled Vibrations .............................................................. 11 2.6.1. Coupled Bending-Bending Vibrations ......................................................................... 11 2.6.2. Coupled Bending-Torsion Vibrations ............................................................................ 12 CHAPTER 3: SOLUTION OF EIGENPROBLEMS ................................................................... 14 3 1 Introduction 14 4.1. Introduction ............................................................................................................................... 22 4.2. Galerkin Method ..................................................................................................................... 22 4.3. Approximation Functions ....................................................................................................... 26 4.4. Derivation of System Eigenproblem ....................................................................................... 28 4.5. Numerical results ..................................................................................................................... 32 4.5.1. Pure Bending Vibrations of Uncoupled Beam ............................................................. 32 4.5.2. Coupled Bending-Torsion Vibrations ............................................................................ Table of Contents: 34 Vll List of Tables: Table 4.1 : The first five natural frequencies of out-of-plane vibration of a cantilever beam 33 Table 4.2: The first six natural frequencies of coupled bending-torsion vibrations of a cantilever 36 beam Table 4.3: The mechanical and geometrical parameters of the three-beam wing 36 Table 4.4: The first five natural frequencies of the three-beam aircraft wing 37 Table 4.5: The bending-torsion natural frequencies of the cantilever beam (no axial load {T = 0)) 40 Table 4.6: The bending-torsion natural frequencies of the axially loaded cantilever beam 41 (r=-1790) Table 4.7: The bending-torsion natural frequencies of the pre-twisted clamped-free beam 42 Table 4.8: The properties of the scaled propeller blade model WADC S-5, with hub radius ej = 6 44 (in) and blade length L = 18 (in) Table 4.9: The natural frequencies (Hz) of the first three modes of vibrations of propeller blade 45 WADC S-5 Table 4.10: The uncoupled bending, bending and torsion natural frequencies of the clamped-free 49 beam Table 4.11: The coupled bending-bending-torsion natural frequencies of the pre-twisted cantilever 51 beam Table 4.12: The bending-bending-torsion natural frequencies of the pre-twisted cantilever rotating 53 blade, 0 = 0 (rpm) Table 4.13: The bending-bending-torsion natural frequencies of the pre-twisted cantilever rotating 53 blade, 0 =360 (rpm) Table 5.1: The first five natural frequencies of out-of-plane vibration of a cantilever beam 69 calculated by DFE Table 5.2: The first six natural frequencies of coupled bending-torsion vibrations of a cantilever 71 beam Table 5.3: The first five natural frequencies of the three-beam aircraft wing obtained by DFE 72 Table 5.4: The bending-torsion coupled natural frequencies of the clamped-free beam 73 Table 5.5: The bending-torsion natural frequencies of the cantilever beam (no axial load (7= 0)) 77 Table 5.6: The bending-torsion natural frequencies of the axially loaded cantilever beam 77 (7=-1790 N) Table 5.7: The uncoupled in- and out-of-plane bending natural frequencies of the cantilever 80 beam IX Table 5.8: The coupled bending-bending natural frequencies of a rotating cantilever beam, 82 ft = 5 (rad/s) Table 5.9: The coupled bending-bending natural frequencies of the rotating beam 84 Table 5.10: The first three natural frequencies (Hz) of vibrations of propeller blade WADC S~5 86 (DFE method) Table 5.11: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted 90 clamped-free beam Table 5.12: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted 94 rotating beam, ft = 360 (rpm) Table 5.13: The triply coupled bending-bending-torsion natural frequencies of the twisted 96 rotating blade , ft =360 (rpm) Table 5.14: The triply coupled bending-bending-torsion natural frequencies of the piecewise 97 uniform twisted rotating blade, ft = 360 (rpm) Table A. Table of Contents: 1 : The weighted and normalized natural frequencies, ctfJL and co„, for a cantilever beam 106 obtained by analytical solution List of Figures: Figure 2.1 : The beam configuration and geometrical parameters 8 Figure 3.1: The determinant of stiffness matrix versus frequency 18 Figure 3.2: The sign count of stiffness matrix versus frequency 19 Figure 3.3 : The variation o f v e r s u s frequency 19 Figure 4.1: The element’s nodal variables 25 Figure 4.2: The transformation from physical coordinate to reference element coordinate 27 Figure 4.3: The first three modes of out-of-plane vibrations of a cantilever beam based on FEM 34 method Figure 4.4: The three-beam airplane wing with coordinate system and geometry 37 Figure 4.5: The FEM convergence results for three-beam wing 38 Figure 4.6: The dually coupled bending-torsion coupled beam cross-sectional geometry 40 Figure 4.7: The first eight modes of the WADC S-5 propeller blade for 12 = 0 46 Figure 4.8: The first eight mode shapes of the WADC S-5 propeller blade for 12 = 1567 rpm 47 Figure 4.9: The first eight mode shapes of the WADC S-5 propeller blade for 12 = 5884 rpm 48 Figure 4.10: The FEM convergence results for non-rotating triply coupled beam 51 Figure 4.11: The effect of rotary speed on frequencies and modes of triply coupled rotating blade 54 Figure 5.1: The beam bending shape functions calculated at its first four natural frequencies 61 Figure 5.2: The first three out-of-plane modes of vibrations for a cantilever beam based on DFE 69 method Figure 5.3: The DFE method convergence results for coupled bending-torsion vibrations 73 Figure 5.4: The comparison between the DFE and FEM convergence for coupled bending-torsion 74 free vibrations XI Figure 5.5: The DFE results for the first five modes of coupled bending-torsion vibrations for a cantilever beam; flap (bending) displacements 74 Figure 5.6: The DFE results for the first five modes of coupled bending-torsion vibrations for a cantilever beam; torsional displacements 75 Figure 5.7: The DFE convergence results for axially loaded cantilever coupled beam (T = -1790N) 78 Figure 5.8: The effect of axial force on natural frequencies of a clamped-free beam bending- bending vibrations 81 Figure 5.9: The DFE convergence results for bending-bending vibrations of a rotating beam 82 Figure 5.10: The effect of rotating speed on natural frequencies for clamped-free beam bending- bending vibrations 83 Figure 5.11: The first eight mode shapes of bending-bending vibrations of a clamped-free rotating beam 85 Figure 5.12: The comparison between the DFE and FEM convergence for non-rotating triply coupled beam 91 Figure 5.13: The first three modes of vibrations of non-rotating triply coupled beam 92 Figure 5.14: The DFE convergence results for rotating triply coupled beam 94 Figure 5.15: The modes of vibrations for a triply coupled rotating beam 95 Figure 5.16: The model for piecewise uniform beam 97 Figure B.l: The Algorithm of main program of FEM method for calculation of natural frequencies 111 Figure B.2: The Algorithm of the program which calculates the mode shapes, used for the FEM method 112 Figure B.3: The main DFE fimction bisection algorithm 114 Figure B.4: The algorithm of calculation ofJ(j(a) 116 XII NOMENCLATURE: W , {Ov}, {at} Non-nodal parameters ^<fh Constant parameters in uncoupled stijfness matrix Dfw> Dfv Denominators of element stijfness matrices DEV Deviator matrix e Off set beam root ei Eccentricity between elastic center and center o f mass E Modulus o f elasticity {F} Perturbed force GJ Torsion rigidity I y > -^z > ^ xy ’ ^a Mass moments o f inertia j> jOt jm> jh jf Parameters used in Wittrick-Williams method J Jacobian r^e ire jre jre A. } A \| , j A j Element stiffness matrices K Beam stiffness matrix W Dynamic (frequency dependent) stiffness matrix L Length o f beam l e ,l Element length m Mass per unit length M" Element mass matrix M Beam mass matrix Mx Torque along x axis M ^M x Bending moments Ns-. Shape function for bending in z direction Fg-v Shape function for bending in y direction Shape function for torsion along x axis <P(ÿ>f, <P(ÿ>t Vectors of basis functions for flexural and torsional di: s { K m Sign count o f matrix K(w) T, T(x) Axial load / Centrifugal force {«} Nodal displacement {Un} Element nodal displacement V Cord-wise displacement (in y direction) Vy,V, Shear forces Xlll Xlll w Flap-wise displacement (in z direction) W Total virtual work Wint Virtual work done by internal forces (stresses) Wext Virtual work done by external forces Wf.w, fVfv, Virtual work of flexural and torsion displacements X Physical coordinate Kmi, Hm2. Km Radius o f gyration ÔU, ÔW, ÔV, Ô4> Virtual displacements a, P Element frequency dependent parameters for bending vibrations 4> Torsion displacement 6 Pre-twisting angle T Element frequency dependent parameter for torsional vibrations CO, CO/., w* Natural frequencies CO * Trial natural frequency CO/. Lower natural frequency co„ Upper natural frequency Q Angular (rotating) velocity of beam ^ Element coordinate w Flap-wise displacement (in z direction) W Total virtual work Wint Virtual work done by internal forces (stresses) Wext Virtual work done by external forces Wf.w, fVfv, Virtual work of flexural and torsion displacements X Physical coordinate Kmi, Hm2. Km Radius o f gyration ÔU, ÔW, ÔV, Ô4> Virtual displacements a, P Element frequency dependent parameters for bending vibrations 4> Torsion displacement 6 Pre-twisting angle T Element frequency dependent parameter for torsional vibrations CO, CO/., w* Natural frequencies CO * Trial natural frequency CO/. Lower natural frequency co„ Upper natural frequency Q Angular (rotating) velocity of beam ^ Element coordinate XIV 1.1. Introduction The study of the dynamic behaviour of flexible structures is an intrinsic part of design of such systems. The determination of dynamic characteristics of structures is very important in many terrestrial engineering applications, such as automotive industries, industrial robots design and automations, buildings and bridges, and aerospace structural problems such as fixed and rotary aircraft wings and structures, control surfaces, satellite antenna and solar and many more simple or complicated systems. In many cases, when there is a source of vibration with variable frequency, the vibration behaviour of a system is studied to avoid resonance in transient response, where design engineer is to find the frequency spectrum of the system and check the interference of working frequencies and the system natural frequencies. Besides, in many structural problems, the system modal analysis is also important, especially when the design is subject to geometric considerations. For example, the nodes locations in the natural modes could be used to define the supports. Also, the changes in the boundary conditions can considerably affect the system’s natural frequencies. Beams vibration analysis plays a significant role in the investigation of the dynamic behaviour of flexible structures, since many systems can be simply represented by a beam model. Bridges, slender propellers blades, airplane wings and satellite antennas are modeled, at least for the first few natural frequencies, as beam structures. Therefore, many research works have been carried out on the new analytical and numerical methods for beam vibrations with different mechanical and geometrical characteristics [1-13,15-25,27-30]. Beams exhibit different vibrational behaviours depending on their mechanical and geometrical properties. A beam can undergo single or multiple bending (lateral) vibrations, torsional vibrations, longitudinal vibrations and/or a combination of some or all displacements. For the coupled systems, the differential equations governing the structural vibrations are coupled. The coupled equations are widely used in modeling and analysis of aeronautical systems, namely the aircraft wings and stator and rotary 1 compressor and turbine blades, etc. exhibiting coupled bending-bending, bending-torsion or bending-bending-torsion vibrations. Houbolt and brooks (1956) [1] investigated the beam coupled vibrations by formulating a rotating beam model with coupling between flap-wise bending, cord-wise bending and torsion, where the beam has variable cross-sectional properties, twist angle and is subjected to external aerodynamic lift and drag forces. The Euler-Bemoulli bending and St. Venant torsion beam theories were considered where the shear deformation and rotary inertia effects are neglected. 1.1. Introduction Computers opened a new horizon to engineering problems and many researchers then focused their efforts to develop numerical methods where the exact analytical solutions to the system’s governing equations are not known. Murthy (1976) [2] used the Transmission Matrix Method (TMM) to solve the triply coupled differential equations governing the vibrations of twisted non-uniform blades. The state vector (satisfying the differential equations) and backward transmission matrix were employed to form the system’s eigenproblem. The eigenproblem was then solved to obtain natural frequencies and mode shapes of the blade. In another attempt, Murthy used the Integrating Matrix Method (IMM) to solve the bending-torsion coupled equations of rotating beams [3,4], where equations are written in matrix form and are integrated to develop the system eigenproblem. Lang and Nemat-Nasser (1979) [5] proposed a new quotient method to investigate the out-of-plane, in-plane and torsional vibrations of pre-twisted non-uniform blades. This method is based on a variational statement of equations and can be used to find the blade’s natural frequencies and mode shapes. Magari et al. (1987) [6] introduced a Finite Element Method (FEM) and the Hermite bending shape functions were employed to solve the equations of motion for triply coupled beam vibrations. The resulting equations were then solved using MSC/NASTRAN program. The development of numerical methods was continued by Surace et al. [16,17] and they investigated the bending-bending-torsion coupled beam vibrations using the integral formulation based on Green functions (structural influence functions). Besides the numerical approaches, there have also been some attempts to find the analytical solutions of the differential equations governing the coupled beam dynamics. Based on the Dynamic Stiffness Matrix (DSM) method, Baneijee introduced the exact solutions for different coupled bending-torsion beam vibrations [7-13] where the Wittrick-Williams (W-W) root counting algorithm [14,15] was used to find the system’s natural fi-equencies. The exact DSM solution is limited to simple problems and it cannot be used to solve the complex beam geometries and configurations. Furthermore, many researches have also been focused on more advanced beam theories to incorporate the effect of warping, shear deformation and rotary inertia, etc. Arpaci et al. [18] introduced an exact solution method for thin-walled open cross section beams considering warping and rotary inertia effects. Subrahmanyam et al. [19] presented an approach for beam vibrations analysis including the shear deformation, rotary inertia, warping and thermal effects. 1.1. Introduction This model can be used for turbine blades where the beam does not satisfy Euler-Bemoulli assumptions and the thermal effects must be taken into account [19]. The effect of warping on the axially loaded coupled bending-torsion beam was investigated by Jun et al. [20]. As shown in this research, ignoring the warping effect causes decrements in natural frequencies. Also, for higher modes, the warping effect is more pronounced. The Dynamic Finite Element (DFE) method, first introduced by Hashemi (and his coauthors) [21-25], represents an intermediate method between analytical exact DSM and numerical FEM methods. In this approach, the Dynamic (frequency dependent) Trigonometric Shape Functions (DTSF’s), employed as approximation functions, are formulated based on the solution of beam uncoupled governing differential equations. Similar to the DSM method, the DFE produces a non-linear eigenvalue problem and the Wittrick-Williams algorithm [14,15,26] can therefore be used to extract the natural frequencies of the system. The DFE has been applied to the uncoupled flexural and bending-torsion vibrations of uniform and non-uniform beams where the equations of motion are geometrically and/or materially coupled, resulting in excellent convergence rates [21-25]. It has been proven that the DFE combines the advantages of the FEM and DSM methods and results in an accurate, flexible, and systematic method to determine the natural frequencies of beams and beam-like structures. The DFE can be advantageously used for preliminary design of mechanical systems made of flexible beam assemblies, where designer needs to portrait the general dynamic behaviour of the system with acceptable accuracy before starting the detailed analysis and design. Complete modeling of complex systems takes relatively long time and is expensive, and employing the DFE especially for systems with repeated sub-structures can provides a versatile tool to depict the dynamic characteristics of the system. 1.2. Objective The objective of this thesis is to develop and validate a new DFE formulation for triply coupled bending-bending-torsion vibrations of rotating and non-rotating beams. A classical FEM approach is also presented and the two methods are compared. Both DFE and FEM formulations are developed based on Galerkin-type weighted residual method (WRM). Due to the nature of the DFE method, the resulting eigenproblem is nonlinear. A dedicated Wittrick-Williams root counting algorithm [14,15,26] which provides a powerful tool to evaluate the natural frequencies of nonlinear eigenproblems is introduced. The method can then be implemented to investigate the natural frequencies and corresponding modes of free vibrations of dually and triply coupled beams. The method presented in this research is able to calculate the eigenvalue (natural frequency) of the resulting nonlinear eigenproblem. This research also investigates the coupled vibrations of flexible beams and the effect of various geometric and dynamic parameters on the system behaviour. The dynamic coupling (caused by rotating speed and/or constant axial load) and geometrical coupling (caused by pre-twist angle and/or distance between mass and elastic center) are studied by several illustrative examples and the natural frequencies and modes of free vibrations of beams and rotating blades are evaluated. These examples provide a general understanding and a better insight to the free vibrations of beams and blades. The DFE and FEM formulations presented in this research can be used to evaluate the fundamental natural frequencies and the corresponding modes of a beam structure. In many engineering design problems, the designer needs to get a general idea about the dynamic behaviour of the system even before a detailed and rigorous FEM analysis starts. In such cases, having a reliable, simple and accurate simulation tool is helpful. 1.3. Thesis Organization In order to construct an accurate DFE formulation for the free vibration analysis of blades, a progressive procedure is adopted. The modeling starts with the well-known classic FEM method for beam's pure bending vibration and finally comes to an end with a DFE model for coupled bending-bending-torsion vibrations of rotating blades. In Chapter 1, the importance of mechanical vibrations and the vibration analysis of flexible structures, in general, and the coupled beam vibrations, in particular, are briefly discussed. Some of the numerical methods used in analysis of rotating and non rotating beams are reviewed, and the DFE as a bridge between exact solution method (DSM) and finite element method (FEM) is outlined. In Chapter 2, the equations of motions for the coupled flap-wise, cord-wise and torsional vibrations of rotating beams along with the boundary conditions are presented. The Euler-Bemoulli bending and St. Venant torsion beam theories are considered where the shear deformation, rotary inertia and warping are neglected. The equations are coupled due to the geometrical and mechanical properties and the dynamics of the beam. Then equations of motion and the boundary conditions for coupled bending-torsion and bending-bending vibrations are extracted from the triply coupled equations. In Chapter 3, a dedicated Wittrick-Williams root counting algorithm for calculation of natural frequencies of a flexible structure is discussed. The solution methodology is explained for the non-linear eigenvalue problems resulting from the frequency dependent element dynamic stiffness matrices. As it is then briefly discussed, the corresponding modes of coupled vibration can be extracted using a perturbation technique. In Chapter 4, the Galerkin based finite element methodology is described and the coupled equations of motion along with static Hermite polynomial shape functions are used to derive the FEM method. The FEM approach then is applied to the triply coupled beams as well as coupled bending-torsion and bending-bending beams to verify the solution method. In Chapter 5, the Dynamie Finite Element (DFE) formulation is introduced. The Dynamic Trigonometric Shape Functions (DTSF’s) are used to form the frequency dependent stiffness matrix. The Wittrick-Williams root counting method is then employed to find the natural fi-equencies. Some illustrative examples of dually and triply coupled beams are then discussed to prove the validity of the method. In concluding Chapter 6, the formulations introduced in this research work are reviewed. A comparative study between DFE and FEM methods is then provided, where the advantages of each method are highlighted. 1.3. Thesis Organization At the end, the direction and future of the research is stated. In appendix A, the solutions of uncoupled bending and torsion vibrations of a cantilever beam are derived. The solutions are used to develop Dynamic Trigonometric Shape Functions for DFE method. Appendix B introduces the program logic and the algorithm for FEM and DFE methods, and explains the differences between two programs. Also the functions which have been developed and used in the FEM and DEF programs are discussed. 2.1. Introduction The governing differential equations of motion for coupled vibrations of rotating beams incorporating different levels of complexity bave been introduced in several occasions [1,16,22,23,25]. The proposed models bave been developed using different methods and consist of one or more geometric/dynamic parameters leading the coupling between equations of motion. Regardless of the derivation method, the resulting general equations can be easily reduced to special cases such as constant twisting angle, non rotating beam with constant tension, rotating beam without eccentricity, coupled bending- torsion [2,16], bending-bending [2,16,27] and the simple case of uncoupled bending and torsion vibrations of a beam [34]. 2.2. Model A cantilever rotating beam (Figure 2.1) with length L, and an offset ej from rotating axis (i.e. bub radius), distance e between mass centroid and elastic (shear) center and rigid cross-section is the basis of the model. All rigidities are assumed to be constant, or piecewise constant, along x-axis. The rigidities are; flexural rigidities EI^, Ely, Ely, and Torsional rigidity GJ. The rigidities can be determined experimentally or theoretically. The beam has a pre-twist angle 6. 2.3. Assumptions The Euler-Bemoulli bending and St. Venant shear beam theories are exploited. The shear deformation, the rotary inertia effect, warping and thermal effects are neglected. The small linear displacements are considered, the axial displacements are neglected and the bending slope is set equal to the derivative of bending displacement with respect to spatial variable x. (a) (a) C.E. c .a (b) (c) Figure 2.1: The beam configuration and geometrical parameters. C.E. c .a (b) (c) (b) (a) (b) Figure 2.1: The beam configuration and geometrical parameters. The beam undergoes three displacements: rv and v are out-of-plane and in-plane displacements, respectively, associated with bending vibrations in two directions and ^ is the rotational displacement associated with torsion. 2.4. Governing Differential Equations of Motion There have been many studies to derive the differential equations governing the free vibrations of rotating blades. Houbolt and Brooks [1] followed the stress-strain analysis in which the longitudinal strains are expressed in terms of two perpendicular bending displacements and torsional displacement. The stress is then integrated over the beam cross sectional area to evaluate the flap-wise, cord-wise and torsion moments at any section of the beam. The resulting internal and external moments and forces are used in Newton’s second law to relate the forces with accelerations. The Newton’s second law results in the final differential equations of motion. Magari et al. [6] employed the Hamilton’s principle in which the action function is minimized in the time interval, and the internal potential energy and kinetic energy are calculated in terms of displacements. Using any of these two methods, the displacements are functions of time (t) and .v and final differential equations of motion consist of time and spatial partial derivatives. Using separation of variables technique and based on the simple harmonic motion assumption, one can get the final spatial differential equations, governing the free vibrations of rotating beams as follow: (Ely + El y y - (Twy - [çyem(x + e, )<t> cos 0]' - aym(w+e(j) cos 0) = 0 2.1 2.1 (Ely + El y ” - (Tv')' + [Q^em(x + e, )f)sin 0]' + Çy em(j) sin 0 - y m(v-e(j)S\n 9 ) -Ç y m v = 0 2.2 (Ely + El y ” - (Tv')' + [Q^em(x + e, )f)sin 0]' + Çy em(j) sin 0 - y m(v-e(j)S\n 9 ) -Ç y m v = 0 2.2 2.2 - (GJ<j)'y - Q^em(x + e^ )(v' sin 0 - w ' cos 0) + Cyemvsin0 + Q^m(Kl2 -K"2,)^cos2^ 2.3 -û)^7«/£r^^ + <y^effj(vsin^-vvcos^) = 0 2.3 where “ ' ” represents the differentiation with respect to x, 0^ is a result of two times differentiation with respect to time, ft is the angular (rotating) velocity of the beam about z-axis, m is the mass per unit length of the beam and T, the tensile axial force acting on the beam, is the centrifugal force and is calculated at any point of beam from: T(x)= ^ Q^m(x + e, )dx 2.4 2.4 One can see that the differential equations (2.1) to (2.3) are coupled due to the geometric parameters e and 9, and the dynamic parameters fl. 2.6. Equations of Motion for Dually Coupled Vibrations As already stated, the triply coupled differential equations of beam vibrations (2.1) to (2.3) and the boundary conditions (2.5) to (2.11) can be used to derive the differential equations of motions for dually coupled vibrations of a beam. 2.5. Boundary Conditions For free vibrations of a cantilever beam, all displacements at the clamped end, and the internal forces and moments at free end are equal to zero. The boundary conditions for a clamped-free beam are: v = w = ^ = v' = w' = 0 25 = My = =Vy =V^ =0 2.6 v = w = ^ = v' = w' = 0 25 v = w = ^ = v' = w' = 0 Atx = 0: 25 25 And at jc = Z,: = My = =Vy =V^ =0 2.6 = My = =Vy =V^ =0 2.6 2.6 where Mx, My and are internal moments about x, y and z axes, respectively, and Vy and Fj represent internal shear forces inj; and z directions. where Mx, My and are internal moments about x, y and z axes, respectively, and Vy and Fj represent internal shear forces inj; and z directions. For uncoupled beam equations, shear forces and moments expressions are reported in any solid mechanics textbook, but for coupled vibrations, shear forces and moments are to be calculated considering the coupling terms. The internal forces and moments at any point along the beam length are then calculated from following equations [1]: Fj = —{ElyW + El^yVy + Tw' + {Çî^em{x + )ÿ)cos^) 2 7 2 7 2 7 Vy = + E l y y +Tv’ - +e, )ip sin 6) 2.8 = GJ(j)' 2.9 My = ElyW" + E l y 2.10 M ^ = E i y + E i y 2.11 = GJ(j)' My = ElyW" + E l y M ^ = E i y + E i y My = ElyW" + E l y 10 The equations (2.1) to (2.3) and the boundary conditions (2.5) to (2.11) fully define the dynamic behaviour of flexible linear rotating beams and are exploited in next chapters to find the natural frequencies and mode shapes of the triply coupled beams vibrations. 2.6.1. Coupled Bending-Bending Vibrations The coupled flap-wise and cord-wise bending vibrations of a rotating beam are governed by the following differential equations: {ElyW + - (Tw'y - m^mw = 0 2.12 {Ely + EI^^w)" - {Tv'y - y m v - Q}mv = 0 2.13 {ElyW + - (Tw'y - m^mw = 0 2.12 {Ely + EI^^w)" - {Tv'y - y m v - Q}mv = 0 2.13 Obtained by setting ç to zero in equations (2.1) and (2.2). The boundary conditions at free end, in this case, reduce to following form: = -{E ly -h E l^v”y + Tw' = 0 2.14 Vy = -{EI^w" + E i y y + Tv' = 0 2.15 M y = E i y + EI^v" = 0 2.16 = -{E ly -h E l^v”y + Tw' = 0 2.14 Vy = -{EI^w" + E i y y + Tv' = 0 2.15 M y = E i y + EI^v" = 0 2.16 Vy = -{EI^w" + E i y y + Tv' = 0 M y = E i y + EI^v" = 0 2.16 11 M ^ = EI^v” + EI^w" = 0 2.17 M ^ = EI^v” + EI^w" = 0 2.17 One can see that the angular velocity of the beam, ft, doesn’t have any contribution in coupling terms and only appears in the expression for centrifugal force (see equation 2.9), T, and the coupling between two in-plane and out-of-plane of rotation lateral displacements is related to asymmetric term El^y. Also, the eccentricity e disappears when torsional displacement <j> vanishes. It means that the eccentricity between centroid and elastic center doesn’t affect the bending-bending coupled equations. It is also to be noted that out-of-plane flap vibrations are only affected by centrifugal effect (TwY, whereas the in-plane displacement is related to the centripetal term, -ft^wv, as well. The centrifugal term increases the beam stiffness in both directions, while the centripetal term, due to the negative sign, decreases the stiffness in in-plane direction and causes decrement in cord-wise mode frequencies. 2.6.2. Coupled Bending-Torsion Vibrations The differential equations governing the coupled bending-torsion vibrations can be extracted from the triply coupled general case by setting v = 0: (El y y - (Tw'y - [n^em(x+e, )^cos ey — a>^m(w+e(f> cos 0) = O 2.18 2.18 -{GJ(f>y + Ç1 em{x + e^)Wcos9 -CO emwcosO + Q}m(Kl^ -Kly)<l)cos2e-ûymKl(l> = 0 2.19 -{GJ(f>y + Ç1 em{x + e^)Wcos9 -CO emwcosO + Q}m(Kl^ -Kly)<l)cos2e-ûymKl(l> = 0 -{GJ(f>y + Ç1 em{x + e^)Wcos9 -CO emwcosO + Q}m(Kl^ -Kly)<l)cos2e-ûymKl(l> = 0 2.19 where, in this case, the boundary conditions at free end are: where, in this case, the boundary conditions at free end are: 12 =-(-£’/j.w)' + rw' + (Q^e/;j(x + e,)^cos0) = O 2.20 =-(-£’/j.w)' + rw' + (Q^e/;j(x + e,)^cos0) = O =-(-£’/j.w)' + rw' + (Q^e/;j(x + e,)^cos0) = O 2.20 M ^ = GJ(f)' = 0 2.21 My=EIyW" = 0 2.22 M ^ = GJ(f)' = 0 2.21 M ^ = GJ(f)' = 0 2.21 My=EIyW" = 0 2.22 My=EIyW" = 0 2.22 2.22 The coupling occurs because of non-coincident shear and mass axes {e ?^0) and the beam rotating speed 0 . Even though the pre-twist angle d appears in coupling term, but it doesn’t have direct contribution in these terms. In words, if 0 vanishes, the nature of equations will not change. In this chapter the differential equations of motion governing the triply and dually coupled vibrations of rotating beams were presented. In Chapter 3, the Wittrick-Williams root counting method, used to evaluate the natural frequencies of an eigenproblem, will be presented. Then, in Chapter 4 and 5, the governing differential equations of motion introduced in this chapter are solved, using the FEM and DFE methods, respectively, to evaluate the natural frequencies and modes of rotating and non-rotating beams. 13 3.2. Wittrick-Williams (W-W) Method As already stated, both the DSM and DFE methods lead to a nonlinear eigenproblem due to the fact that the approximation functions and the element stiffiiess matrices are frequency dependent. In this case, the coefficient matrix in (3.3) is the nonlinear frequency dependent dynamic stiffness matrix, [.4(X)] = [Æ(w)]. The Wittrick- Williams (W-W) root counting algorithm [14,15] can then be exploited to find all natural frequencies lying below a trial frequency u. The advantage of W-W approach is that for the periodic structures and systems composed of sub-structures, the natural frequencies of sub-structures and poles of the system can also be calculated. Besides, the constrained nodes and special boundary conditions which produce repeated frequencies can also be studied [15]. 3.1. Introduction Many engineering problems, such as buckling and vibration analyses of flexible structures, lead to one of the following equations; = 3.1 [A]{x} = X{x} 3.2 [^(A)]{x} = 0 3.3 = 3.1 [A]{x} = X{x} 3.2 [^(A)]{x} = 0 3.3 3.3 known as eigenvalue problems or simply eigenproblems. Here [A], [B] and [^(X)] are /ix« matrices; {x} is an n dimensional vector; {x} and Xare eigenvector and eigenvalue, respectively. When li4] and [B] are constant, the above equation is called a linear eigenproblem (see (3.1) and (3.2)), whereas X-dependent components lead to a nonlinear eigenproblem (3.3). In vibration analysis of flexible structures, the eigenvalues and eigenvectors are equivalent to natural frequencies and mode shapes, respectively, where X= co^. There exist several well-known iterative numerical algorithms established to find the eigenvalues and eigenvectors of the linear eigenproblems. The approach introduced in this chapter, developed by Wittrick and Williams [14, 15, 27], provides a powerful and robust tool for, but is not limited to, nonlinear eigenproblems of form (3.3), where the coefficient matrix [i4(co)j is frequency dependent and other algorithms may skip some natural frequencies. 14 iy = the largest integer < a^/jt iy = the largest integer < a^/jt 3.11 Df,y and are the denominators of the element stiffness matrix for in-plane, v, and out- of-plane, w, flexural displacements, respectively and a and r are element frequency dependent parameters [21]. Df,y and are the denominators of the element stiffness matrix for in-plane, v, and out- of-plane, w, flexural displacements, respectively and a and r are element frequency dependent parameters [21]. Thus, by exploiting the equations (3.4) to (3.11), it is possible to converge on any required natural frequencies, given the fact that the expressions for the dynamic stiffness matrix and the clamped-clamped natural frequencies are known. 3.2.1. Theory For a flexible structure, there are two possible solutions pertaining to the equation \K„((o)\{un}=0: \K„((o)\{un}=0: a) \\Kn(o})\\ = 0, where {u„} ^Ois one set of solution, b) \[K„((o)]\ = 00, where {«„} = 0 corresponds to the case where displacements {u„} are ‘constrained nodes’ or nodes in the natural modes of vibrations. [K„((o)]\ = 00, where {«„} = 0 corresponds to the case where displacements b) \[K„((o)]\ = 00, where {«„} = 0 corresponds to the case where displacements {u } are ‘constrained nodes’ or nodes in the natural modes of vibrations ) [ (( )] , { „} p p {u„} are ‘constrained nodes’ or nodes in the natural modes of vibrations. {u„} are ‘constrained nodes’ or nodes in the natural modes of vibrations. Suppose that (o denotes the natural frequency of the structure. Then, it is known that j, the number of natural frequencies of the system between zero and o), as (o increases, is given by: 15 where lK(û)J] is the overall dynamic (frequency dependent) stiffiiess matrix evaluated at Û) = Û), and s([K(û>)]) is the number of negative elements on the leading diagonal of and [K('coJ]^ is the upper triangular matrix obtained by applying the standard Gauss elimination method to the [li'fû))] with no column interchange. Jo is the number of natural frequencies of the structure still lying between co = 0 and co = cd when displacement components to which [K(co)] corresponds are all zero (in such a case, the beam can still have natural frequencies when all its nodes are clamped, because the formulation allows each individual (beam) element to have an infinite number of degrees of freedom between nodes). Thus; NE Jo - ^ J m 3.5 m=l 3.5 where ]„ is the number of natural frequencies between co= 0 and o)= co* for an element with its ends clamped, while the summation extends over all elements. where ]„ is the number of natural frequencies between co= 0 and o)= co* for an element with its ends clamped, while the summation extends over all elements. It can be shown [14] that ]„ for the case of coupled bending-bending-torsion vibrations is calculated as: where: Jt = the largest integer < x/n 3.7 /h. = the largest integer < a j n 3.9 /h. 3.2.1. Theory = the largest integer < a j n 16 jf,v - K ~ ~ [1 + (-I)'' sgn(Z)^ J ] 3 3.10 iy = the largest integer < a^/jt 3.11 3.2.2. Application to the Beam Vibrations A beam with uncoupled out-of-plane vibrations is used to demonstrate Wittrick- Williams method. The equation of motion for a cantilever beam with out of plane flexural vibrations is: {ElyW"y — oP'mw = 0 3.12 3.12 After applying the natural (force) boundary conditions, using the DFE or DSM method [21], the final eigenproblem can be written as: [K(co)]{u} = 0 3.13 3.13 For a one-element beam model, the natural frequencies are obtained when the determinant of stiffiiess matrix, {K(a))\, vanishes. In other words, when the \K((o)] 17 determinant is plotted versus frequency (Figure 3.1), the intersections between the curve and o-axis are the natural frequencies. It can be seen from Figure 3.1 that between the second and third natural frequencies there exist two poles, where oo. ' 1 1 , ' 1 1 1 1 1 1 0 10 20 30 40 50 60 omega (fad/s) , ‘ . . - ":.%70 : .^.1000 • ( 0 f •'t -1000 • -20Ô0 -■S(D -3000 <D ■Si xjî: rig -4000 -5000 j, , . -6000 ‘ r-7000 # # # ; ' -8000 Figure 3.1: The determinant of stiffness matrix versus frequency. Figure 3.1: The determinant of stiffness matrix versus frequency. Investigation of the parameters s[K(w)\ and shows that these numbers can change independently. The variation of sign count of stiffiiess matrix, s\K(i^)\, for a beam with one element is shown in Figure 3.2: 18 _ ^ ,:-2 . L 1.8 i 1.4 . % * ^ ,. ■ s 1-2 • ; :< I " . ,.3:0.6 0.4 -, ' 0.2 ' O'—T- •>„-'5 101 : , ' 15 - 20': ' 25 ilKSO; - . - i-> rZ ' ^ omega (rad/s) ; . 'j; \ ^ Figure 3.2: The sign count of stiffness matrix versus frequency. Figure 3.2: The sign count of stiffness matrix versus frequency. and jf,w varies as shown in Figure 3.3: and jf,w varies as shown in Figure 3.3: 10 i 20 ; . : 30 ' : 40 ' ■ :v - , ) . .'omega(rad/s) -Sfli Figure 3.3: The variation of jf,„ versus frequency. 10 i 20 ; . : 30 ' : 40 ' ■ :v - , ) . .'omega(rad/s) -Sfli Figure 3.3: The variation of jf,„ versus frequency. 19 Gloser look at Figures (3.1), (3.2) and (3.3) shows that the first and second natural frequencies can be obtained by checking the sign count, that means before the first natural frequency, the sign count is zero and at w = W; it jumps to 1. This jump is repeated after the second natural fi’equency but immediately after sign count decreases as one unit, and this decrement is compensated by j/,w (which is equal to jo when there is only one bending element) which jumps to 1. The summation of sign count and jf,w however remain equal to 2. It means that regardless of individual changes of s[Æ(w)] and jo, the total j changes only at each natural frequency by one unit [15]. The Wittrick-Williams method, demonstrated in this section, can be used for cases of linear and/or non-linear eigenproblems. This method provides a powerful tool especially for nonlinear problems, for the periodic structures and those exhibiting repeated natural frequencies [15]. 3.3. Solution of the Eigenproblem By implementing the Wittrick-Williams approach, a simple bisection algorithm can be employed to converge to the natural frequency [14]. The algorithm has been introduced in Appendix B and starts with two arbitrary initial upper and lower frequencies, w„ and w,. A trial frequency w* then is selected and the j number, corresponding to the natural frequency, is calculated. The co * changes in the way that the difference between co„ and co/ decreases and they converge with any desirable accuracy to the i'* natural frequency co,-. Once the natural frequencies are known, the corresponding desired mode shapes can then be calculated by perturbation method. By definition, the dynamic stiffiiess matrix cannot be inverted due to its zero determinant at any natural fi'equencies. To obtain a non-trivial solution, the frequency variable is manipulated so that the fi-equency dependent stiffness matrix is altered slightly (in this case 10''° is considered as perturbation factor which guarantees the desirable accuracy). This perturbation must be small enough to prevent significant deviation in solution: 2 0 û) , =<y,.(l + 10-‘°) 3.14 û) , =<y,.(l + 10-‘°) 3.14 where w / is the altered natural frequency, and eigenvalue equation can be written: where w / is the altered natural frequency, and eigenvalue equation can be written: where w / is the altered natural frequency, and eigenvalue equation can be written: where w / is the altered natural frequency, and eigenvalue equation can be written: where w / is the altered natural frequency, and eigenvalue equation can be written: [^ (6)‘,)]{w,} = {F} 3.15 3.15 where Fj{ j= \ to /i), the components of zero force function {F}, are slightly altered from zero. where Fj{ j= \ to /i), the components of zero force function {F}, are slightly altered from zero. F% =10-'° 3.16 F% =10-'° 3.16 3.16 F% 10 3.16 The eigenvectors can be evaluated by manipulating equation as: The eigenvectors can be evaluated by manipulating equation as: The eigenvectors can be evaluated by manipulating equation as: } 3.17 3.17 The order of perturbation of the natural frequency, «/, and the force vector depends on the numerical precision. Using double precision, the lO"’ order perturbation is acceptable to accurately describe the modes of deformation. The Wittrick-Williams algorithm provides an accurate and robust tool to evaluate the natural frequencies of a nonlinear eigenproblem as well as linear eigenproblems. 4.1. Introduction Finite Element Methodology (FEM) is one of the most powerful numerical methods widely used to solve the problems of complicated geometry and/or mechanical properties, where the analytical solutions are not always available. In vibration analysis of a beam there are many different approaches using the FEM (see for example [6,17,28,29,30]). Each of these methods transforms the differential equations of motion into an algebraic eigenvalue problem. The approach presented in this work is based on the Galerkin method and a one dimensional 2-node beam element. 3.3. Solution of the Eigenproblem Using this algorithm, one can approximate any individual natural frequency and the solution can converge to the theoretical value by any desirable accuracy. This method will be used in Chapter 5, where the DFE formulation produces a nonlinear dynamic (frequency dependent) stiffness matrix. In next chapter. Chapter 4, the FEM formulations based on Galerkin-type weighted residual method will be developed to solve the governing differential equations of motion for dually and triply coupled vibrations of beams. 21 4.2. Galerkin Method Galerkin FEM, based on the weighted residual method, is an accurate tool with high rate of convergence used in analysis of mechanical and structural systems. Substituting the approximation functions into the differential equations results in a residual for each equation, due to the fact that the approximation functions are not the exact solutions. In Galerkin method, the approximation functions are forced to be orthogonal to the residual of equations to minimize the error, and the orthogonality conditions result in algebraic equations [31]. In what follows, the FEM formulation with application to the free vibrations analysis of triply coupled rotating beams is established. Equations (2.1) to (2.3) can be written in following general from: Z,(w,v,(^) = 0 L^(w,v,4i) = Q 4.1 L^{yv,v,^) = 0 4.1 2 2 where Li, L2 and L 3 are linear differential operators. As already explained, the approximation solutions, w'', v" and result in a residual for each equation, as: where Li, L2 and L 3 are linear differential operators. As already explained, the approximation solutions, w'', v" and result in a residual for each equation, as: L,(w,vJ) = R, 4.2 Z3 (vv, V, L,(w,vJ) = R, 4.2 Z3 (vv, V, L,(w,vJ) = R, Z3 (vv, V, 4.2 and to satisfy the orthogonality conditions: and to satisfy the orthogonality conditions: and to satisfy the orthogonality conditions: 8w{L^{w,v,(l))}dx = 0 ^ ôv{L 2 {yv,v,(j))}dx = 0 4.3 ^ 5(f> {L^{w,v,(j))}dx = 0 4.3 Closer look at equations (4.3) shows that the LHS of equations represents the virtual work done by each displacement [31]: " ^Sw{L^{^v,v,(\|))}dx Wj-^ = ^ 8 v { l 2 (w,v,^)}dx 4.4 4.4 Since each integral in (4.3) vanishes, the summation of these integrals also vanishes: 23 23 where in the absence of external forces (i.e., free vibrations), the total virtual work is equal to internal virtual work; where in the absence of external forces (i.e., free vibrations), the total virtual work is equal to internal virtual work; IF = - 0 4.6 IF = - 0 4.6 One can see that the implementation of Galerkin weak formulation also satisfies the principle of virtual work. One can see that the implementation of Galerkin weak formulation also satisfies the principle of virtual work. 4.2. Galerkin Method After substituting the equations (2.1), (2.2) and (2.3) into flexural and torsional virtual work of equations (4.4), and to satisfy the natural boundary conditions, the appropriate number of integration by parts should be applied to the terms which produce the shear forces and moments of (2.7) to (2.11): = I" {Sw\EIyW'' +El ^v'') + ôw'{Tw’) + Sw'[n^em{x + e^)^ cose] - Sw[o)^m{w + cos 9)'\}dx + [5w {{E iy + EI^v")'- (Tw’) - (n^em(x + e, cos 0)]]^" + [-^w'(£/,w'' + £/^^v")]J 4.7 - Sw[o)^m{w + cos 9)'\}dx + [5w {{E iy + EI^v")'- (Tw’) - (n^em(x + e, cos 0)]]^" + [-^w'(£/,w'' + £/^^v")]J 4.7 4.7 {Sv"(E/^v'' + EI^w") + 5v\Tv') - Sv'[Q^em{x + e,)f) sin 9] + 5v[€l^em <l>sm 6 - œ ^m{v - e<j) sin 6) - Çl^mv]]dx + + El^w”)' - {Tv') + {Çl^em{x + e,)(^ sin 0)]]^ + [-S v '{E iy + EI^w'')\ 4.8 {Sv"(E/^v'' + EI^w") + 5v\Tv') - Sv'[Q^em{x + e,)f) sin 9] + 5v[€l^em <l>sm 6 - œ ^m{v - e<j) sin 6) - Çl^mv]]dx + + El^w”)' - {Tv') + {Çl^em{x + e,)(^ sin 0)]]^ + [-S v '{E iy + EI^w'')\ 4.8 4.8 W, ^ = ^ {0<l>'{GJ<l>') - S<j>[D.^em{x + e, )(v'sin 9 - w'cos 9) - Cl^emv sin 9 - Q^w(v^2 - /c^,)^cos 29 + ymKl^cj) - ûJ^e7M(vsin 9 - wcos 9))}dx 4.9 W, ^ = ^ {0<l>'{GJ<l>') - S<j>[D.^em{x + e, )(v'sin 9 - w'cos 9) - Cl^emv sin 9 - Q^w(v^2 - /c^,)^cos 29 + ymKl^cj) - ûJ^e7M(vsin 9 - wcos 9))}dx 4.9 4.9 The terms produced in [ ] are the boundary terms represented by product of a displacement and its corresponding force/moment and vanish at both clamped and free 24 ends. After applying the natural boundary conditions, the system equations can be discretized into element equations considering the fact that the inter-element continuity conditions must be satisfied. 4.2. Galerkin Method The equations (4.7), (4.8) and (4.9) then can be written as: [0w\EI^)w'' + S w \E I^ )v’ + Sw\T)w' + Sw'{Çï^etn{x + e, ) cos 6)<j> - 5w{o)^m)w - Sw(co^em cos 6)(j>]dx 4.10 [0w\EI^)w'' + S w \E I^ )v’ + Sw\T)w' + Sw'{Çï^etn{x + e, ) cos 6)<j> - 5w{o)^m)w - Sw(co^em cos 6)(j>]dx 4.10 4.10 ^/.v = [Sy"{EI, )v" + 5v'\EI ^ )w" + Sv'(T)v' - ôv'(Çl ^em (% + e,) sin 6)^ - ^v(Q ^m)v + Sv{Q^em sin 0)^ - Sv{o)^m)v + Sv{a>^em sin 0)<j)'\dx 4.11 ^/.v = [Sy"{EI, )v" + 5v'\EI ^ )w" + Sv'(T)v' - ôv'(Çl ^em (% + e,) sin 6)^ - ^v(Q ^m)v + Sv{Q^em sin 0)^ - Sv{o)^m)v + Sv{a>^em sin 0)<j)'\dx 4.11 4.11 W,% = p " [ 5 ( l > \ G J ) < t , ' e m (x + e ,) sin 0)v' + (Q^e/M(x + e, ) cos B)w' + S(!>{Q.^em sin 9)v + ô(t>{Çï^ m{Kl^ -\-^ ,)cos 10)(j) - S(j>{œ^ mKl)(p + S<l>{o)^em sin 6)v - 8<j){co^em cos 9)w^dx 4.12 W,% = p " [ 5 ( l > \ G J ) < t , ' e m (x + e ,) sin 0)v' + (Q^e/M(x + e, ) cos B)w' + S(!>{Q.^em sin 9)v + ô(t>{Çï^ m{Kl^ -\-^ ,)cos 10)(j) - S(j>{œ^ mKl)(p + S<l>{o)^em sin 6)v - 8<j){co^em cos 9)w^dx 4.12 Satisfaction of inter-element continuity conditions depends on the approximation functions selected for each displacement. The element nodal variables and nodal coordinates are shown in Figure 4.1 : node i <Pi node Xi Xm / element Figure 4.1: The element’s nodal variables. Figure 4.1: The element’s nodal variables. 4.3. Approximation Functions The approximation functions are used to calculate the element displacements inside the element domain, and should satisfy the continuity conditions at the element boundary. For flexural displacements h> and v, the approximation functions should satisfy continuity, and for torsional displacement (j>, the function should satisfy Cf continuity. “Hermite” cubic shape functions and linear shape functions have been developed for flexural and torsional displacements, respectively, such that they satisfy the inter-element continuity. These polynomial shape functions have been introduced in FEM text books [32, 33] as follow: 2-3^+ ^' 2+ 3^-^' > 4.13 AT,=< I z i > 4.14 2 2 4.13 AT,=< I z i > 4.14 2 2 4.14 where the relation between physical coordinate, x, and element coordinate can be written as (see Figure 4.1): 2 I •^y+i 2 I •^y+i and and so and dx = Jd^ so dx = Jd^ 26 J in equation (4.17) is called Jacobian and is taken into account where there is integration and differentiation with respect to and / is the length of element. The transformation from the physical coordinate to the reference element coordinate is iso parametric for torsional displacement and sub-parametric for bending displacements (Figure 4.1). ^=-1 ^ = 0 ^=+1 X ■J- ■J Figure 4.2: The transformation from physical coordinate to reference element coordinate. Figure 4.2: The transformation from physical coordinate to reference element coordinate. Along the length of beam element, the displacements can be calculated by the ( - dependent shape functions as (i.e., nodal approximations): W (^) =< N g < w, w, 1 ^ 2 J ' = < >w 4.19 V(^) =< Ng > , = < N g > , {V} 4.20 4.19 4.20 2 7 where < N b > w /v and < N t > are flexural and torsional shape functions respectively, and {w}, {v} and are nodal displacements. where < N b > w /v and < N t > are flexural and torsional shape functions respectively, and {w}, {v} and are nodal displacements. 4.4. Derivation of System Eigenproblem Substituting virtual and element displacements by approximation functions (4.19) to (4.21) into (4.10), (4.11) and (4.12), leads to: = £' [(£/,) <&,> < K > . M +(^4) <^ > {NIL <K>. M + (n < & '> {iv ;},.< K > . w + (Q W ^ + e i)c o s 0 < a v > < N ^ > { < j > } 4.22 - {(o^m) < à v > {Ng}^ < Ng >„ {w}- (co^emcosû) < à v > [Ng}„ <Nj.> {(j>}'\j\d^ 4.22 ^;.V = £' [(^4 )<SV>{N'g},<N’g>A^) + (^ 4 )<SV>{N"g},<N'’g>, {w} + (r) <<5v> {iv;}, < iv; >, M + {flLmsine)<0v>{Ng}^<N,> {$)} 4 23 - (0^g7M(%+e,)sin^)< & > „ <Nj >{^}-{co^ni)<ôv>{Ng}^ <Ng {v} + (A)^emsin0) < & > {//g }, <N.j- > {^}-{0}m ) <ôv> {Ng}^ <Ng >{v}]\|y\|d^ 4 23 w,:* = C [+(G/) < > {A^;} < A^; > w - (Q^e/n(x + e, ) sin 9)<ô(f)> {Nj}< N'g>^ {v'} + (fi^e/n(x + e,)cos0)<<5ÿ>{7/;.)< A^g {w'j - (fi"w(/c^2 - kI,)cos29)<(j» {N^} <Nr> (f)} ^ 24 + {co^em sin 9)<(j>> {Nj.} < A^g >v {v) + {p}em sin 9)<S<j>> {A^j.} < A^b >v {v} - )<<j»{Nr}<N^>{<l)} - {co^emcos9 ) < ^ > { Nj } < A^b >„ (w}]\|j\|d^ w,:* = C [+(G/) < > {A^;} < A^; > w - (Q^e/n(x + e, ) sin 9)<ô(f)> {Nj}< N'g>^ {v'} + (fi^e/n(x + e,)cos0)<<5ÿ>{7/;.)< A^g {w'j - (fi"w(/c^2 - kI,)cos29)<(j» {N^} <Nr> (f)} ^ 24 + {co^em sin 9)<(j>> {Nj.} < A^g >v {v) + {p}em sin 9)<S<j>> {A^j.} < A^b >v {v} - )<<j»{Nr}<N^>{<l)} - {co^emcos9 ) < ^ > { Nj } < A^b >„ (w}]\|j\|d^ ^ 24 2 8 The products of shape functions in equations (4.22) to (4.24) produce different size matrices, i.e. {N'b}< N' b > produces a 4x4 matrix, while {A'b}< N t > produces a 4x2 matrix. Similarly, the nodal displacements and virtual displacements are not of same size. 4.4. Derivation of System Eigenproblem In order to write all three equations in a unified format, the following nodal displacement vector and shape functions are introduced: ATg, 0 0 0 N,, Ng, 0 0 0> 4.26 <A^fi-v>=<0 0 Ng, Ng; 0 0 0 Ng3 Ng, 0> 4.27 <Nr_^>=<0 0 0 0 N„ 0 0 0 0 Nr; > 4.28 <Nr_^>=<0 0 0 0 N„ 0 0 0 0 Nr; > 4.28 where Nbi, Nb2, Nbs and Nb4 are the flexural shape functions and N ti and N t2 are torsional shape functions. Using nodal parameters (4.25) to (4.28), the components of element virtual work can be written as: where Nbi, Nb2, Nbs and Nb4 are the flexural shape functions and N ti and N t2 are torsional shape functions. Using nodal parameters (4.25) to (4.28), the components of element virtual work can be written as: where Nbi, Nb2, Nbs and Nb4 are the flexural shape functions and N ti and N t2 are torsional shape functions. Using nodal parameters (4.25) to (4.28), the components of element virtual work can be written as: = f,' >+(E/,,){N;_}<N%> +(T){N;.J<N'.„ >+(n^em(x+e,)cos0){N;.J<Nr.^ > 4.29 -(<a^w){Ng.„}<Ng.„ >-[o)^emcos9){Ng.J<N.j..^ >]{uY\j\d^ = f,' >+(E/,,){N;_}<N%> +(T){N;.J<N'.„ >+(n^em(x+e,)cos0){N;.J<Nr.^ > 4.29 -(<a^w){Ng.„}<Ng.„ >-[o)^emcos9){Ng.J<N.j..^ >]{uY\j\d^ 4.29 K y = f,'< ^ [(^4){N%} < N;_, > +{EI^){NIJ< Nl„ > + iCl^emsm9){Ng,J<Nj.,^ >-(fl^ew(x + e,)sin0){N;.U <Nr_^ > - (ûJ^TTi){N g .J < Ng.„ >+(û>^em sin 0 ){N g .J < N r .^ > + iT){N’g_,}<N'g.^>-(n^m){Ng.,}<Ng., >]{»}' K y = f,'< ^ [(^4){N%} < N;_, > +{EI^){NIJ< Nl„ > + iCl^emsm9){Ng,J<Nj.,^ >-(fl^ew(x + e,)sin0){N;.U <Nr_^ > - (ûJ^TTi){N g .J < Ng.„ >+(û>^em sin 0 ){N g .J < N r .^ > + iT){N’g_,}<N'g.^>-(n^m){Ng.,}<Ng., >]{»}' 2 9 = £ <âu >' [(GJ){N;_,} < N'r.^ > -{a>^mKl){Nr.,} < Nr., > - {Ci^em{x + e , ) sin 6 ) { N r . , } < N'g_^ > + (fî^em(x + e,) cos (9){ N r . , } < N'b.„ > + (n^em sm 0){N r., }< N ,., > 4.31 + (œ^em sin 0 ) { N r . , } < Ng.^ > + - k I , ) cos 2 0 ) { N r . , } < N r . , > - {co'^em cos 0 ) { N r . 4.4. Derivation of System Eigenproblem , } < N >]{uY\j\d^ = £ <âu >' [(GJ){N;_,} < N'r.^ > -{a>^mKl){Nr.,} < Nr., > - {Ci^em{x + e , ) sin 6 ) { N r . , } < N'g_^ > + (fî^em(x + e,) cos (9){ N r . , } < N'b.„ > + (n^em sm 0){N r., }< N ,., > 4.31 + (œ^em sin 0 ) { N r . , } < Ng.^ > + - k I , ) cos 2 0 ) { N r . , } < N r . , > - {co'^em cos 0 ) { N r . 4.4. Derivation of System Eigenproblem , } < N >]{uY\j\d^ 4.31 In order to obtain the unified governing equation of the element, equations (4.29) to (4.31) are added together to form the total element virtual work representing the discretized form of principle of virtual work (4.5): 4.32 4.32 4.32 Leading to the following matrix form: Leading to the following matrix form: Leading to the following matrix form: Leading to the following matrix form: 11^; =< Su >' (-û}^[MY H K Y){uY 4.33 4.33 where \M\" and \KY are the element mass and stiffiiess matrices, respectively, and are calculated from following equations: where \M\" and \KY are the element mass and stiffiiess matrices, respectively, and are calculated from following equations: {MY = £' [(/«){^B-».} < > +(e»« cos 0){Ng_J <Nr.,> + i^n){NB-y) < ^B-y > -(^"' slu 0){Ng_^} < Nr., > + (mK^l){Nr.,) < Nr-, > -{em sin 0){Nr.,} < N > + {em cos 0){Nr.,} < N >]\|j\|d^ 4.34 {MY = £' [(/«){^B-».} < > +(e»« cos 0){Ng_J <Nr.,> + i^n){NB-y) < ^B-y > -(^"' slu 0){Ng_^} < Nr., > + (mK^l){Nr.,) < Nr-, > -{em sin 0){Nr.,} < N > + {em cos 0){Nr.,} < N >]\|j\|d^ 4.34 4.34 [KY =[KJ +{Ty[K,Y +{x + e,y[K,Y 4.35 [KY =[KJ +{Ty[K,Y +{x + e,y[K,Y 4.35 4.35 3 0 [K,y = £ ' < N i „ > h e i ^){n ;.j < n ;., > + (E I^){N IJ < N l^ > H E I^ ){N l^ } < N l ^ > + {GJ){Ny^} < > +(Q^em sin 6){Ng_^) < Nj-_^ > 4.36 + {Q^emsm0){Nj._^}<Ns_^ > - ( Q ^ i n ) { N < N b_, > + - 'c^,)cos 20){Nr.^} < >]\j\d^ [K,y = £ ' < N i „ > h e i ^){n ;.j < n ;., > + (E I^){N IJ < N l^ > H E I^ ){N l^ } < N l ^ > + {GJ){Ny^} < > +(Q^em sin 6){Ng_^) < Nj-_^ > 4.36 + {Q^emsm0){Nj._^}<Ns_^ > - ( Q ^ i n ) { N < N b_, > + - 'c^,)cos 20){Nr.^} < >]\j\d^ 4.36 [K^Y = f ' [ { N I J < N l„ > + {N IJ < 4.37 [K^Y = f ' [ { N I J < N l„ > + {N IJ < 4.37 [^ 3]' = f ,' m^ern cos e){N',.J < N^., > -(Q^em sin < N^., > + (Q^em sin d){Nj._^} < N'g,^ > -(Q^em cos 6){Nt.^} < N'g.^ >p\d^ 4.38 [^ 3]' = f ,' m^ern cos e){N',.J < N^., > -(Q^em sin < N^., > + (Q^em sin d){Nj._^} < N'g,^ > -(Q^em cos 6){Nt.^} < N'g.^ >p\d^ 4.38 4.38 The system eigenproblem is then obtained by assembling the elements equations (using the well-known FEM assembly algorithm and connectivity table [32,33]): NE i r ^ = 'Z '^ M = o 4.39 4.39 or w.. 4.5. Numerical Results To verify the formulation introduced in this chapter, the free vibrations of a certain number of illustrative beam configurations, exhibiting different couplings, were investigated and their natural frequencies and mode shapes were evaluated. 4.4. Derivation of System Eigenproblem =<âu> ([Æ] - Û)^ [M]) {«} = 0 4.40 where NE and NE [M] = Y ,W T 4.42 and Since the virtual displacement < > is arbitrary, then one can write: Since the virtual displacement < > is arbitrary, then one can write: 31 { [ K ] - c û \ M ] ) { u ) = 0 { [ K ] - c û \ M ] ) { u ) = 0 4 .4 3 The equation (4.43) is a linear eigenvalue problem in which the natural frequencies are eigenvalues and the vectors of nodal variables, mode shapes, are the eigenvectors. Equation (4.43) can be solved using any numerical method, and in this thesis MATLAB® and MAPLE® programs have been used to calculate the natural frequencies and mode shapes. 4.5.1. Bending Vibrations of Uncoupled Beam First Three Modes of Out-Of-Plane Vibrations of a Cantilever Beam obtained by FEM 0.5 1 -0.5 Length of the beam -^Mode 1 -♦-Mode 2 -•-Mode 3 First Three Modes of Out-Of-Plane Vibrations of a Cantilever Beam obtained by FEM Length of the beam Figure 4.3: The first three mode of out-of-plane vibrations of a cantilever beam based on FEM method. 4.5.1. Bending Vibrations of Uncoupled Beam As the first example, the out-of-plane flexural vibration of a beam was studied. The analytical solution for this beam is available (see Appendix A) and the natural frequencies and mode shapes can be found in Vibrations text books [34]. The FEM results can then be compared with the exact analytical solution, and the convergence of the FEM method can therefore be evaluated. The beam parameters for the following example are considered as: Ely =1 ; /« = 1 ; I, = 1 Ely =1 ; /« = 1 ; I, = 1 and the pre-twist angle of beam is zero. The differential equation of motion (2.1) for single uncoupled out-of-plane vibrations of beam reduces to: 3 2 { E l y V / y - fy^wjvv = 0 4 .4 4 { E l y V / y - fy^wjvv = 0 4 .4 4 By implementing the Galerkin-type weighted residual fomiulation, the element discretized form of equation (4.44) can be written as: {àw"EIyW" - Swco^mw}Jd^ 4.45 4.45 The cubic Hermite shape functions were used and the FEM results along with the exact values obtained by analytical solution (see Appendix A) for the first five natural frequencies are shown in table 4.1. Table 4.1: The first five natural frequencies of out-of-plane Table 4.1: The first five natural frequencies of out-of-plane vibration of a cantilever beam Table 4.1: The first five natural frequencies of out-of-plane vibration of a cantilever beam Natural Frequency (rad/s) Exact analytical method [34] FEM With 5 elements Error (%) for n = 5 FEM With 20 elements Error (%) for n ^ 20 CO; 3.51602 3.51601 0.000 3.51603 0.000 (02 22.0345 22.0455 0.050 22.0345 0.000 COj 61.6972 61.9188 0.359 61.6982 0.002 (0^ 120.9019 122.3196 1.173 120.9094 0.006 (05 199.8595 203.0202 1.574 199.8933 0.010 The mode shapes corresponding to the first three natural frequencies were calculated by the FEM method and are shown in Figure 4.2. 33 First Three Modes of Out-Of-Plane Vibrations of a Cantilever Beam obtained by FEM 0.5 1 -0.5 Length of the beam -^Mode 1 -♦-Mode 2 -•-Mode 3 Figure 4.3: The first three mode of out-of-plane vibrations of a cantilever beam based on FEM method. 4.5.2. Coupled Bending-Torsion Vibrations For the case of coupled bending-torsion, the governing differential equations of motion (2.18) and (2.19) and the shear force and moments, equations (2.20) to (2.22), are coupled due to the rotating speed fi and eccentricity e effects. The mass and stiffiiess matrices, in this case, both contain coupling terms. Following a procedure similar to the pure bending case, the FEM final element equation changes to: IFJ. = < & > ' {[K Y-(o\M Y){uY 4.46 4.46 where: where: [MY = £' [M iN e-J < > +(e»i cos < Nj._^ > + (f»K-m){^r-^} < > Hem cos < Ng_^ >]\j\d4 4 .4 7 [MY = £' [M iN e-J < > +(e»i cos < Nj._^ > + (f»K-m){^r-^} < > Hem cos < Ng_^ >]\j\d4 4 .4 7 4 .4 7 3 4 and [KY =[KJ+ (Ty[K, Y + {X + e,y [ K J [KY =[KJ+ (Ty[K, Y + {X + e,y [ K J [KY =[KJ+ (Ty[K, Y + {X + e,y [ K J 4.48 with with + -< ,)c o s 20){Nr.^}<Nr.^ >]\j\d^ 4.49 {K,Y = f ' >]\j\d4 4.50 [^ 3]' = f m^ern cos 0){N',_J < > -(Q^em cos N',.„ >]\j\d^ 4.51 [^ 3]' = f m^ern cos 0){N',_J < > -(Q^em cos N',.„ >]\j\d^ 4.51 [^ 3]' = f m^ern cos 0){N',_J < > -(Q^em cos N',.„ >]\j\d^ 4.51 4.5.2.I. Non-rotating Beam Aircraft wings and thin-walled open section beams fall in this category, since, in these cases, the equations governing bending and torsion displacements are coupled due to the eccentricity between the mass and elastic axes. As the first example, the natural frequencies of a typical cantilever uniform aircraft wing were calculated. The beam specifications are: L = 6.0 m e = 0.18 m m = 35.75 kg/m E l =9.15 MN.m^ (77=0.988 MN.m^ nt.km^ = 8.65 kg.m 35 and the FEM results were compared with the exact solution obtained from the DSM method [22] (Table 4.2): Table 4.2: The first six natural frequencies of coupled bending-torsion vibrations of a cantilever beam Natural Frequency (rad/s) Exact Method (DSM) FEM n = 30 Error of FEM (%) CO; 49.62 49.62 0.01 (02 97.04 97.05 0.01 COj 248.87 249.07 0.08 (0^ 355.59 355.78 0.05 (05 451.46 452.58 0.25 (06 610.32 613.59 0.54 Table 4.2: The first six natural frequencies of coupled bending-torsion vibrations of a cantilever beam As the second example, an aircraft wing, modeled by three uniform beams with different cross section parameters, was studied to show the flexibility and applicability of the FEM method. The beam is shown in Figure 4.3, and the beam parameters are given in Table 4.3. Table 4.3: The mechanical and geometrical parameters of the three-beam wing Section I Section II Section III L = 6.0 tn /; =2m I2 =2m 1} =2m m = 35.75 kg/m m j = m m2 = (2/3)* m m} = (l/3)m fff.Ary = 8.65 kg.m (m .k j ), = (m .k j )i=(2/3)m.kj‘ (m .kj)}= (l/3)m .kj‘ E l =9.15 MN.m^ E I,= E I El2 = (2/3)EI El3 = (l/3)EI (77=0.988 MN.m^ GJ1 — GJ GJ2 = (2/3)GJ GJ3 = (1/3)GJ ^ = 0.18 m ^ = 0.18 m c = 0.18 m c = 0.18 m 3 6 Figure 4.4: The three-beam airplane wing with coordinate system and geometry. Figure 4.4: The three-beam airplane wing with coordinate system and geometry. The coupled natural frequencies obtained from the FEM and the exact DSM method results [22] are presented in Table 4.4. The convergence test results are also shown in Figure 4.4. Table 4.4: The first five natural frequencies of the three-beam aircraft wing Natural Frequency (rad/s) Exact Method (DSM) FEM n = 15 Error of FEM (%) CO/ 74.43 74.43 0.00 0)2 128.57 128.58 0.00 0)3 253.40 253.56 0.06 0)4 376.59 376.96 0.10 0)5 431.29 431.97 0.16 Table 4.4: The first five natural frequencies 3 7 Convergence test for ilrsi five natural frequencies for 3-beam wing model 3.00 2.70 2.40 2.10 1.80 I 1.50 0.90 0.60 0.30 0.00 27 30 21 24 12 15 18 6 9 3 -omega 1 - omega 2 -omega 3 - omega 4 -omega 5 Number of elements Figure 4.5: The FEM convergence results for three-beam wing. Convergence test for ilrsi five natural frequencies for 3-beam wing model 3.00 2.70 2.40 2.10 1.80 I 1.50 0.90 0.60 0.30 0.00 27 30 21 24 12 15 18 6 9 3 -omega 1 - omega 2 -omega 3 - omega 4 -omega 5 Number of elements Convergence test for ilrsi five natural frequencies for 3-beam wing model Number of elements Figure 4.5: The FEM convergence results for three-beam wing. As it can be seen, the FEM method exhibits satisfactory convergence rates, where the error for the first natural frequency obtained by a six-element model is 0.06%, and for the first five frequencies a model made of 30 elements ends up with errors less than 0.2%. 4.S.2.2. Axially Loaded Beam The analysis of an axially loaded beam can be considered as the first step in the modeling of rotating systems such as helicopter, propeller and turbine blades, where the element centrifugal force is replaced by a constant axial load. The differential equations of motion governing coupled bending-torsion vibrations of an axially loaded beam have been introduced in [23]; E l y - (Tw'y + T e f - n>^/n(w-ef)) = 0 4.52 4.52 ■ (GJ + T K j ) f + Tew" - (o^m(Kl(t> - ew) = 0 4.53 4.53 4.53 3 8 where the force boundary conditions at free end, in this case, are: K = -E ly \v " + r(w ' - e(f>') = 0 4 54 K = -E ly \v " + r(w ' - e(f>') = 0 4 54 = {GJ + TkJ)(!)' - Tew' = 0 4.55 My = ElyW" = 0 4.56 My = ElyW" = 0 4.56 In order to investigate the vibrational behaviour of axially loaded beams, a cantilever beam with semi-circular cross section (Figure 4.5) and the following properties was studied [20,23]: L = 0.82 m g = 0.0155 m m = 0.835 kg/m £■/= 6380.14 n W C?/= 43.46 N.m^ nukm = 0.000501 kg.m L = 0.82 m g = 0.0155 m m = 0.835 kg/m £■/= 6380.14 n W C?/= 43.46 N.m^ nukm = 0.000501 kg.m L = 0.82 m g = 0.0155 m 39 z 15.5 mm Figure 4.6: The bending-torsion coupled beam cross-sectional geometry. Figure 4.6: The bending-torsion coupled beam cross-sectional geometry. The free vibrations of beam in two different situations were studied. First, the axial force was considered to be zero, T = 0, and the first five natural frequencies of the beam were evaluated (Table 4.5): Table 4.5: The bending-torsion natural frequencies of the cantilever beam (no axial load (r=0)) Natural Frequency (Hz) Reference Frequency [20] FEM with 20 elements Error of FEM (%) U] 62.60 62.61 0.02 130.18 130.21 0.02 Wi 261.15 261.66 0.20 0)4 421.36 423.59 0.53 0)5 612.09 618.52 1.05 Table 4.5: The bending-torsion natural frequencies of the cantilever beam (no axial load (r=0)) 4 0 If the axial force is applied to the beam, then the natural frequencies will change. Including a compressive force T = -1790 (N), the natural frequencies have been sought and shown in Table 4.6. 4.S.2.2. Axially Loaded Beam Table 4.6: The bending-torsion natural frequencies of the axially loaded cantilever beam (T= -1790 N) Natural Frequency (Hz) Reference Frequency 120] FEM with 20 elements Error of FEM (%) Ul 60.23 60.24 0.02 ' 0)2 128.42 128.45 0.02 Us 257.96 258.46 0.19 U4 415.54 417.74 0.53 Us 604.60 611.12 1.08 Table 4.6: The bending-torsion natural frequencies of the axially loaded cantilever beam (T= -1790 N) One can see that the natural frequencies for a beam with compressive axial force are smaller than those of same beam without axial load. This can be explained by the fact that the compressive axial force decreases the flexural rigidity of the beam, while tensile axial force increases the so-called geometric stiffness of the system [23]. 4.S.2.3. Non-rotating Pre-twisted Beam For pre-twisted beam the coupling between flap-wise bending and torsion displacements, equations (2.18) and (2.19), occurs because of eccentricity, e, and pre twist angle, 0, appear in coupling terms. As an example, the FEM analysis of a beam with following characteristics was studied; L = 40.0 in e = 0.4 in 6 = 0 degree L = 40.0 in e = 0.4 in 6 = 0 degree L = 40.0 in e = 0.4 in 6 = 0 degree «1 = 0.0015 slug/in = 0.18 in^ km2^ = 0.71 in^ £ /= 25000 Ih.ir? (?J=9000 Ih.in^ «1 = 0.0015 slug/in = 0.18 in^ km2^ = 0.71 in^ £ /= 25000 Ih.ir? (?J=9000 Ih.in^ The FEM results for the first five natural fi'equencies were calculated and compared to the reference values obtained from the Integrating Matrix Method (IMM) [2] (see Table 4.7). Table 4.7: The bending-torsion natural frequencies of the pre-twisted clamped-free beam Natural Frequency (rad/s) IMM Method (15 stations) FEM element With 30 elements Error of FEM method (%) CO; 31.05 31.059 0.029 C02 193.74 193.748 0.004 COj 390.87 390.923 0.014 co^ 539.54 539.598 0.011 ^5 1043.94 1044.306 0.035 All examples presented in this section confirmed the applicability and good convergence of the FEM formulation developed for coupled bending-torsion beam vibrations. 4.5.3. Coupled Bending-Bending Vibrations For the coupled flap-lag vibrations, the equations of motion (2.1) and (2.2) reduce to (2.12) and (2.13), where the natural (force) boundary conditions at fi-ee end can be written as equations (2.14) to (2.17). 42 42 As already stated, asymmetric geometry leads to the coupling terms in the equations. The rotating speed, ft, appears in both centrifugal force, T, and centripetal term, -ft^/«v. The centrifugal force increases the system stiffhess and the centripetal force, due to its negative sign, decreases the rigidity of the beam for in-plane vibrations [16,21]. The resulting element mass and stiffhess matrices produced by FEM approach can then be written as: {MY = > +(/h){A^5-v} < >V\d^ 4.57 4.57 nd [^ r= [^ ,r+ (T )'[^ ,r 4 .5s and [^ r= [^ ,r+ (T )'[^ ,r 4 .5s [^ r= [^ ,r+ (T )'[^ ,r 4 .5s where where [7^,]' = £ ' < Nl.„ > < N l , > + (EI,){N ;_J < > + (EI^){N l^} < > 4.59 > p\d^ and and [K,Y = £' < a ;., >]\|7\|d^ 4.6O The rotating beam analyzed in this section is a scaled model of propeller blade WADC S-5 [2,16,27], which is 24" long with a hub radius (offset) of 6". The mechanical and geometrical properties of the blade are given in table 4.8. 43 Table 4.8: The properties of the scaled propeller blade model WADC S-5, with hub radius e/ = 6 (In) and blade length L = 18 (In) Sect/on X, m, El[, El„, B, number In Ib.s^/in^ Ib.in^ Ib.ln^ deg 1 0.0 1.026x10^ 0.200x10 63x10* 30.5 2 2.0 0.696 0.110 49 25.2 3 4.0 0.660 0.083 46 20.1 4 6.0 0.608 0.058 44 14.8 5 8.0 0.564 0.042 43 9.6 6 10.0 0.535 0.031 43 4.7 7 12.0 0.520 0.027 44 0.0 8 14.0 0.506 0.026 47 -4.2 9 16.0 0.498 0.025 51 -7.5 10 18.0 0.498 0.024 56 -10.0 Table 4.8: The properties of the scaled propeller blade model WADC S-5, with hub radius e/ = 6 (In) and blade length L = 18 (In) The flexural rigidities in global coordinates at each point along the length of the beam can be obtained by following equations [16] (see Figure 2.1). 4.5.3. Coupled Bending-Bending Vibrations EI^ = EI^ cos^ 6 + EI^ sin^ 6 4.61 Ely = EI^ cos^ 6 4- EI^ sin^ 6 4.62 E l^ = {El^ - EI^ ) sin 9 cos 9 4.63 4.63 where the EI^ and El,, flexural rigidities are evaluated with respect to the element principal coordinates whereas Ely and EI^ are those calculated with respect to the global coordinates; d is the pre-twist angle assumed to be constant along each element. The natural frequencies obtained from FEM method are shown in Table 4.9. The first three natural frequencies for different angular velocities were obtained and compared with the experimental values [2,5,16,27]. It can be seen that comparing with other numerical approaches [2] and [5], the frequencies obtained from the FEM are close enough to the experimental results. This verifies the validity of FEM formulations and its good convergence for coupled bending-bending vibrations. The mode shapes for three different angular speeds of blade were investigated and shown in Figures 4.6 to 4.8. where the EI^ and El,, flexural rigidities are evaluated with respect to the element principal coordinates whereas Ely and EI^ are those calculated with respect to the global coordinates; d is the pre-twist angle assumed to be constant along each element. The natural frequencies obtained from FEM method are shown in Table 4.9. The first three natural frequencies for different angular velocities were obtained and compared with the experimental values [2,5,16,27]. It can be seen that comparing with other numerical approaches [2] and [5], the frequencies obtained from the FEM are close enough to the experimental results. This verifies the validity of FEM formulations and its good convergence for coupled bending-bending vibrations. The mode shapes for three different angular speeds of blade were investigated and shown in Figures 4.6 to 4.8. 4.5.3. Coupled Bending-Bending Vibrations 44 Table 4.9: The natural frequencies (Hz) of the first three modes of v of propeller blade WADC S-5 Blade rotary speed (rpm) Mode numbe r FEM n = 9 Result of Reference [2] Result of Reference [5] Experlme nt [27] 1 40.32 40.96 39.89 40.08 1567 2 108.49 109.22 107.40 3 279.27 279.79 276.32 1 40.70 41.35 40.26 1589 2 109.01 109.77 107.93 107.53 3 279.92 280.47 276.97 1 58.99 60.07 58.05 58.73 2609 2 137.18 139.52 135.99 3 316.82 309.40 313.98 1 59.08 60.16 58.14 2614 2 137.33 139.68 136.14 137.02 3 317.03 319.62 314.19 1 76.87 78.34 75.30 76.52 3583 2 168.08 166.25 170.60 3 361.38 357.70 1 93.48 91.42 93.07 4486 2 197.91 3 407.24 1 94.42 4537 2 199.61 196.41 202.53 3 409.91 1 119.17 116.07 117.50 5884 2 244.05 3 481.05 e 4.9: The natural frequencies (Hz) of the first three modes of vibrations of propeller blade WADC S-5 45 First mode I6 Flap Lag Second mode - - Flap Third mode 1o Flap Lag Fourth mode I Flap Lag Fifth mode Sixth mode • ................ 1 1 % • - Flap — Lag - - Flap — Lag Seventh mode • Flap Eighth mode 1Q Flap Lag Figure 4.7: The first eight modes of the WADC S-5 propeller blade for 0 = 0 . First mode I6 Flap Lag Second mode - - Flap Fourth mode I Flap Lag I Sixth mode 1 1 % - - Flap — Lag 1 1 Eighth mode 1Q Flap Lag 1Q Figure 4.7: The first eight modes of the WADC S-5 propeller blade for 0 = 0 . 4 6 First mode Î IJÛ Q Flap Lag Second mode Third mode Q Flap Lag Fourth mode I iQ Flap Lag Fifth mode 1 Q Flap Lag Sixth mode § IaO Flap Lag Seventh mode • • Flap Eighth mode I 5 Flap Lag Figure 4.8: The first eight mode shapes of the fVADC S-5 propeller blade for ft = 1567 rpm. First mode Î IJÛ Q Flap Lag Second mode Fourth mode I iQ Flap Lag I iQ Sixth mode § IaO Flap Lag Fifth mode 1 Q Flap Lag § IaO Eighth mode I 5 Flap Lag I 5 Figure 4.8: The first eight mode shapes of the fVADC S-5 propeller blade for ft = 1567 rpm. 4.5.4. Coupled Bending-Bending-Torsion Vibrations In previous sections, the coupling between out-of-plane (flap) bending and torsion displacements and also the coupling between flap and in-plane (lag) displacements for rotating and non-rotating beam structures were studied. In this section, the free vibration analysis of the triply coupled bending-bending-torsion beams is investigated. 4.5.3. Coupled Bending-Bending Vibrations 4 7 Displacements Displacements Displacements Displacements 4Æ- 3 0 Î0 II 1 fDf 3O CLfD I f I £ &fD Displacements Displacements Displacements Displacements Displacements Displacements 4Æ- 3 0 Î0 II 1 fDf 3O CLfD I f I £ &fD Displacements Displacements Displacements Displacements Displacements One can see that by increasing the angular velocity modes exhibit less deformations since the centrifugal force increases the blade stiffness. Comparing flap mode shapes for two cases, li = 1567 (rpm) and fl = 5884 (rpm), shows that increasing the angular velocity of the blade leads to different mode shapes in higher frequencies (see for example the 8'’ mode shapes). 4.5.4.I. Vibrations of Non-rotating Uncoupled Beam If none of the parameters H, e, 6 appears in the equations of motion, for a symmetric uniform beam, the equations are uncoupled and reduce to the well-known forms of pure bending vibration and pure torsion vibration of a beam (see Appendix A). To find dimensionless natural frequencies, the beam parameters Ely, EI^, GJ, m and K„, are considered to be unit. The FEM results for such a beam are presented in table 4.10. Table 4.10: The uncoupled bending, bending and torsion natural frequencies of the clamped-free beam Natural Frequency (rad/s) CO/ 0)4 0)5 0)6 0)7 0)8 0)p (0/%// FEM element With 20 elements 1.571 3.516 4.723 7.905 11.135 14.433 17.820 21.315 22.035 4 9 Table 4.10: The uncoupled bending, bending and torsion natural frequencies of the clamped-free beam Natural Frequency (rad/s) CO/ 0)4 0)5 0)6 0)7 0)8 0)p (0/%// FEM element With 20 elements 1.571 3.516 4.723 7.905 11.135 14.433 17.820 21.315 22.035 Table 4.10: The uncoupled bending, bending and torsion natural frequencies of the clamped-free beam Natural Frequency (rad/s) CO/ 0)4 0)5 0)6 0)7 0)8 0)p (0/%// FEM element With 20 elements 1.571 3.516 4.723 7.905 11.135 14.433 17.820 21.315 22.035 4 9 It can be seen that the natural frequencies evaluated from uncoupled bending- bending-torsion equations are equal to the one calculated by separate equations. It means that U2,3 and Ui0,n are repeated natural frequencies corresponding to the first and second frequencies of the pure lateral vibrations of a cantilever beam, and w / and are the first two frequencies corresponding to the torsional vibrations of the beam. It is also to be mentioned that since a mesh of only 20 elements has been used, then the third and higher frequencies of torsion vibrations are not accurate enough. Investigation in error shows that for Wj = 7.905 the error is 0.65%, for Wg = 11.135 the error is 1.27% and the error increases up to repeated frequencies U]o,ii = 22.035, for which the error decreases to 0.005%. This means the first two frequencies of each uncoupled vibrations are obtained accurately by only 20 elements. 4.S.4.2. Vibrations of Non-rotating Coupled Beam A good aircraft wing-beam model exhibits the triply coupled flap-lag-torsion Vibrational behaviour. To verify the validity of the presented FEM method for triply coupled vibrations, a beam with the following properties was investigated [2,16,28,29]: L = 40.0 in e= 1.414 in 6 —45 deg m = 0.0015 slug/in kmi = k„2 = 1.0 in £ /, = 25000 Ib.in^ ET( = 75000 Ib.in^ (7/= 9000 Ib.in^ L = 40.0 in e= 1.414 in 6 —45 deg m = 0.0015 slug/in kmi = k„2 = 1.0 in £ /, = 25000 Ib.in^ ET( = 75000 Ib.in^ (7/= 9000 Ib.in^ e= 1.414 in 6 —45 deg m = 0.0015 slug/in 5 0 The flexural rigidities EI^, Ely and EI^, are evaluated from equations (4.56), (4.57) and (4.58). The FEM results for natural frequencies of the coupled beam are shown in table 4.11. The convergence rates of the FEM method for the first four frequencies were also investigated and are shown in Figure 4.9. Table 4.11: The coupled bending-bending-torsion natural frequencies of the pre-twisted cantilever beam Natural Frequency (rad/s) TMM[2] FEM n = 5 Error (%) fern = 5 FEM n - 1 0 Error (%) for 10 CO; 30.8295 30.8320 0.008 30.8300 0.002 CO2 53.8277 53.8285 0.001 53.8278 0.000 COi 184.6175 185.1379 0.282 184.7376 0.065 co^ 337.3333 337.5015 0.050 337.3440 0.003 CO5 484.3373 493.1382 1.817 486.4762 0.442 Convergence test of first four frequencies for non-rotating triply coupled beam 1.00 0.80 - ^ 0.60 - ÏUJ 0.40 - 0.20 - 0.00 10 9 8 7 6 5 4 3 2 -Iomega 1 omega 2 omega 3 -♦-omega 4 Number of elements Figure 4.10: The FEM convergence results for non-rotating triply coupled beam. Table 4.11: The coupled bending-bending-torsion natural frequencies of the pre-twisted cantilever beam Natural Frequency (rad/s) TMM[2] FEM n = 5 Error (%) fern = 5 FEM n - 1 0 Error (%) for 10 CO; 30.8295 30.8320 0.008 30.8300 0.002 CO2 53.8277 53.8285 0.001 53.8278 0.000 COi 184.6175 185.1379 0.282 184.7376 0.065 co^ 337.3333 337.5015 0.050 337.3440 0.003 CO5 484.3373 493.1382 1.817 486.4762 0.442 Convergence test of first four frequencies for non-rotating triply coupled beam 1.00 0.80 - ^ 0.60 - ÏUJ 0.40 - 0.20 - 0.00 10 9 8 7 6 5 4 3 2 -Iomega 1 omega 2 omega 3 -♦-omega 4 Number of elements Figure 4.10: The FEM convergence results for non-rotating triply coupled beam. 4.S.4.2. Vibrations of Non-rotating Coupled Beam Table 4.11: The coupled bending-bending-torsion natural frequencies of the pre-twisted cantilever beam Natural Frequency (rad/s) TMM[2] FEM n = 5 Error (%) fern = 5 FEM n - 1 0 Error (%) for 10 CO; 30.8295 30.8320 0.008 30.8300 0.002 CO2 53.8277 53.8285 0.001 53.8278 0.000 COi 184.6175 185.1379 0.282 184.7376 0.065 co^ 337.3333 337.5015 0.050 337.3440 0.003 CO5 484.3373 493.1382 1.817 486.4762 0.442 Table 4.11: The coupled bending-bending-torsion natural frequencies of the pre-twisted cantilever beam Convergence test of first four frequencies for non-rotating triply coupled beam 1.00 0.80 - ^ 0.60 - ÏUJ 0.40 - 0.20 - 0.00 10 9 8 7 6 5 4 3 2 -Iomega 1 omega 2 omega 3 -♦-omega 4 Number of elements Figure 4.10: The FEM convergence results for non-rotating triply coupled beam. Convergence test of first four frequencies for non-rotating triply coupled beam 1.00 0.80 - ^ 0.60 - ÏUJ 0.40 - 0.20 - 0.00 10 9 8 7 6 5 4 3 2 -Iomega 1 omega 2 omega 3 -♦-omega 4 Number of elements Convergence test of first four frequencies for non-rotating triply coupled beam Number of elements Figure 4.10: The FEM convergence results for non-rotating triply coupled beam. 51 51 One can see the FEM convergence rates are quite satisfactory where for first three natural frequencies as a five-element model results in an error less than 0.1%. 4.S.4.3. Vibrations of Rotating Triply Coupled Beam Triply coupled rotating beam can be used in low frequency calculation of some helicopter blades, propeller blades and also compressor/turbine blades. The coupled bending-bending-torsion vibrations of a uniform rotating beam with a pre-twist angle are governed by equations (2.1), (2.2) and (2.3). In order to investigate the dynamic behaviour of such beams, a helicopter blade reported in literature [6] was investigated. The blade in this section is considered to be cantilever, while in the real model the blade is connected to rotating hub with some load paths [6]. The FEM results are shown in Tables 4.12 and 4.13 for non-rotating (fl = 0) and rotating (fl = 360 rpm), where the properties of beam are: L = 208 in e = -0.6 in ei = 52 in 6 = 15.026 deg m = 0.0015 Ib.sec^/in^ »i./r„/ = 0.89545x10’^ Ib.sec^ m.km2^ = 0 .0A Ib.sec^ .E/, = 0.2977x10^ Ib.in^ E/(=10^ Ib.in^ ( 7 J = 0 .2 x 1 0 Ib.in^ m = 0.0015 Ib.sec^/in^ 5 2 Table 4.12; The bending-bending-torsion natural frequencies of the pre-twisted cantilever rotating blade, 0 = 0 (rpm) Frequency (rad/s) Triple load path IMM method [6] FEM n = 40 Error (%) t o r n - 5 CO/ 11.4 11.4 0.17 0)2 65.8 66.4 0.79 0)3 70.4 71.7 1.94 Table 4.13: The bending-bending-torsion natural frequencies of the pre-twisted cantilever rotating blade ft = 360 rpm Frequency (rad/s) Triple load path TMM method [6] FEM n = 40 Error (%) for n^ 5 CO/ 45.1 46.7 3.52 0)2 72.3 73.7 1.91 0)3 125.0 133.1 6.47 Table 4.13: The bending-bending-torsion natural frequencies of the pre-twisted cantilever rotating blade ft = 360 rpm One can see that the difference between triple load path and cantilevered configurations for the first few frequencies of non-rotating case is negligible, while adding rotating speed to the blade causes slightly different dynamic behaviour. Higher frequencies exhibit larger differences between cantilevered blade and triple load path blade configurations. In order to study the effect of rotating speed on vibration frequencies and modes, the variation of the first 10 frequencies of the helicopter blade [6] (with cantilever boundary conditions) versus blade rotating speed is shown in Figure 4.10. 4.S.4.3. Vibrations of Rotating Triply Coupled Beam 53 Natural Frequencies of rotating blade versus rotating speed 900 Torsion 3 800 F laps 700 600 Torsion 2 Flap 4 Lag 2 (A 500 oo §3U" 2 400 300 LL Flap 3 200 Torsion 1 Flap 2 L agl Flap 1 100 0 50 100 150 200 250 300 350 Rotating speed (rpm) Figure 4.11: The effect of rotary speed on frequencies and modes of triply coupled rotating blade. Natural Frequencies of rotating blade versus rotating speed 900 Torsion 3 800 F laps 700 600 Torsion 2 Flap 4 Lag 2 (A 500 oo §3U" 2 400 300 LL Flap 3 200 Torsion 1 Flap 2 L agl Flap 1 100 0 50 100 150 200 250 300 350 Rotating speed (rpm) Natural Frequencies of rotating blade versus rotating speed Figure 4.11: The effect of rotary speed on frequencies and modes of triply coupled rotating blade. Figure 4.10 shows that, as expected, the rotating speed of beam can change the blade’s natural frequencies. One can see that due to the centripetal effect, the stiffening effect of rotating speed for lag vibrations decreases. The flap frequencies increase as rotating speed increases, but the increments in lag frequencies are less pronounced. A closer look at “Flap 4” and “Lag 2” modes reveals that by increasing the rotating speed flap frequency passes the lag frequency. Besides, the torsion frequencies are almost constant. The vibrational behaviour of the cantilever blade shown in Figure 4.10 is very similar to real triple load path blade model [16]. All examples of this chapter verified the applicability of FEM method as it has already been used in last decades for analysis of many flexible structures including beams. The convergence rates of the FEM methodology introduced in this chapter are very good, and for preliminary design purposes and to get the few first frequencies and modes, a model of only 10 to 30 elements can be exploited. 54 54 In next chapter, the DFE formulation based on Galerkin-type WRM will be discussed and the Dynamic Trigonometric Shape Functions (DTSF’s) will be employed to solve the governing differential equations of motion of dually and triply coupled vibrations of beams and rotating blades. 55 55 5.1. Introduction The Dynamic Finite Element (DFE) method is an intermediate formulation between exact DSM and classic FEM methods. The main difference between the classical FEM method and the DFE is “the definition of approximation functions”. In the traditional FEM (Chapter 4) the cubic polynomial shape functions for flexural displacements and linear shape function for torsional displacement are used, where theses shape functions are the solutions of static deformation of a beam in elastic area. In the DFE, the solutions of the differential equations governing the uncoupled vibrations of the beam are chosen as the basis functions of approximation space. The dynamic interpolation (shape) functions of approximation are then obtained based on the standard FEM approach. 5.2. Frequency Dependent Approximation Functions In general, the DFE and FEM methods follow the same formulation that means the DFE approach starts with Galerkin weak formulation and integral form of equations. Then the same integration by parts are applied to the equations to satisfy the natural (force) boundary conditions which results in equations (4.10) to (4.12). The major difference between two methods is the basis functions from which the shape functions are calculated. As already mentioned, the basis functions in the DFE method are dynamic (frequency dependent) trigonometric shape functions (DTSF’s). The basis functions are extracted from differential equations governing uncoupled vibrations of the beam. <P(^>f and <P(^>h the basis functions for flexural and torsional displacements, respectively, then can be defined as: 5 6 P(^) . cosh(/?\|) - cos(g^) _ sinh(y?\|) - sin(«^) ^ < P(^) >,=< cos(r^) — > 5.2 5.2 where or, ^ (separately calculated for v and »v) and t can be obtained by solving the uncoupled equations of beam for bending and torsion vibrations (see Appendix A); -4AC a = 1 2A ; j3=^ ^B+^\|B^-4AC 2A 5.3 r = col. mK„ 1 GJ 5.4 El T A = — ; B =------ ; C = -ml^a 5.5 -4AC a = 1 2A ; j3=^ ^B+^\|B^-4AC 2A 5.3 r = col. mK„ 1 GJ 5.4 El T A = — ; B =------ ; C = -ml^a 5.5 -4AC a = 1 2A ; j3=^ ^B+^\|B^-4AC 2A 5.3 5.3 r = col. mK„ 1 GJ 5.4 5.4 El T A = — ; B =------ ; C = -ml^a 5.5 5.5 The basis functions bave been manipulated to reduce to Hermite and linear basis functions for bending and torsion, respectively, as jg, % O- The Hermite basis functions bave been widely used in finite elements for many years, for two main reasons, they satisfy completeness and continuity. Completeness is satisfied by including the lowest order admissible term. Continuity condition is met such that the shape functions are continuous across all inter-element boundaries. With these conditions satisfied the DFE with its Hermite based trigonometric basis functions is guaranteed to converge monotonically to the exact solution. 5.2. Frequency Dependent Approximation Functions The classic basis functions of approximation space for standard “Hermite” beam element (flexural displacements) are 1, and the linear basis functions (torsional displacements) are 1, The so-called non-nodal approximation of displacements can then be written in terms of basis functions: 57 w{^)=<P{4)>f.^ {a j v(#)=<P(^)>^_, K ) f,(a = < f(a > , {«,} 5.6 5.7 5.8 w{^)=<P{4)>f.^ {a j v(#)=<P(^)>^_, K ) f,(a = < f(a > , {«,} 5.6 5.7 5.8 5.6 5.7 5.8 where the constant vectors {an-}, {ov} and {a,} can be calculated using the nodal displacements: =[-7n]/,v{^v} 5.9 5.10 5.11 5.9 5.10 5.11 5.11 where {h»„}, {v„} and are the nodal displacements, and {P n ]f-w /v and [ P „ ] , are: where {h»„}, {v„} and are the nodal displacements, and {P n ]f-w /v and [ P „ ] , are: where {h»„}, {v„} and are the nodal displacements, and {P n ]f-w /v and [ P „ ] , are: 1 0 cos(a„,J 0 0 sin(a„,J cosh%,,J-cosOz^„) wIvJ +Pw, ^ Pwlv ^w!v ^wlv Pw IV smh09^/v)-sm((z^/v) ^wtv ^ Pw IV wiv ^ w fv ^ H w t v n cinr/y ^ \ Æ/>sinh(g,„)+a,,,sin(g„,,) P„„cosh(JJ„i,)-a„i,cos(pc„i,) - a ^ / y S i i n a ^ / v ; 2 ^ 2 ^w/v PwIV ^wlv Pw IV 5.12 and and [p j. = 1 cos(r)r _____ sin(r) sin(r) 0 r 5.13 5.13 58 By determining the coefficient vectors {ok,}, {a,.} and {a,} in terms of nodal displacements, the element displacements can be written in terms of shape functions and nodal displacements (i.e., nodal approximations): =< >/.w bK) =< > {'vj 5.14 v(^) =<P(^) {v„} =< > {v„} 5.15 m = < P {^ )> , {PnV\%}-<Nr{^)>{(Pn) 5.16 =< >/.w bK) =< > {'vj 5.14 v(^) =<P(^) {v„} =< > {v„} 5.15 v(^) =<P(^) {v„} =< > {v„} 5.15 m = < P {^ )> , {PnV\%}-<Nr{^)>{(Pn) 5.16 where the bending and torsion shape functions are: < (^ ) > 1 = {- cos(a^) + cos(a(l - 1 )) cosh(/?) + cos(a) cosh(/? ( 1 - ^)) Pf - cosh(/9^) - —sin(a(l - ^)) sinh(y3) + ^ sin(a) sinh(/? 5.2. Frequency Dependent Approximation Functions ( 1 - ^))} a p 5.17 < (^) > 2 = - ^ {y^[cosh(/?(l - ^)) sin(a) - sin(a(l - ^)) cosh(/î) - sin(a^)] Dj- 5.18 + a[cos(a(l - ^))sinh(/?) - cos(a)sinh(y3(l - ^)) - sinh(y5^)]} < (^) > 2 = - ^ {y^[cosh(/?(l - ^)) sin(a) - sin(a(l - ^)) cosh(/î) - sin(a^)] Dj- 5.18 + a[cos(a(l - ^))sinh(/?) - cos(a)sinh(y3(l - ^)) - sinh(y5^)]} < ^ 5 (^) > 3 = — {- cos(a(l - ^)) + cos(a^) cosh(/?) - cosh(/3(l - ^)) Pf < ^ 5 (^) > 3 = — {- cos(a(l - ^)) + cos(a^) cosh(/?) - cosh(/3(l - ^)) Pf + cos(a) cosh(y0^) ——sin(a^) sinh(y3) + —sin(a) sinh(/7^)} 5.19 < ^ 5 (^) > 3 = — {- cos(a(l - ^)) + cos(a^) cosh(/?) - cosh(/3(l - ^)) Pf + cos(a) cosh(y0^) ——sin(a^) sinh(y3) + —sin(a) sinh(/7^)} 5.19 : >,= — {y9[-cosh(y3(^)sin(a) - sin(a(l - ^)) + sin(a^)cosh(/?)] P f + a[-cos(a^)sinh(/î) - sinh(yî(l - ^)) + cos(a)sinh(^^)]} and 59 < //j. (^) >, = ^ {sin(r) cos(r^) - cos(r)sin(r^)} 5.21 < //j. (^) >, = ^ {sin(r) cos(r^) - cos(r)sin(r^)} 5.21 < //j. (^) >, = ^ {sin(r) cos(r^) - cos(r)sin(r^)} 5.2 < //j. (^) >, = ^ {sin(r) cos(r^) - cos(r)sin(r^)} 5.21 The parameters D/and Dt are: The parameters D/and Dt are: a ^ -p ^ ap Dj. = aP{2[cos(a)cosh(P) - 1] + sin(a)sinh(>9)} 5.23 D, =sin(r) 5.24 a ^ -p ^ ap Dj. = aP{2[cos(a)cosh(P) - 1] + sin(a)sinh(>9)} 5.23 5.23 D, =sin(r) 5.24 5.24 where a and P in equations (5.17) to (5.24) can be replaced by «h- and P„ or % and py to obtain the shape functions of out-of-plane or in-plane displacements, respectively. where a and P in equations (5.17) to (5.24) can be replaced by «h- and P„ or % and py to obtain the shape functions of out-of-plane or in-plane displacements, respectively. The shape functions (5.17) to (5.22) are called Dynamic (frequency dependent) Trigonometric Shape Functions (DTSF). The bending shape functions have been shown in Figure 5.1 for the first four natural frequencies of a non-rotating beam with pure flap vibrations. 5.2. Frequency Dependent Approximation Functions 60 Shape functions of single bending vibrations for first natural frequency I 0<xK1 Shape function 1 Shape functions -<Shape functions Shape function 4 Shape functions of single bending vibrations for second natural frequency ê •10 0<xl<1 Shape function 1 Shape function 2 Shape function 3 Shape function 4 Shape functions of single bending vibrations for third natural frequency 40 Ir 5 -20 •60 0<xK1 Shape function 1 "O Shape function 2 Shape function 3 Shape function 4 Shape functions of single bending vibrations for fourth natural frequency I •10 •15 0<xK1 -«-Shape function 1 -O’ Shape function 2 Shape function 3 Shape function 4 Figure 5.1: The beam bending shape functions calcu at Its first four natural frequencies. 61 Shape functions of single bending vibrations for first natural frequency I 0<xK1 Shape function 1 Shape functions -<Shape functions Shape function 4 0<xK1 Shape functions of single bending vibrations for second natural frequency ê •10 0<xl<1 Shape function 1 Shape function 2 Shape function 3 Shape function 4 Shape functions of single bending vibrations for second natural frequency 0<xl<1 Shape functions of single bending vibrations for third natural frequency 40 Ir 5 -20 •60 0<xK1 Shape function 1 "O Shape function 2 Shape function 3 Shape function 4 Shape functions of single bending vibrations for fourth natural frequency I •10 •15 0<xK1 -«-Shape function 1 -O’ Shape function 2 Shape function 3 Shape function 4 Figure 5.1: The beam bending shape functions calculated at Its first four natural frequencies. 61 5.3. Derivation of System Eigenproblem Implementing the Galerkin-type Weighted Residual Method (WRM) on the governing differential equations (2.1) to (2.3) and applying the natural boundary conditions, the element virtual works (after discretization and performing appropriate integration by parts) is written in following form: (•) El + + 5w\Ci^em{x + e^)cos6)<f) 5.25 5.25 - ôw{û)^eml cos 9)^}d^ El El T + [5w”(r-^)w ' - 5 w '\-^ )w + 0w \j)w ]\ ^/.v = f ' - Sv"{j)v - 6v{co ^ml )v}df () E l. + {<^v"( ^3^ )w" - ^v'(Q ^em (x + e, ) sin d)^ 5.26 E l. + {<^v"( ^3^ )w" - ^v'(Q ^em (x + e, ) sin d)^ 5.26 + < 5 v[(fl" +(o^)eml sin 9)]^ - S v{n h n l )v}d^ FT FT T + [ S v ''i-^ )v ' - S v " 'i-^ )v + <5v'(y)v]:, E l. + {<^v"( ^3^ )w" - ^v'(Q ^em (x + e, ) sin d)^ E l. + {<^v"( ^3^ )w" - ^v'(Q ^em (x + e, ) sin d)^ 5.26 + < 5 v[(fl" +(o^)eml sin 9)]^ - S v{n h n l )v}d^ FT FT T + [ S v ''i-^ )v ' - S v " 'i-^ )v + <5v'(y)v]:, + < 5 v[(fl" +(o^)eml sin 9)]^ - S v{n h n l )v}d^ FT FT T + [ S v ''i-^ )v ' - S v " 'i-^ )v + <5v'(y)v]:, ( ...) ( ...) + ^ {0^{Q^eni(x + e^)cos 9)w' - S^(D.^em(x + e^)sin 9)v' + ^ {0^{Q^eni(x + e^)cos 9)w' - S^(D.^em(x + e^)sin 9)v' + + (o^)eml sin 9)\v - ô^{û)^eml cos 9)w + 5 ( j ) { Q . ^ m l c o s 29)^}d^ 5.27 5.27 + + (o^)eml sin 9)\v - ô^{û)^eml cos 9)w 5.27 + 5 ( j ) { Q . ^ m l c o s 29)^}d^ 6 2 where 0 <1. 5.3. Derivation of System Eigenproblem The approximations for >t», bw, v, bv, (j) and b<j> are made such that the integral terms (), () and () in the above equations (5.25) to (5.27) vanish, or: EL T [ a / '( - ^ ) - & v\j) - &iiû}hn[)]w= 0 5.28 El T [ J v " " (^ ) - 6v"{j) - 6v{o)^ml)]v = 0 5.29 + 0(/>(co^mlKl )](j> = 0 5.30 5.28 The equations (5.28) to (5.30) can be used to determine the parameters /3v and t following the approaches introduced in Appendix A. The components of element virtual work then reduce to the following forms: ~ r ' + 5w\0}em{x + e, ) cos 0)^ - 8\v{o)^eml cos 6 )<l>}d^ 5.31 El El T + [ S w \-^ )w ' - 0w \ - ^ ) w + 5w\-j)w][ 5.31 El El T + [ S w \-^ )w ' - 0w \ - ^ ) w + 5w\-j)w][ = J^' {6v"{^—^ )w " - 6v'{0.^em{x + e^)s\n 6)(f> + Sv{{Çl^ + (o^)eml sin 6 )\(l) - 5v{Çl^ ml )v)d^ 5.32 FT FI T + [Sv”{ ^ ) v ' - 8v”'{-jf-)v + 8 v \j)v ] [ = J^' {6v"{^—^ )w " - 6v'{0.^em{x + e^)s\n 6)(f> + Sv{{Çl^ + (o^)eml sin 6 )\(l) - 5v{Çl^ ml )v)d^ 5.32 FT FI T + [Sv”{ ^ ) v ' - 8v”'{-jf-)v + 8 v \j)v ] [ {8^(Q^em{x + e J c o s d ) w ' -8i^{Q^em(x + ef)smû)v' + 8<!>[{0.^ + (O^)eml sin 9)]v - 8(f>{co^eml cos 0)w + 8(^iQ^ml{Kl^ - Kl^)cos 20)(^}d^ 5 .3 3 63 and the element virtual work can be written as: and the element virtual work can be written as: and the element virtual work can be written as: Wf,=<Su>‘ [K{co)Y{uY 5.34 Wf,=<Su>‘ [K{co)Y{uY 5.34 Wf,=<Su>‘ [K{co)Y{uY Wf,=<Su>‘ [K{co)Y{uY Wf,=<Su>‘ [K{co)Y{uY 5.34 where the element nonlinear dynamic (frequency dependent) stiffness matrix, [ff(co)]^ can be written as: where the element nonlinear dynamic (frequency dependent) stiffness matrix, [ff(co)]^ can be written as: [K{(o)Y = [Kmvncoupua + 5.35 5.35 The coupled and uncoupled element stiffness matrices are extracted from equations (5.31), (5.32) and (5.33) considering the nodal parameters of equations (4.21) to (4.24): \.^i.^)\vncouplal ~ 5.36 + [{0}ml{Kl,-Kl)cos29){N,_^}<N,_^>+{-Q}ml){N,J<N,_^ >]d^ + [{0}ml{Kl,-Kl)cos29){N,_^}<N,_^>+{-Q}ml){N,J<N,_^ >]d^ = r < N l, > +{Nl^} < Nl„ >) -(o)^emlcose)({Ng_J < Nj.,^ > +{Nj.,^} < >) +(0^e/M(x+e,)cosg)({Ng_,,} < > +{A^r-p) < ■^b-h. The system equation then can be obtained by the standard assembly process: The system equation then can be obtained by the standard assembly process: The system equation then can be obtained by the standard assembly process: NE » :.= Z W i.'= 0 5.39 NE » :.= Z W i.'= 0 5.39 5.39 » :.= Z W i.'= 0 5.39 or à [K( )]{ } 0 5 40 or =< ài > [K(<y)] {m} = 0 5.40 =< ài > [K(<y)] {m} = 0 where: where: NE [X(®)] = 2][A:(®)] 5.41 1 5.41 The virtual displacements vector < ô« > in equation (5.40) is arbitrary, then the rest of equation should vanish: [K(co)]{u} = 0 5.42 5.42 The stiffhess matrix is frequency dependent and the equation (5.42) represents a nonlinear eigenproblem. The frequencies of this eigenproblem are calculated by Wittrick- Williams root counting algorithm [14,15,26] introduced in Chapter 3. 5.3. Derivation of System Eigenproblem > ) +[(0' +ûJ^)m/sin0)]({A^3.,} >+{Nr_^}<Ng,^ >) -(QYem{x+eY)sme){{N'g,^<N^_^ >-{N^.^} <N'g_^ >)]d# 5.37 5.37 where: 64 — j r : =— ; c? = y 5.38 5.38 5.4. Refined Dynamic Finite Element (RDFE) The parameters EIj,, EI^, T, m, GJ and ihkJ in equations (5.28) to (5.30) are assumed to be constant inside each element domain. If the beam specifications vary along the length of the beam, this assumption is not valid and the equations (5.28) to (5.30) 65 cannot result exactly the assumed basis functions. For small number of elements, where the variation of parameters are considerably large, vanishing (), () and (*) in equations (5.25), (5.26) and (5.27) results some errors. In order to avoid this error a compensating term can be defined for each variable parameter to be added to the equations, called deviator term. The variable parameters can be written as: E m = EI + EI^,y n ^ ) = T + T^,y m(4) = m + m^Ev 5.43 GJ{^) = GJ + GJ^,y E m = EI + EI^,y n ^ ) = T + T^,y m(4) = m + m^Ev 5.43 GJ{^) = GJ + GJ^,y 5.43 where El, T, m, GJ and ntK„^ are average values in each element, and the parameters with subscript “DEV” are the deviation of average values from exact values [21]. The uncoupled stiffness matrix then can be rearranged as: where El, T, m, GJ and ntK„^ are average values in each element, and the parameters with subscript “DEV” are the deviation of average values from exact values [21]. The uncoupled stiffness matrix then can be rearranged as: \.K.(p))Yu„coupled ~ , , !:! . + f ' [(Ü'm/(;v^,-Kl,)cos29){Nr_^} < > +(-Q^ml){Ns.^} < >]d^ +DEV where: where: 6 6 - < N i^ > +{A^;.J < yv;.„ >) ^ - 1(0 [m{Q - ;;/]({A^g_.} < N > +{/Vg_,} < Ng., >) + ^ ) (A^;. ^} < A^;.^ > -1(0^[/H/r„' (^) - hikJ ] {A^r-„ ) < A^r-„ ^ Adding DEV matrix drastically increases the aceuracy of the model and provides faster convergence. It can be seen from equation (5.45) if any parameter in beam specification is constant, the corresponding term will not appear in DEV matrix. 5.4. Refined Dynamic Finite Element (RDFE) For uniform rotating beam all geometrical deviator terms vanish and the deviator matrix reduces to following form in which the effect of variable axial force, centrifugal force, is taken into account: DEV = f ' J < >)]d^ 5.46 5.46 As it will be discussed later, without considering the deviator matrix, the convergence rate of DFE and FEM are nearly the same, but by “refining” the DFE element equations, the DFE convergence rates are effectively improved. The methodology presented here is called Refined DFE and will be used later in numerical examples where variable parameters and rotating speed are to be considered. 5.5. Numerical Results The DFE method has already been applied to many bending-torsion coupled vibration problems including axially loaded or materially coupled composite beams [21-25]. In this thesis, some illustrative numerical examples are discussed to verify the DFE formulation introduced in this chapter. The natural frequencies, mode shapes and 6 7 6 7 the convergence rate of DFE is investigated and compared with the existing results in literature and those presented in Chapter 4. 5.5.2. Coupled Bending-Torsion Vibrations The DFE analysis of coupled bending-torsion vibrations of beams for uniform beams as well as non-uniform composite beams, with or without axial forces has been well established [21-25]. In this section, some illustrative examples are studied to confirm the correctness of the presented DFE formulation. 5.5.1. Bending Vibrations of Uncoupled Beam As the first step, the natural frequencies and mode shapes of lateral vibrations of a non-rotating uniform beam is investigated. To implement the DFE, the integration by parts is applied to (4.41) and final equation will be in following form: If/,, =[àv\EI^)w' - 5.47 5.47 and the uncoupled stiffness matrix, equation (5.36), reduces to the following form: ________ ijO________ ________ £^i_________ c J N lJ ] Using the Wittrick-Williams (W-W) root counting algorithm [14,15,26], the natural frequencies can be evaluated from a one-element model. The first five frequencies of a uniform cantilever beam are shown in Table 5.1. The beam parameters are considered to be unit, Ely = /n = Z = 1. As already stated, the DFE formulation for uncoupled bending is equivalent to the exact DSM method, due to the fact that the basis functions are the solutions of uncoupled bending vibrations. That is the reason why using a one-element mesh results in the exact frequencies. 68 Table 5.1: The first five natural frequencies of out-of-plane vibration of a cantilever beam calculated by DFE Natural Frequency (rad/s) Exact analytical value [34] DFE With 1 elements Error (%) CO/ 3.51602 3.51602 0.000 C02 22.0345 22.0345 0.000 0)3 61.6972 61.6972 0.000 0)4 120.9019 120.9019 0.000 0)5 199.8595 199.8593 0.000 Table 5.1: The first five natural frequencies of out-of-plane vibration of a cantilever beam calculated by DFE The first three mode of vibration were also calculated by the DFE method and are plotted in Figure 5.2. First Three Modes of Out-Of-Plane Vibrations of a Cantilever Beam obtained by DFE 0.5 c I Mode 1 — - Mode 2 Mode 3 I -0.5 Length of the beam Figure 5.2: The first three out-of-plane modes of vibrations of a cantilever beam based on DFE method First Three Modes of Out-Of-Plane Vibrations of a Cantilever Beam obtained by DFE c II Length of the beam Figure 5.2: The first three out-of-plane modes of vibrations of a cantilever beam based on DFE method From Table 5.1 and Figure 5.2, one can see that the natural frequencies and mode shapes confirm the validity and complete agreement of the DFE method and exact results for pure bending vibrations of a cantilever beam. 69 5.5.2.I. Non-rotating Beam: As the first coupled bending-torsion case, the dynamic behaviour of untwisted uniform coupled beams is studied. The equations of motion, in this case, can be extracted from (2.18) and (2.19). The element stiffiiess matrix, equations (5.35), is then written as: [K(co)Y = + [K{<v)Ycour,e, 5.49 where i‘0 4=1 5.50 5.49 where 5.50 and Here, and {A^r-ç,} are the bending and torsional shape functions. A uniform cantilever wing [22] with following mechanical properties is considered: Here, and {A^r-ç,} are the bending and torsional shape functions. A uniform cantilever wing [22] with following mechanical properties is considered: Here, and {A^r-ç,} are the bending and torsional shape functions. A uniform cantilever wing [22] with following mechanical properties is considered: 7 0 L = 6.0 m e = 0.18 m m = 35.75 kg/m E/=9.75 MN.m^ GJ= 0.988 MN.m^ m.km^ = 8.65 kg.m L = 6.0 m e = 0.18 m m = 35.75 kg/m E/=9.75 MN.m^ GJ= 0.988 MN.m^ m.km^ = 8.65 kg.m The natural frequencies obtained from the DFE and FEM formulations along with those obtained from the exact DSM method [22] are presented in Table 5.2. The natural frequencies obtained from the DFE and FEM formulations along with those obtained from the exact DSM method [22] are presented in Table 5.2. Table 5.2: The first six natural frequencies of coupled bending-torsion vibrations of a cantilever beam Natural Frequency (rad/s) Exact Method (DSM) DFE method With 5 element Error of DFE method (%) FEM element With 30 elements Error of FEM 30-element (%) CO; 49.62 49.62 0.00 49.62 0.01 0)2 97.04 97.05 0.01 97.05 0.01 0)3 248.87 249.00 0.05 249.07 0.08 0)4 355.59 357.54 0.55 355.78 0.05 0)5 451.46 452.57 0.24 452.58 0.25 0)6 610.32 610.63 0.05 613.59 0.54 Table 5.2: The first six natural frequencies of coupled bending-torsion As it can be seen (Table 5.2), the comparison between higher modes obtained from the FEM and DFE (for example the fifth and sixth frequencies) shows the DFE’s higher convergence rates. A three-beam piecewise uniform wing, introduced in Table 4.3 and Figure 4.3, was also studied. The vibration analysis shows that DFE has better convergence rate than FEM, as a 6-element DFE model leads to the same results as a 15-element FEM model (Table 5.3). 5.5.2.I. Non-rotating Beam: 71 Table 5.3: The first five natural frequencies of the three-beam aircraft wing obtained by DFE Frequency (rad/s) Exact Method (DSM) DFE n = 6 Error of DFE (%) FEM n - 1 5 Error of FEM (%) CO; 74.43 74.44 0.02 74.43 0.01 CO2 128.57 128.59 0.01 128.58 0.06 COi 253.40 253.44 0.01 253.56 0.32 0^4 376.59 378.36 0.47 376.96 0.37 CO5 431.29 433.42 0.49 431.97 0.67 Table 5.3: The first five natural frequencies Table 5.3: The first five natural frequencies of the three-beam aircraft wing obtained by DFE Table 5.3: The first five natural frequencies As another example, the DFE method was also implemented to the beam of following properties [2,16]: Z, = 40.0 in g = 0.4 in m = 0.0015 slug/in ^m/ = 0.18 in^ km2^ = 0.71 in^ E7= 25000 Ib.in^ C /=9000 Ib.in^ Z, = 40.0 in g = 0.4 in m = 0.0015 slug/in ^m/ = 0.18 in^ km2^ = 0.71 in^ E7= 25000 Ib.in^ C /=9000 Ib.in^ The first five natural frequencies are calculated by DFE method and have been compared with Integrating Matrix Method [16] and the FEM (Chapter 4) as the reference values (see Table 5.4). 72 Table 5.4: The bending-torsion coupled natural frequencies of the clamped-free beam Natural IMM DFE method Error of DFE FEM element Error of FEM Frequency Method With method With method (rad/s) (15 stations) 5 element (%) 30 elements (%) CO; 31,05 31,06 0,02 31,059 0.03 C02 193,74 193,79 0,02 193,748 0.00 390,87 391,15 0,07 390,923 0.01 0)4 539,54 540,34 0,15 539,598 0.01 0)5 1043,94 1048,97 0,48 1044,306 0.04 Table 5.4: The bending-torsion coupled natural frequencies of the clamped-free beam The DFE convergence rates for the first five natural frequencies are shown in F The DFE convergence rates for the first five natural frequencies are shown in Figure 5.3. Figure 5.4 presents the comparison between the convergence rates of DFE and FEM for the third and fifth frequencies to highlight the difference between DFE and FEM in higher modes. Figure 5.4 presents the comparison between the convergence rates of DFE and FEM for the third and fifth frequencies to highlight the difference between DFE and FEM in higher modes. 5.5.2.I. Non-rotating Beam: Convergence test of DFE for bending-torsion vibrations 0.20 0.18 - 0.16 - 0.14 - 3 - k 0.10 - HI 0.08 - 0.06 - 0.04 - 0.02 - 0.00 30 25 20 15 10 5 ■ omega 1 -omega 2 -omega 3 -omega 4 - omega 5 Number of elements Figure 5.3: The DFE method convergence results for coupled bending-torsion vibrations. Convergence test of DFE for bending-torsion vibrations Convergence test of DFE for bending-torsion vibrations 0.20 0.18 - 0.16 - 0.14 - 3 - k 0.10 - HI 0.08 - 0.06 - 0.04 - 0.02 - 0.00 30 25 20 15 10 5 ■ omega 1 -omega 2 -omega 3 -omega 4 - omega 5 Number of elements Figure 5.3: The DFE method convergence results for coupled bending-torsion vibrations. 73 Convergence test of FEM a nd DFE for third and fifth frequencies for coupled bending-torsion vibrations 1.0 DFE-omega 3 FEM-omega 3 DFE-omega 5 FEM-omega 5 0.9 - 0.8 - 0.7 - 0.6 - 5 0.5 UJ 0.4 - 0.3 - 0.2 - 0.0 10 20 30 5 15 25 Number of elements Figure 5.4: The comparison between the DFE and FEM convergence for coupled bending-torsion free vibrations. Convergence test of FEM a nd DFE for third and fifth frequencies for coupled bending-torsion vibrations 1.0 DFE-omega 3 FEM-omega 3 DFE-omega 5 FEM-omega 5 0.9 - 0.8 - 0.7 - 0.6 - 5 0.5 UJ 0.4 - 0.3 - 0.2 - 0.0 10 20 30 5 15 25 Number of elements Convergence test of FEM a nd DFE for third and fifth frequencies for coupled bending-torsion vibrations f for coupled bending-torsion vibrations Figure 5.4: The comparison between the DFE and FEM convergence for coupled bending-torsion free vibrations. The mode shapes of flap and torsion vibrations of the beam have been calculated and shown in Figures 5.5 and 5.6. Mode shapes of out-of-plane vibrations of bending-torsion coupled beam a Length of beam -^ o m e g a l omega2 omegas omega4 -^omegas Figure 5.5: The DFE results for the first five modes of coupled bending-torsion vibrations for a cantilever beam; flap (bending) displacements. 5.5.2.I. Non-rotating Beam: Mode shapes of out-of-plane vibrations of bending-torsion coupled beam a Length of beam -^ o m e g a l omega2 omegas omega4 -^omegas Mode shapes of out-of-plane vibrations of bending-torsion coupled beam a Length of beam Figure 5.5: The DFE results for the first five modes of coupled bending-torsion vibrations for a cantilever beam; flap (bending) displacements. 7 4 First five modes of torsional vibrations of bending-torsion coupled beam 1.2 0.8 - —♦“ Mode 1 — — Mode 2 —A— Mode 3 • Mode 4 Mode 5 0.6 - a 0 .4 - 0.2 -0.2 Length of beam Figure 5.6: The DFE results for the first five modes of coupled bending-torsion vibrations for a cantilever beam; torsional displacements. First five modes of torsional vibrations of bending-torsion coupled beam First five modes of torsional vibrations of bending-torsion coupled beam Length of beam Figure 5.6: The DFE results for the first five modes of coupled bending-torsion vibrations for a cantilever beam; torsional displacements. The examples of this section show that the results obtained by the DFE are in complete agreement with the results of the FEM (Chapter 4) and those of literature. Also the mode shapes calculated by the DFE exhibit the expected behaviour of the coupled bending-torsion vibrations of the non-rotating beams. 5.S.2.2. Axially Loaded Coupled Beam: Considering the equations of motion introduced in expressions (4.48) and (4.49) [23], the element stiffness matrix for an axially loaded coupled bending-torsion beam can be written in following form: 5.52 5.52 5.52 where 75 _______________^ _______________ M______________ 5 _______________^ _______________ M______________ 5.53 and [^W]L,W = r [-(y)({A^;-J < K _, > +{N'r_,} < >) . + i o } ^ e m l ) i { N g . J < Nr_^ > + { N ^ . ^ } < Ng_ „ > )]d ^ 5.54 5.54 The same semi-circular beam configuration as presented in Figure 4.6 was then selected to investigate the DFE application to free vibration analysis of axially loaded beams. The beam properties are [20,23]: L = 0.82 m g = 0.0155 m m = 0.835 kg/m E /= 6380.14 N.m^ (7J= 43.46 N.m^ m.krn^ = 0.000501 kg.m For two cases of T = 0 and T= -1790 (N), the natural frequencies of beam were calculated, and the comparisons between DFE, FEM (Chapter 4) and reference values [20] are presented in Tables 5.5 and 5.6. 7 6 Table 5.5: The bending-torsion natural frequencies of the cantilever beam (no axial load (7^0)) F r e \| y % % % g S l 2 3 : 2 s «/ 62.60 62.60 0.00 62.61 0.02 130.18 130.75 0.44 130.21 0.02 « 5 261.15 261.56 0.16 261.66 0.20 0)4 421.36 423.25 0.45 423.59 0.53 0)5 612.09 616.03 0.64 618.52 1.05 Table 5.6: The bending-torsion natural frequencies of the axially loaded cantilever beam (J= -1790 N) Natural Frequency (rad/s) Reference Frequency [20] DFE with 6 elements Error of DFE (%) FEM with 20 elements Error of FEM (%) 0)1 60.23 60.25 0.04 60.24 0.02 0)2 128.42 128.74 0.25 128.45 0.02 0)3 257.96 258.48 0.20 258.46 0.19 0)4 415.54 416.79 0.30 417.74 0.53 0)5 604.60 607.87 0.54 611.12 1.08 The DFE convergence results for the beam subjected to a compressive axial 1790 (N) are shown in Figure 5.7. One can see that for the first four frequencies or for a 4-element model is less than 0.8% (the error for the first frequency obtaine model is less than 0.1%). 5.S.2.2. Axially Loaded Coupled Beam: Therefore, using 4 to 6 elements, one could accura Table 5.5: The bending-torsion natural frequencies of the cantilever beam (no axial load (7^0)) F r e \| y % % % g S l 2 3 : 2 s «/ 62.60 62.60 0.00 62.61 0.02 130.18 130.75 0.44 130.21 0.02 « 5 261.15 261.56 0.16 261.66 0.20 0)4 421.36 423.25 0.45 423.59 0.53 0)5 612.09 616.03 0.64 618.52 1.05 Table 5.5: The bending-torsion natural frequencies of the cantilever beam (no axial load (7^0)) Table 5.5: The bending-torsion natural frequencies Table 5.6: The bending-torsion natural frequencies of the axially loaded cantilever beam (J= -1790 N) Natural Frequency (rad/s) Reference Frequency [20] DFE with 6 elements Error of DFE (%) FEM with 20 elements Error of FEM (%) 0)1 60.23 60.25 0.04 60.24 0.02 0)2 128.42 128.74 0.25 128.45 0.02 0)3 257.96 258.48 0.20 258.46 0.19 0)4 415.54 416.79 0.30 417.74 0.53 0)5 604.60 607.87 0.54 611.12 1.08 Table 5.6: The bending-torsion natural frequencies of the axially loaded cantilever beam (J= -1790 N) The DFE convergence results for the beam subjected to a compressive axial load of 1790 (N) are shown in Figure 5.7. One can see that for the first four frequencies the error for a 4-element model is less than 0.8% (the error for the first frequency obtained by this model is less than 0.1%). Therefore, using 4 to 6 elements, one could accurately evaluate the beam’s first few frequencies. 77 Convergence test of DFE for Bending-Torsion coupled vibrations of an axially loaded beam, P = -1790 1.40 - 1.20 - 1.00 - r 0.80 - 0.60 - 0.40 - 0.20 - 0.00 4 6 8 10 12 16 14 18 20 -omega 1 -omega 2 -omega 3 -omega 4 -omega 5 Num ber of elem ents Figure 5.7: The DFE convergence results for axially loaded cantilever coupled beam (T = -1790 N). 5.S.2.2. Axially Loaded Coupled Beam: Convergence test of DFE for Bending-Torsion coupled vibrations of an axially loaded beam, P = -1790 1.40 - 1.20 - 1.00 - r 0.80 - 0.60 - 0.40 - 0.20 - 0.00 4 6 8 10 12 16 14 18 20 -omega 1 -omega 2 -omega 3 -omega 4 -omega 5 N b f l t Convergence test of DFE for Bending-Torsion coupled vibrations of an axially loaded beam, P = -1790 Num ber of elem ents Figure 5.7: The DFE convergence results for axially loaded cantilever coupled beam (T = -1790 N). 5.5.3. Coupled Bending-Bending Vibrations The DFE formulation was then applied to the free vibration analysis of coupled bending-bending rotating beams. The element stiffness matrix, equation (5.35), reduces to: to: [K(co)Y 5.55 [K(co)Y 5.55 5.55 where Uncoupled f=0 K . - Cr J ; - c J ; - C, {iv;.,}; ------------------------------------------------------------------------------f:!------------------------------------------------------------------------------, Cr { } - c... { } ; C„. } ; c, { { N t ^.} ; c„ ] 5.56 Uncoupled 5.56 78 and where the vectors of nodal variables {w} and shape functions < N b-w > and < Nb.v > arc written as follow: { w }= < U ', w\ V, V,' \\>2 w'2 V2 v'2>^ 5.58 ^B2 0 0 A^B4 0 0> 5.59 <Ng_^,>=<0 0 Ngf Ng2 0 0 Ng^ > 5.60 { w }= < U ', w\ V, V,' \\>2 w'2 V2 v'2>^ 5.58 ^B2 0 0 A^B4 0 0> 5.59 <Ng_^,>=<0 0 Ngf Ng2 0 0 Ng^ > 5.60 In next sections coupled bending-bending beams with constant axial load and centrifugal forces will be investigated to validate the DFE for this category of structures. 5.5.3.I. Axially Loaded Beam: The uncoupled bending-bending vibrations of a cantilever beam with symmetric cross section under constant axial load was first studied. The beam geometric and mechanical properties are as follow: L = 7.0 m m = 4.5 kg/m T= 1000 = 3000 N.m^ Elr, = 3000 n W 79 Since the flexural rigidities in both directions are the same, it is expected to obtain repeated frequencies for in-plane and out-of-plane free vibrations. Table 5.7 shows the first six natural frequencies of the Beam. As already mentioned, the DFE for imcoupled beam is an exact method and the error for DFE with one element is zero. One can see that the FEM errors for the first few natural frequencies are also within the acceptable range and the FEM follows the general pattern such that the error increases for higher frequencies. Table 5.7: The uncoupled in- and out-of-plane bending natural frequencies of the cantilever beam Natural Frequency (rad/s) Exact value DFE with 1 element Error of DFE (%) FEM with 10 elements Error of FEM CO; 4.4821 4.4821 0.000 4.4822 0.001 0)2 4.4821 4.4821 0.000 4.4822 0.001 0)3 16.5835 16.5835 0.000 16.5843 0.005 0)4 16.5835 16.5835 0.000 16.5843 0.005 0)5 37.4918 37.4918 0.000 37.5011 0.025 0)6 37.4918 37.4918 0.000 37.5011 0.025 Table 5.7: The uncoupled in- and out-of-plane bending Table 5.7: The uncoupled in- and out-of-plane bending natural frequencies of the cantilever beam If a pre-twist angle 6 is added to the system (i.e., rotation of principle axes around longitudinal axes x) then the beam will experience the coupled bending-bending vibrations. The pre-twist angle causes geometric coupling between two bending displacements by producing the coupling term The frequencies for the per-twisted beam, in this case, are the same as the uncoupled beam due to the fact that rotation of the coordinate system doesn’t change the nature of the system. The natural frequencies in coupled case also confirm the DFE’s higher convergence rates comparing to FEM, where the DFE errors with only one element for the first six frequencies for this example are zero. Even though constant force can be used to estimate the effect of centrifugal force, as it was used for coupled bending-torsion beam, but there is a principal difference between bending-torsion coupled vibrations and bending-bending coupled vibrations. 5.5.3.I. Axially Loaded Beam: In 80 the latter case, the centripetal term decreases the rigidity of the beam in j’-direction, which cannot be taken into account when the axial force is simulated by constant force. In Figure 5.8 the effect of a constant axial force on the first six natural frequencies of the beam are shown. Frequency Force cuvre for coupled bendina«bendlng axially loaded beam, S 25.0 ♦ omega 1 omega 2 ▲ omega 3 omega 4 ■ omega 5 — ■ omega 6 0 100 200 300 400 500 600 700 800 900 1000 Axial force (N) Figure 5.8: The effect of axial force on natural frequencies of clamped-free beam bending-bending vibrations. q y coupled bendina«bendlng axially loaded beam, Axial force (N) Figure 5.8: The effect of axial force on natural frequencies of clamped-free beam bending-bending vibrations. One can see (Figure 5.8) that by constant axial force the flap frequencies and lag frequencies inerease with the same rate (for this special case they are exactly the same). It will be shown later that when the effect of centripetal force is taken into account, the increment rate of flap and lag frequencies are not the same. 5.S.3.2. Coupled Bending-Bending Rotating Beams: For rotating beams, the equations (5.55) to (5.57) are used to build the system’s frequency dependent (nonlinear) stiffness matrix. In this case the centrifugal and centripetal terms both contribute in stiffhess matrix, when the former increases and the latter decreases the stiffiiess of the system. 81 Using the same beam configuration as discussed in the previous section, ^ d incorporating a constant pre-twist angle B =30°, the beam natural frequencies for rotating = 5 (rad/s) were calculated and shown in Table 5.8: Table 5.8: The coupled bending-bending natural frequencies Table 5.8: The coupled bending-bending natural frequencies Table 5.8: The coupled bending-bending natural frequencies of a rotating cantilever beam, ft = 5 (rad/s) Natural Frequency Reference DFE with Error of DFE (rad/s) FEM, n = 500 5 element (%) (0; 2.619 2.608 0.422 «2 5.645 5.640 0.079 Wj 16.494 16.479 0.094 U4 17.235 17.221 0.085 38.409 38.396 0.035 38.733 38.720 0.035 of a rotating cantilever beam, ft = 5 (rad/s) Table 5.8 shows excellent convergence of DFE in comparison with FEM, where the error for 5*’ and 6'’ frequencies is less than 0.04%. The error percentage versus number of elements for DFE method are presented in Figure 5.9: Convergenc test of DFE for coupled bending-bending rotating beam 1.40 - 1.20 - 1.0 0 - 0.80 - w 0.60 - 0.40 - 0 .2 0 - 0.00 6 5 7 10 2 3 4 8 9 -omega 1 -omega 2 -omega 3 -omega 4 - omega 5 -omega 6 Number of elemenets 8 2 Convergenc test of DFE for coupled bending-bending rotating beam 1.40 - 1.20 - 1.0 0 - 0.80 - w 0.60 - 0.40 - 0 .2 0 - 0.00 6 5 7 10 2 3 4 8 9 -omega 1 -omega 2 -omega 3 -omega 4 - omega 5 -omega 6 Number of elemenets 8 2 Figure 5.9: The DFE convergence results for bending-bending vibrations of a rotating beam. Figure 5.9: The DFE convergence results for bending-bending vibrations of a rotating beam. Figure 5.9: The DFE convergence results for bending-bending vibrations of a rotating beam. The first three flap and lag natural frequencies of the rotating beam are shown in Figure 5.10. The effect of rotary speed on natural frequencies for coupled bending-bending rotating beam 50 =§ 40 2 & g 30 s* LL 20 22 10 0 8 10 0 2 4 6 -omega 1 (lag) -omega 2 (flap) -omega 3 (lag) -omega 4 (flap) -omega 5 (lag) -omega 6 (flap) Rotary Speed (rad/s) Figure 5.10: The effect of rotating speed on natural frequencies for clamped-free beam bending-bending vibrations. The effect of rotary speed on natural frequencies for coupled bending-bending rotating beam 50 =§ 40 2 & g 30 s* LL 20 22 10 0 8 10 0 2 4 6 -omega 1 (lag) -omega 2 (flap) -omega 3 (lag) -omega 4 (flap) -omega 5 (lag) -omega 6 (flap) Rotary Speed (rad/s) The effect of rotary speed on natural frequencies for coupled bending-bending rotating beam Figure 5.10: The effect of rotating speed on natural frequencies for clamped-free beam bending-bending vibrations. Figure 5.10 shows that the growth of lag frequencies is slower than flap frequencies due to the centripetal effect on the in-plane vibrations which plays a softening role and decreases the stiffness of beam in lag direction. On the other hand, the difference between flap and lag frequencies decreases for higher modes. Also one can see that higher frequencies grow faster than lower frequencies. In order to investigate the natural modes of coupled bending-bending rotating beam, a uniform beam with following properties is considered in which the flexural rigidities in two directions are different: 83 L = 7.0 m m = 4.5 kg/m 0 =30 degree Elr, = 3000 N.m^ EI^ = 7500 N W 0 = 5 rad/s L = 7.0 m m = 4.5 kg/m 0 =30 degree Elr, = 3000 N.m^ EI^ = 7500 N W 0 = 5 rad/s The first five natural frequencies were calculated and compared with FEM method. Reference frequencies have been obtained from a 600-element FEM model. The results are summarized in Table 5.9. Figure 5.9: The DFE convergence results for bending-bending vibrations of a rotating beam. Table 5.9: The coupled bending-bending natural frequencies of the rotating beam Natural Frequency (rad/s) Reference frequency FEM, n = 600 DFE with 3 element Error of DFE (%) 03] 3.307 3.301 0.202 t02 5.795 5.796 0.013 Wj 17.032 17.038 0.032 0)4 21.940 21.952 0.058 U s 38.648 38.748 0.257 Table 5.9: The coupled bending-bending natural frequencies of the rotating beam Table 5.9: The coupled bending-bending natural frequencies Table 5.9 shows the excellent convergence rate for the DFE, where using only three elements results in an error less than 0.3%. The mode shapes corresponding to the first eight frequencies are shown in Figure 5.11. 8 4 Mode 1 Mode 2 — Flap Lag — Flap Lag Mode 3 Mode 4 — Flap Lag — Flap Lag Mode 5 Mode 6 — Flap Lag — Flap Lag Mode 7 Mode 8 Lag Lag Figure 5.11: The first eighth mode shapes of bending-bending vibrations of a clamped-free rotating beam. Mode 1 — Flap Lag Mode 2 — Flap Lag Mode 8 Lag Figure 5.11: The first eighth mode shapes of bending-bending vibrations of a clamped-free rotating beam. The mode shapes (Figure 5.11) exhibit the expected behaviour, where for higher modes the number of nodes (zero displacement point on beam) increases. 85 5.5.3.3. Rotating Blade with Variable Cross Section: In order to demonstrate the applicability of the DFE approach to complex problems, the rotating coupled bending-bending blade with variable cross section, introduced in section 4.5.3, was then studied. The first three natural frequencies at different rotating speed fl were calculated and are shown in Table 5.10. Table 5.10: The first three natural frequencies (Hz) of vibrations of propeller blade WADC S-5 (DFE method) Blade rotary speed (rpm) Mode number DFE n = 9 FEM n = 9 Result of Reference 12] Result of Reference [5] Experiment [27] 1 40.82 40.32 40.96 39.89 40.08 1567 2 109.54 108.49 109.22 107.40 3 280.30 279.27 279.79 276.32 1 41.22 40.70 41.35 40.26 1589 2 110.10 109.01 109.77 107.93 107.53 3 280.98 279.92 280.47 276.97 1 59.74 58.99 60.07 58.05 58.73 2609 2 139.47 137.18 139.52 135.99 3 319.42 316.82 309.40 313.98 1 59.84 59.08 60.16 58.14 2614 2 139.63 137.33 139.68 136.14 137.02 3 319.63 317.03 319.62 314.19 1 77.78 76.87 78.34 75.30 76.52 3583 2 171.49 168.08 166.25 170.60 3 365.76 361.38 357.70 1 94.53 93.48 91.42 93.07 4486 2 202.27 197.91 3 413.43 407.24 1 95.48 94.42 4537 2 204.02 199.61 196.41 202.53 3 416.20 409.91 1 120.41 119.17 116.07 117.50 5884 2 249.67 244.05 3 490.10 481.05 Table 5.10: The first three natural frequencies (Hz) of vibrations of propeller blade WADC S-5 (DFE method) 86 The DFE results are in good agreement with those of Transfer Matrix Method (TTM) [2], where the error for angular speed of 1567 (rpm) and 1589 (rpm) is less than 0.2%. This example shows the applicability of DFE method for complex geometries where the exact method cannot be used. Reviewing all examples of this section, one can see that the DFE method can be advantageously used to calculate the natural frequencies and mode shapes of vibrations of bending-bending coupled beam with different levels of complexity. 5.5.4. Coupled Bending-Bending-Torsion Vibrations Bending-bending-torsion coupled beams can be used in simulation of the turbine or propeller blades or aircraft wings. For short beams, the shear deformations and rotary inertia must be considered to provide a more accurate model, and in some cases it is necessary to consider the thermal effect where the temperature is considerably higher than ambient temperature [19]. For propeller blades and aircraft wings the geometry of beam allows using Euler-Bemoulli assumptions and the results are accurate enough for vibration analysis of the first few modes of vibrations. The coupling between differential equations governing the flap-wise bending, cord-wise bending and torsion occurs because of the beam’s cross-sectional geometry, due to the distance between mass and elastic (shear) centers, e, and asymmetry of the cross section. In this section, by a progressive approach, the free vibrations of triply coupled bending-bending-torsion beams for non-rotating and rotating cases are discussed. 5 5.4.1. Uncoupled Beam Vibrations As already stated, for uncoupled equations of motion, the DFE method results in the exact solutions. The uncoupled equations of motion for lag (in-plane) bending, flap (out-of-plane) bending and torsion vibrations of a beam can be written as: 87 87 {EIyW"y — û) mw = 0 5.61 {E iy y -û }^ m v = 0 5.62 -{GJ(/>y-û)^mK:l,(/> = 0 5.63 {EIyW"y — û) mw = 0 5.61 {E iy y -û }^ m v = 0 5.62 5.62 -{GJ(/>y-û)^mK:l,(/> = 0 5.63 5.63 By implementing the DFE method to the equations (5.61), (5.62) and (5.63), the exact uncoupled in- and out-of-plane bending and torsion natural frequencies as discussed in Chapter 4, were obtained. These results are also in agreement with FEM (see section 4.5.4.1 and Table 4.10). 5.S.4.2. Vibrations of Non-rotating Coupled Beam For a non-rotating beam, the eccentricity e and the asymmetry produce the coupling terms, and the element stiffness matrix expressions (5.35) to (5.37) reduces to the following form: [/^(u>)]^ = [K{CO)Yvncoup,., + 5.64 5.64 5.64 Where [ K m 'Uncoupled and 88 = r [ ( ^ ) ( W - j < > +{Ni^} < N i^ >) -ico^emlcosû)({N^_J<N^_^>+{N^_^}<Ng,„ >) + (ty^e/H/sin g)((#g_J < > +{N,.^} < >)]d^ In order to investigate the applicability of DFE to the triply coupled vibrations of a non-rotating pre-twisted beam, the beam configuration studied in section 4.5.4.2 re investigated [28,29]. The geometric and material properties of the beam are: L = 40.0 in e= 1.414 in d =45 deg (constant along the beam length) m = 0.0015 slug/in kmi= k„2= 1.0 in Elr, = 25000 Ib.in^ = 75000 Ib.in^ GJ=9000 Ib.in^ d =45 deg (constant along the beam length) m = 0.0015 slug/in where the pre-twist angle, 6, assumed to be constant along the beam length. The beam natural frequencies obtained from the proposed DFE method are shown in Table 5.11. The reference values for beam natural frequencies, in this case, are those obtained from ’Transfer Matrix Method (TMM)” published by Murthy [2]. The comparison between DFE and FEM results confirms the superiority of the proposed DFE method (Table 5.11). The convergence results for the FEM and DFE methods for the first, third and fifth frequencies are also compared in Figure 5.12. 89 Table 5.11: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted clamped-free beam Table 5.11: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted clamped-free beam Natural Error for Error for Frequency TMM [2] DFE " T n (rad/s) (%) " ■ (%) CO; 30.8295 30.8300 0.001 30.8300 0.002 (02 53.8277 53.8278 0.000 53.8278 0.000 ^3 184.6175 184.7138 0.052 184.7376 0.065 (0^ 337.3333 337.3351 0.001 337.3440 0.003 CO5 484.3373 485.7737 0.297 486.4762 0.442 90 1st natural frequency Oj DFE 0.15 0.1 - o 0.05 1 2 3 4 5 6 7 8 10 9 Number of elements 3rd natural frequency Number of elements 5th natural frequency 0.8 ^ 0.6 0.4 - 0.2 9 10 5 6 7 8 3 4 2 Number of elements DFE -ei-FEM 2.5 1.5 I 0.5 10 8 7 5 8 4 3 Figure 5.12: The comparison between the DFE and FEM convergenc for non-rotating triply coupled beam. 5.S.4.2. Vibrations of Non-rotating Coupled Beam 1st natural frequency Oj DFE 0.15 0.1 - o 0.05 1 2 3 4 5 6 7 8 10 9 Number of elements 3rd natural frequency Number of elements 0.8 ^ 0.6 0.4 - 0.2 9 10 5 6 7 8 3 4 2 1st natural frequency Oj DFE 0.15 0.1 - o 0.05 1 2 3 4 5 6 7 8 10 9 Number of elements 1st natural frequency 1st natural frequency Oj DFE 0.15 0.1 - o 0.05 1 2 3 4 5 6 7 8 10 9 Number of elements 3rd natural frequency Number of elements 5th natural frequency 0.8 ^ 0.6 0.4 - 0.2 9 10 5 6 7 8 3 4 2 Number of elements DFE -ei-FEM 2.5 1.5 I 0.5 10 8 7 5 8 4 3 Figure 5.12: The comparison between the DFE and FEM convergence for non-rotating triply coupled beam. Number of elements 3rd natural frequency 3rd natural frequency Figure 5.12: The comparison between the DFE and FEM convergence for non-rotating triply coupled beam. 91 Investigation in mode shapes reveals that the modes obtained by DFE and FEM are identical. The first five modes corresponding to the first five natural frequencies are shown in Figure 5.13 (Note: the first, second and fourth torsion modes have been magnified hy 10,10" and iO'* respectively to be visible). magnified hy 10,10" and iO'* respectively to be visible). Mode 1 Mode 2 Mode 3 Mode 4 Mode 5 Figure 5.13: The first three modes of vibrations of non-rotating triply coupled beam. Mode 1 Mode 2 Mode 3 Mode 4 Mode 2 Mode 1 Mode 4 Mode 3 Mode 5 Figure 5.13: The first three modes of vibrations of non-rotating triply coupled beam. Figure 5.13: The first three modes of vibrations of non-rotating triply coupled beam. 9 2 S.5.4.3. Vibrations of Rotating Triply Coupled Beam S.5.4.3. Vibrations of Rotating Triply Coupled Beam For a rotating pre-twisted beam, the governing equations of motion are in the form of (2.1), (2.2) and (2.3). These equations can be used to model the low frequency vibrations of high aspect ratio compressor, turbine, propeller and helicopter blades. The element dynamic stiffness matrix, consisting of coupled and uncoupled stiffness matrices, can be written as introduced in equations (5.35) to (5.37). By including the deviator matrix, the uncoupled stiffness matrix then changes to equation (5.45). Here, the free vibration of a triply coupled pre-twisted cantilever helicopter blade is studied. The following geometric, mechanical and material properties, as reported in the literature [4,6,16] (see also section 4.5.4.3) were used: L = 208 in g = -0.6 in ei = 52 in 0 = 15.026 deg (constant along the blade length) (« = 0.0015 Ib.sec^/in^ «i.^„/ = 0.89545x10'^ Ib.sec^ in^hif,2 = 0.04 lb.sec E /, = 0.2977x10^ Ib.in^ JB'/{= 10x10® Ib.in^ (?J= 0.2x10® Ib.in^ l i = 360 rpm L = 208 in g = -0.6 in ei = 52 in L = 208 in g = -0.6 in ei = 52 in 0 = 15.026 deg (constant along th (« = 0.0015 Ib.sec^/in^ «i.^„/ = 0.89545x10'^ Ib.sec^ in^hif,2 = 0.04 lb.sec E /, = 0.2977x10^ Ib.in^ JB'/{= 10x10® Ib.in^ (?J= 0.2x10® Ib.in^ l i = 360 rpm 0 = 15.026 deg (constant along the blade length) «i.^„/ = 0.89545x10'^ Ib.sec^ in^hif,2 = 0.04 lb.sec E /, = 0.2977x10^ Ib.in^ JB'/{= 10x10® Ib.in^ (?J= 0.2x10® Ib.in^ l i = 360 rpm «i.^„/ = 0.89545x10'^ Ib.sec^ Based on the DFE method, the natural frequencies of the cantilever blade were then calculated and compared with those obtained from the FEM (Table 5.11). Since the exact results are not available, an FEM model based on 700-element mesh was used to evaluate the reference values. As it can be seen from Table 5.11, a coarse mesh of only 93 seven DFE elements, leads to an infinitesimal average error of less than 0.02% when the first 5 natural frequencies are to be evaluated. The DFE convergence results compared with an FEM model (700-element mesh) are shown in Figure 5.14. S.5.4.3. Vibrations of Rotating Triply Coupled Beam Table 5.12: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted rotating beam, fl = 360 (rpm) Natural Frequency (rad/s) Reference FEM n - 700 DFE with 7 element Error of DFE (%) U l 46.125 46.096 0.013 W2 73.253 73.225 0.001 Wj 130.864 130.757 0.023 0)4 171.963 171.965 0.000 U s 271.435 271.452 0.060 Natural frequemcies of DFE analysis of a triply coupled rotating beam, Omega = 360 rpm Number of elements 0.500 0.400 -♦-om ega 1 -"-om ega 2 -Io m e g a 3 -"-om ega 4 -Iomega 5 ^ 0.300 - 0.200 - 0.100 0.000 6 7 3 4 5 8 9 10 Figure 5.14: The DFE convergence results for rotating triply coupled beam. Table 5.12: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted rotating beam, fl = 360 (rpm) Natural Frequency (rad/s) Reference FEM n - 700 DFE with 7 element Error of DFE (%) U l 46.125 46.096 0.013 W2 73.253 73.225 0.001 Wj 130.864 130.757 0.023 0)4 171.963 171.965 0.000 U s 271.435 271.452 0.060 Natural frequemcies of DFE analysis of a triply coupled rotating beam, Omega = 360 rpm Number of elements 0.500 0.400 -♦-om ega 1 -"-om ega 2 -Io m e g a 3 -"-om ega 4 -Iomega 5 ^ 0.300 - 0.200 - 0.100 0.000 6 7 3 4 5 8 9 10 Figure 5.14: The DFE convergence results for rotating triply coupled beam. S.5.4.3. Vibrations of Rotating Triply Coupled Beam Table 5.12: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted rotating beam, fl = 360 (rpm) Natural Frequency (rad/s) Reference FEM n - 700 DFE with 7 element Error of DFE (%) U l 46.125 46.096 0.013 W2 73.253 73.225 0.001 Wj 130.864 130.757 0.023 0)4 171.963 171.965 0.000 U s 271.435 271.452 0.060 Table 5.12: The triply coupled bending-bending-torsion natural frequencies of the pre-twisted rotating beam, fl = 360 (rpm) Natural frequemcies of DFE analysis of a triply coupled rotating beam, Omega = 360 rpm Number of elements 0.500 0.400 -♦-om ega 1 -"-om ega 2 -Io m e g a 3 -"-om ega 4 -Iomega 5 ^ 0.300 - 0.200 - 0.100 0.000 6 7 3 4 5 8 9 10 Natural frequemcies of DFE analysis of a triply coupled rotating beam, Omega = 360 rpm Number of elements 0.500 0.400 -♦-om ega 1 -"-om ega 2 -Io m e g a 3 -"-om ega 4 -Iomega 5 ^ 0.300 - 0.200 - 0.100 0.000 6 7 3 4 5 8 9 10 Figure 5.14: The DFE convergence results for rotating triply coupled beam. Natural frequemcies of DFE analysis of a triply coupled rotating beam, Omega = 360 rpm Number of elements 6 7 Figure 5.14: The DFE convergence results for rotating triply coupled beam. The mode shapes of the triply coupled rotating beam were also investigated (see Figure 5.15). The results confirm the expected modal behaviour for given configuration 9 4 (the first, second, third and fifth torsion modes have been magnified by 10^, 10^, 10 and 10^ respectively to be visible). 10^ respectively to be visible). Mode 1 Mode 2 Mode 3 Mode 4 Mode 5 Figure 5.15: The modes of vibrations for a triply coupled rotating beam. Furthermore in order to validate the proposed method and to verify the Mode 2 Mode 1 Mode 1 Mode 4 Mode 3 Mode 5 Figure 5.15: The modes of vibrations for a triply coupled rotating beam. Mode 5 Figure 5.15: The modes of vibrations for a triply coupled rotating beam. Figure 5.15: The modes of vibrations for a triply coupled rotating beam. Furthermore, in order to validate the proposed method and to verify the applicability of DFE method to more complicated cases, a uniform linearly twisted blade 95 was also studied. S.5.4.3. Vibrations of Rotating Triply Coupled Beam A constant rate of twist is considered, where 0 = 0” at root and 6 = 15.026° at free end (tip). The frequencies of free vibrations of blade are calculated and compared to the reference values obtained from a 600-element mesh model based on classical FEM (Table 5.13). Based on a six-element DFE analysis, and evaluating the first 5 natural frequencies, an excellent average error <0.1% was obtained. Table 5.13: The triply coupled bending-bending-torsion natural frequencies of the twisted rotating blade, fl = 360 (rpm) Natural Frequency (rad/s) FEM n = 600 DFE n = 6 Error of DFE (%) CO; 47.56 47.51 0.10 «2 71.67 71.64 0.03 Wj 132.60 132.26 0.26 CO.; 172.25 172.25 0.00 W5 268.72 268.90 0.07 Table 5.13: The triply coupled bending-bending-torsion natural frequencies of the twisted rotating blade, fl = 360 (rpm) Finally, in order to validate the DFE method for blades and beams of more complex geometries, the blade as discussed in the previous example was re-investigated when the uniform geometry and pre-twisted geometry were modified to model a non- uniform pre-twisted beam. The cross-sectional properties vary along the blade length when the blade is divided into five uniform segments with equal lengths. The flexural and torsional rigidities, i?/„ EI^ and GJ, and also inertia parameters, /«, nt.kmr and nt.km2 , are decreased by 5% in each segment as shown in Figure 5.16. “ c ” is the element geometric coefficient multiplied by the reference rigidities and inertia parameters (at root). 9 6 ------u s -------,‘----- L/^ - US «----- U S----, 1 ' Hv ; i; • ( 1 * ^ * t-i? j ? c = 1 c = 0.95 c = 0.90 c = 0.85 c = 0.80 X Figure 5.16: The model for piecewise uniform beam. X Figure 5.16: The model for piecewise uniform beam. The results for the natural frequencies of the beam (Figure 5.16) are calculated and shown in Table 5.13 (0 = 360 (rpm)). Table 5.14: The triply coupled bending-bending-torsion natural frequencie Table 5.14: The triply coupled bending-bending-torsion natural frequencies of the piecewise uniform twisted rotating blade, ft = 360 (rpm) Frequency (rad/s) Reference FEM. S.5.4.3. Vibrations of Rotating Triply Coupled Beam n = 800 DFE n = 10 Error of DFE (%) FEM n = 200 Error of FEM (%) W; 48.54 48.51 0.06 48.63 0.20 (02 79.49 79.46 0.03 79.56 0.09 U j 132.46 132.36 0.08 132.81 0.26 U4 181.00 181.00 0.00 181.00 0.00 U s 272.59 272.52 0.03 272.97 0.14 Table 5.14: The triply coupled bending-bending-torsion natural frequencie of the piecewise uniform twisted rotating blade, ft = 360 (rpm) As it can be seen (Table 5.14) the DFE convergence to the reference values, obtained from an 800-elements FEM model, with much higher rates compared to FEM. As it can be observed (Table 5.14), based on a ten-element DFE model, one can confidently evaluate the first five natural frequencies of the given beam configuration with an average error of les than 0.04%. 97 All examples of this chapter along with numerical results of Chapter 4 demonstrate the precision and the practical applicability of the finite element method (FEM) as a strong tool in investigating the dynamic behaviour of non-rotating and rotating coupled beams. Also a comparison between FEM and DFE reveals that the DFE method, along with Wittrick-Williams root counting approach as a robust and accurate solution tool, provides higher convergence rates and lower computation time and cost, especially when multiple and/or higher frequencies are sought. 9 8 6.1. Concluding Remarks For many engineering problems, dealing with flexible structures, the vibration analysis of the system is a vital part of design. Neglecting the dynamic behaviour of the structure could cause catastrophic results and failure in a system. Finding the first few natural frequencies and mode shapes of a flexible structure helps the designer to have better understanding about dynamic behaviour of the structure and to be able to predict the response of the system to excitation such as periodically variable forces or movements. This research work focused on the free vibration analysis of non-rotating and rotating beams. There are many flexible structures in aerospace engineering which can be modeled by a beam or a combination of beams, such as aircraft wings, propeller blades, satellite antenna and solar panels, compressor, turbine and helicopter blades, etc. All these structure have one character in common; due to the special geometry or material of the structure, they undergo coupled displacements governing coupled differential equations of motion. The analytical solutions for uncoupled equations governing bending and torsion displacements can be found in many vibrations text books, while for the case of coupled vibrations, the analytical solution is not always available. In vibration analysis of rotating beams, the most complete models have three coupled differential equations for in-plane bending, out-of-plane bending and torsional displacements and an uncoupled equation for longitudinal displacements. The equation governing the beam’s axial displacements is uncoupled and can be solved analytically using various methods [24]. For triply coupled beam equations, there have been different numerical approaches proposed by many researchers attempting to find the best solution for the free vibrations response of the system [2,4,5,6,16]. Is this research the FEM method, a powerful and accurate method, was first used to study the coupled beam vibrations. The Galerkin weak (integral) formulation, which also satisfies the principle of virtual work, was employed to transform the coupled differential equations of motion into 99 algebraic equations and an eigenvalue problem, where the eigenvalues are the natural frequencies and the eigenvectors represent the mode shapes of the system. Besides developing a conventional FEM method, this research focused specifically on the development of new Dynamic Finite Element (DFE) for bending- bending coupled beams and triply coupled rotating beams, used as a model representing rotary blade dynamics. 6.1. Concluding Remarks As explained in Chapter 5, after discretizing the beam into elements and applying a certain number of extra integration by parts, the ODE’s governing the uncoupled beam vibrations appear. Vanishing of these terms leads to the dynamic basis functions of approximation space. A closer look at these terms shows that by application of integration by parts, some integral terms transform to boundary terms, and since the integration in DFE is done numerically, this change can decrease the computation time effectively. Because of the nonlinear trigonometric nature of the dynamic interpolation functions in the DFE formulation, the root counting procedure presented by Wittrick and Williams [14,15,26] were exploited in order to evaluate the natural frequencies. The approach provides a powerful solution tool for periodic systems with repeated sub structures and repeated frequencies [15], in particular. Using thee Wittrick-Williams method, one can calculate directly the frequency W/ without calculating the frequencies lying below w,-, whereas in the FEM method, the well-known numerical methods frequently used in frequency calculations do not have this feature, even though one could also adapt the W-W algorithm to the standard FEM analysis [35]. The DFE and FEM were then used to investigate the behaviour of coupled vibrations of non-rotating and rotating beam configurations. The examples investigated in this thesis exhibit different aspects of beam vibrations. First, the vibrations of uncoupled beams were studied, where the results showed that solving two or three uncoupled equations together with one nodal displacement vector results in the same results as analytical solutions. In addition, the effect of an axial load on coupled bending-bending and bending- torsion beams was studied. As shown in Chapters 4 and 5, the axial load can affect the geometric stiffness of the beam and change the natural frequencies and modes, where a 100 tensile force increases the so-called geometric stiffness of the beam and consequently increases the natural frequencies. A compressive axial force, on the other hand, decreases the natural frequencies by softening the beam. Also, when the rotating speed is increased, the centripetal acceleration makes the cord-wise natural frequencies to grow slower than those of beam flap-wise vibration. The DFE method developed in this research was also advantageously used to determine the natural frequencies and modes of triply coupled beam vibrations. 6.1. Concluding Remarks In many engineering design problems, at the preliminary design stage, the designer needs to get a general idea about the dynamic behaviour of the system even before a detailed and rigorous design process or FEM analysis starts. In such cases, having a reliable and accurate simulation tool like DFE helps the designer to better understand the dynamics of a system. Besides, applying the first changes in a trial step is easier and takes much less time with this numerical tool. 6.2. Comparison Between DFE and FEM The DFE, as a finite element approach, follows the same methodology of the conventional FEM, but with some conceptual differences. In the DFE formulation, the Dynamic (frequency dependent) Trigonometric Shape Functions (DTSF’s) are exploited to approximate the displacements, while the FEM uses the static (polynomial) shape functions. These polynomial shape functions, cubic for flexural vibrations and linear for torsional vibration, are the solution of static deformations of the beam produced by external forces and moments. The dynamic basis functions used in the DFE formulation are the solutions to the uncoupled ODE’s governing the free vibrations of the beam and they are altered such that they reduce to static shape functions when w-> 0. The dynamic character of shape functions in the DFE method causes a higher convergence rate, since the nature of approximation functions are closer than Hermite shape functions to the real solutions. For uncoupled vibrations, the DTSF’s are the exact solutions and one can get an infinite number of natural frequencies by a one-element DFE model. 101 There is another important difference between two methods: classic FEM leads to a linear eigenproblem with constant mass and stiffiiess matrices, whereas the DFE formulation results in a nonlinear eigenproblem due to the frequency dependent stiffiiess matrix which represents both inertia and stiffness of the system. In general, for a numerical approach some features are more important than others such as reliability, accuracy (or rate of convergence) and computation time. The FEM based formulations have already been proven as reliable and robust methods, and are widely used in engineering and research areas. The reliability of DFE for single axial or bending vibrations and coupled bending-torsion vibrations has also been approved for beams with geometric and/or material coupling [21-25]. The reproduced results for illustrative examples exhibiting different coupled behaviour reconfirmed the excellent reliability of DFE. Besides, the DFE formulation was also extended to the bending- bending and triply coupled beam vibrations and several illustrative examples were presented. Results showed that, as expected, the DFE provides reliable solution for triply coupled vibrations of a beam, and degenerating the dual coupling from triple coupling problems does not affect the robustness of the method. The accuracy of both DFE and FEM methods is excellent and, as stated in Chapters 4 and 5, in order to obtain more accurate higher frequencies, the number of elements must be increased. 6.2. Comparison Between DFE and FEM Comparing the convergence results for two methods reveals that for the first and second frequencies, both methods result in the same accuracy, but if higher frequencies are sought, the DFE convergence rates are higher than FEM. The difference between the FEM and DFE methods is more significant when refined DFE (RDFE) is used to model rotating beams or beams with variable cross sections. The computation time for both methods depends on the number of elements in the model, and since the frequency calculation in the DFE relies on the bisection method, for the same number of elements the computation time for DFE is higher than FEM. A comparison between a combination of accuracy and computation time shows that DFE provides faster solutions. For example, in the case of the coupled bending-bending- torsion rotating beam (Table 5.11), the average error for the first five natural frequencies obtained from a 7-element DFE model (comparing to a 700-element FEM model) is 0.02 102 percent. The DFE computation time, in this case, is less than two minutes whereas tlie FEM model takes more than two hours to provide the same accuracy. As a result, despite the differences between the DFE and FEM formulations, botli methods can be successfully exploited to carry out the vibration analysis of rotating and non-rotating beams. 6.3. Future Work The research work presented here was focused on the free vibration analysis of homogeneous undamped beams. Euler-Bemoulli bending and St. Venant torsion beam theories were considered in the model. This model neglects the shear deformation and rotary inertia as well as warping and thermal effects. More accurate models consider one, some or all the parameters neglected here; hence, future work can concentrate on increasing the accuracy of the model by including the effects of shear deformation and rotary inertia for triply coupled beams. Also, the warping and thermal effects can be taken into account where needed. To try a realistic model, stmctural damping also must be taken into account, since all materials incorporate a certain degree of damping. 103 A.I. Pure Bending Vibrations of a Clamped-Free Beam The differential equation of motion for vibrations of the Euler-Bemoulli beam, in which the shear deformation and rotary inertia effects are neglected, can be written as: dx dx dx ÔX dt For a clamped-free beam the boundary conditions are: For a clamped-free beam the boundary conditions are: r(0 ,/) = 0 dW{0,t) dx = 0 A.2 b £!2:m , o dx^ OX dx A.2 Using the separation of variables technique, the solution of differential equation is assumed as product of a spatial function w(x) and a harmonic function sincor: = w(%).sinor A.3 = w(%).sinor A.3 1 04 Substituting (A.3) into (A.1): Substituting (A.3) into (A.1): {EIw")" - (Tiv')' - inco^w = 0 A.4 and for a uniform beam: EIw"" — Tw" - mû) \v = 0 EIw"" — Tw" - mû) \v = 0 Assume: At Equation (A.5) becomes: AX' +BÀ^ +C = 0 A = EI ; B = -T ; C = -mco^ Solving (A.7) for X results in: X = ■ B ± ^ B ^ -A A C 2A A.9 K i = -B - ^ B ^ - A A C 2A = ±ia A.10 A.11 By evaluating the \ Equation (A.6) can be written as: Solving (A.7) for X results in: Solving (A.7) for X results in: Solving (A.7) for X results in: By evaluating the \ Equation (A.6) can be written as: By evaluating the \ Equation (A.6) can be written as: By evaluating the \ Equation (A.6) can be written as: w(x) = A sin ccc + B cos ax + C sinh ^x + D cosh fix A.12 w(x) = A sin ccc + B cos ax + C sinh ^x + D cosh fix A.12 Applying the boundary conditions (A.2) to find the constants A, B, C and D results in an eigenvalue problem: 105 0 -1 0 1 A 0 -1 0 1 0 B 0 - sin aZ- - cos aL sinh J3L cosh fiL C o ' - cos aL sinh of, cosh oL sinh pL D 0 A .1 3 A .1 3 The characteristic equation of eigenproblem (A.13) can then be written as: The characteristic equation of eigenproblem (A.13) can then be written as: cos oL. cosh = 1 A. 14 cos oL. cosh = 1 A. 14 cos oL. cosh = 1 A. 14 A. 14 For free vibrations of a uniform beam without axial force, a = /3 and: For free vibrations of a uniform beam without axial force, a = /3 and: For free vibrations of a uniform beam without axial force, a = /3 and: 2 r 2 (o = a L A.15 A.15 The solution of for aL, weighted natural frequency, can be found in vibrations text books [34]. The results have been shown in Table A.1 : The solution of for aL, weighted natural frequency, can be found in vibrations text books [34]. Assume: The results have been shown in Table A.1 : Table A.1: The weighted and normalized natural frequencies, (x„L and co„, for a cantilever beam obtained by analytical solution Weighted Natural Frequency Normalized natural frequency djL 1.87510407 0)7 3.51601527 012L 4.69409113 0)2 22.03449154 cisL 7.85475744 Wj 61.69721922 cCfL 10.99554073 (^4 120.9019159 oisL 14.1376839 0)5 199.8741061 ct„L, n>5 (2n-1)ir/2 0)„ l(2n-1)ir/2f The natural frequencies have been obtained from equation (A.15). For = l,m = 1 and L = 1, the natural frequencies have already been shown in Chapter 4 and 5. Mode shapes of free vibrations of the clamp-free uniform beam without an axial force can then be calculated by substituting coefficients A, B, C and D into the equation A.12 [34]: 1 06 f \ , sinhûr-Z,-smû:_Z, , . , . , w„ W = cos a„ x- cosh a^x---------- — (sinh a .x - sin a„x) cos a„Z, +cosh a„Z, n .A. 16 A.2. Torsional Vibrations of a Cantilever Beam The governing differential equation of free vibration of beam with St. Venant torsion beam theory can be written as: ^ A.17 6% " a/' For a clamped-free beam the boundary conditions are: For a clamped-free beam the boundary conditions are: For a clamped-free beam the boundary conditions are: 0 (0,0 = 0 A.18 00(1,0 dx = 0 0 (0,0 = 0 A.18 00(1,0 dx = 0 0 (0,0 = 0 A.18 00(1,0 dx = 0 A.18 Using the separation of variables, the solution of differential equation can be written as: 0(x, 0 = Ç(x) sin at A. 19 By substituting (A. 19) into (A.17), one can get: By substituting (A. 19) into (A.17), one can get: By substituting (A. 19) into (A.17), one can get: (GJ<p'y + (m a^K j)(p = 0 a.20 (GJ<p'y + (m a^K j)(p = 0 For a uniform beam in element coordinates: For a uniform beam in element coordinates: 107 A.21 GJ(p" ■\-{mKjco^)(p = 0 Assuming an exponential function as the solution of (A.21): Appendix B The formulations presented in this thesis were verified by different illustrative examples discussed in Chapters 4 and 5. Because of some conceptual differences between two approaches, the FEM and DFE method, the algorithms of these two methods are different. The FEM approach, as a well-known and reliable method, has less complexity in programming because of polynomial shape functions and constant mass and stiffness matrices. On the other hand, because of frequency dependent approximation functions, the DFE requires developing more functions and a more complex algorithm. Assuming an exponential function as the solution of (A.21): Assuming an exponential function as the solution of (A.21): A.22 ç{x) = Ae^ and substituting (A.22) into (A.21): and substituting (A.22) into (A.21): G JA '+(m /c„V ) = 0 A.23 G JA '+(m /c„V ) = 0 A.23 A.24 V GJ A.24 A.24 One can see that the parameter t has already introduced in Chapter 5 (equation (5.4)). The term 4 appears in expressions for a, j8 and t in equations (5.3) and (5.4) but not in (A. 10), (A. 11) and (A.24), because in the former equations the solutions are evaluated in terms of element coordinate and 4 (the Jacobian) is produced after each differentiation. Substituting equation (A.24) into (A.22), one can get the mode shapes of torsion vibrations of the beam: çj(a:) = A sinrr + Bcoszx A.25 çj(a:) = A sinrr + Bcoszx A.25 For clamped-free boundary conditions (A.18), an eigenproblem of the following form is obtained: 0 1 cos tL - sin tL Ib C 0 where the characteristic equation can be written as: where the characteristic equation can be written as: where the characteristic equation can be written as: 108 COS Æ = 0 A .2 7 COS Æ = 0 A .2 7 The weighted natural frequencies, j„L, and natural frequencies can be simply calculated as: — (2/1 — 1)— A.28 A.28 =(2«-1)t J .2^^ 2 A.29 niK. 2 \ L A.29 When GJ = 1, L = 1, m = 1 and Km = I, the analytical solution for torsional natural frequencies was compared with numerical values, calculated by FEM and DFE, in Chapter 4 and Chapter 5, respectively. The mode shapes of torsional vibrations of a cantilever beam then can be written as: <Pn W = sin T„x A.30 <Pn W = sin T„x <Pn W = sin T„x A.30 109 B.l. The FEM Program and Algorithm The FEM method has been programmed first by Maple© to develop the shape functions vectors and their derivatives. The matrices produced by vector products of shape functions vectors and their derivatives are integrated over element lengths to develop the elementary matrices which construct the mass and stiffness matrices (these matrices are the terms introduced in equations (4.35) to (4.39)). The element mass and stiffness matrices are then calculated. An assembly loop produces the total mass and stiffness matrices. After applying the boundary conditions, the standard eigenvalue command is used to calculate the natural frequencies. After developing the Maple© program, a similar code was written in the Matlab® environment to calculate the natural frequencies and mode shapes. The Matlab® program consists of two functions, the main program calculates the total mass and stiffness matrices and calculates the natural frequencies, and the second function calculates and plots the mode shapes. The Figures B.l and B.2 show the algorithm of FEM program. 110 Start Reading the beam’s properties, number of elements and DOF per node End Output: Natural Frequencies Applying the boundary conditions Calculating the natural frequencies Assembling the system mass and stiffness matrix Constructing the element mass and stiffness matrices Reading tlie elementary matrices, which form tlie mass and stiffness matrices Algorithm of main program of FEM me of natural frequencies. Start Reading the beam’s properties, number of elements and DOF per node End Output: Natural Frequencies Applying the boundary conditions Calculating the natural frequencies Assembling the system mass and stiffness matrix Constructing the element mass and stiffness matrices Reading tlie elementary matrices, which form tlie mass and stiffness matrices e Algorithm of main program of FEM meth of natural frequencies. Start Reading the beam’s properties, number of elements and DOF per node End Output: Natural Frequencies Applying the boundary conditions Calculating the natural frequencies Assembling the system mass and stiffness matrix Constructing the element mass and stiffness matrices Reading tlie elementary matrices, which form tlie mass and stiffness matrices Figure B.l: The Algorithm of main program of FEM method for calculation of natural frequencies. Reading the beam’s properties, number of elements and DOF per node Reading tlie elementary matrices, which form tlie mass and stiffness matrices Figure B.l: The Algorithm of main program of FEM method for calculation of natural frequencies. B.l. The FEM Program and Algorithm I l l Start Getting the system mass and stiffness matrices, the number of elements, the number of modes, DOF per node and the natural frequencies DGF = 3 DGF = 5 DGF = 4 End Calculation of eigenvectors Storing the mode shapes in separate modal matrices Storing the mode shapes in separate modal matrices Storing the mode shapes in separate modal matrices Separating the nodal displacements of w and V variables Separating the nodal displacements of w, v and (j) variables Separating the nodal displacements of w and (j) variables Plotting the mode shapes of each displacement Perturbation of zero force vector and natural frequencies Figure B.2: The Algorithm of the program which calculates the mode shapes, used for the FEM method. Getting the system mass and stiffness matrices, the number of elements, the number of modes, DOF per node and the natural frequencies Perturbation of zero force vector and natural frequencies Calculation of eigenvectors DGF = 4 DGF = 5 Separating the nodal displacements of w and V variables Separating the nodal displacements of w, v and (j) variables Separating the nodal displacements of w and (j) variables Storing the mode shapes in separate modal matrices Storing the mode shapes in separate modal matrices Plotting the mode shapes of each displacement Figure B.2: The Algorithm of the program which calculates the mode shapes, used for the FEM method. 1 1 2 The DOF in Figure B.2 represents the number of nodal variables, i.e., that means for each bending DOF = 2 (displacement and slope) and for torsion DOF = 1. Then DOF = 3, DOF = 4 and DOF = 5 are corresponding to the bending-torsion, bending- bending and bending-bending-torsion vibrations, respectively. B.2. DFE Program and Algorithm As already stated, the mass and stiffness matrices in FEM are constant, but in DFE the elementary matrices and consequently the stiffness matrix are frequency dependent. This causes that the terms in equation (5.35) should be integrated numerically and for each element. On the other hand the nature of bisection method which is an iterative algorithm, require that the calculation of j to be repeated, which causes more computation time. All these reasons result a different algorithm from FEM. In DFE program, several functions were defined. The main function executes the bisection algorithm to find natural frequencies. In this function another function Jo(w) is called to calculate j number for each trial frequency. The main function algorithm has been shown in Figure B.3. 113 Start Reading the main parameters: Number of frequencies: ttg The accuracy of calculation: 6 Upper and lower trial frequencies: and W/ for i = l t o wj While While ( (j^h- W/) ^ 5 End Store CO in oj/ Output: Natural Frequencies, co,- Figure B.3: The main DFE function bisection algorithm. Reading the main parameters: Number of frequencies: ttg The accuracy of calculation: 6 Upper and lower trial frequencies: and W/ for i = l t o wj While Figure B.3: The main DFE function bisection algorithm. 114 Following the Wittrick-Williams method, the function Jo(w) (used in bisection algorithm) calculates y. All parameters associated with j, such as upper triangular stiffness matrix, its sign count andy„, are calculated in /«(w). The function gets the beam properties, number of elements, and DOF of the element (as input) and calculates the parameters of equations (5.3) to (5.5) using the trial frequency co (co* is the function’s argument which is called from main function). Then the function calculates the uncoupled and coupled element stiffness matrices, equation (5.36) and (5.37). The element stiffness matrix is then calculated along with the jm of the element. After assembling the total stiffness matrix, the geometrical boundary conditions are applied. The result is the matrix, [Æ(co)], which form the nonlinear eigenproblem. [A((o)] is transformed to an upper triangular matrix by a function called UT which gets the matrix and its size as input and returns the upper triangular matrix. Then the sign count of transformed matrix in calculated by another function called SCOUNT. Sign count of total stiffness matrix and jm are added together to calculate y as Vo(w)’s output. B.2. DFE Program and Algorithm The algorithm of function /«(to) is shown in Figure B.4. There are two functions called in Jo(co) to calculate the element uncoupled and coupled stiffness matrix. These functions are shown in Figure B.4 as K COUPLED and K_UNCOUPLED. 115 Start Reading: The properties of beam Angular speed of the beam ft Number of elements n Z)OFper node____________ Assembly loop for i=l to n Calculating: ^W9 Pwj ^V9 ^Vf ^tld T Calculating the element’s jm and stiffness matrix Adding element stiffness matrix to the total stiffness and element jm to the totaly‘;„ Calling K_UNCOUPLED and K_COUPLD: Calculating the coupled and uncoupled stiffness matrices Start Reading: The properties of beam Angular speed of the beam ft Number of elements n Z)OFper node____________ g p Number of elements n Z)OFper node____________ Assembly loop for i=l to n End Calculating: ^W9 Pwj ^V9 ^Vf ^tld T Calling the function “UT” which produces the upper triangular matrix Applying the boundary condition to the total stiffness matrix Calling the function “SCOUNT” which calculates the sign count of a matrix Output: Jo which is used in main program Calculating the element’s jm and stiffness matrix Adding element stiffness matrix to the total stiffness and element jm to the totaly‘;„ Calling K_UNCOUPLED and K_COUPLD: Calculating the coupled and uncoupled stiffness matrices Figure B.4: The algorithm of calculation of Jo(w). Assembly loop for i=l to n Calculating: ^W9 Pwj ^V9 ^Vf ^tld T Applying the boundary condition to the total stiffness matrix Figure B.4: The algorithm of calculation of Jo(w). 116 The DFE algorithm can be used to evaluate any single i** natmal frequency of beam’s free vibrations. Also it is possible to evaluate n natural frequencies, i to (/+«)> which is one of the advantages of this method. When the natural frequencies are evaluated, a function (similar to the one presented in Figure B.2) can be exploited to evaluate the mode shapes corresponding to the natural frequencies. The algorithms and flow charts, presented in this Appendix for the FEM and DFE methods, can be used for analysis of pure bending, dually or triply coupled vibrations of rotating and non-rotating beams and blades. As already stated, the FEM algorithm is simple and easy to implement, because of polynomial shape functions and constant mass and stiffness matrices. On the other hand, the DFE, because of the iterative nature of Wittrick-Williams and bisection method, is more complicated. B.2. DFE Program and Algorithm This algorithm works with more functions, some of them store the symbolic forms of different terms of stiffness matrix. These symbolic matrices are called in numeric integration loop in K_COUPLED and K UNCOUPLED functions. 117 REFERENCES: [1] [1] J.C. Houbolt, and G.W. Brooks, “Differential equations of motion for combined flapwise bending, chordwise bending, and torsion of twisted nonuniform rotor blades.” AC4C/4 Technical Note 3905, report 1346, 1956. [2] V.R. Murthy, “Dynamic characteristics of rotor blades.” Journal of Sound and Vibration, 49(4) 483-500,1976. [3] V.R. Murthy, “Dynamic characteristics of rotor blades: Integrating Matrix Method.” American Institute of Aeronautics and Astronautics Journal, 15(4) 595- 597,1977. [4] V.R. Murthy, and A.M. 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https://openalex.org/W3212504875	https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1010068&type=printable	English	null	Infection, recovery and re-infection of farmed mink with SARS-CoV-2	PLOS pathogens	2,021	cc-by	9,180	RESEARCH ARTICLE Infection, recovery and re-infection of farmed mink with SARS-CoV-2 RESEARCH ARTICLE Thomas Bruun RasmussenID1, Jannik Fonager1, Charlotte Sværke JørgensenID1, Ria LassaunièreID1, Anne Sofie Hammer2, Michelle Lauge QuaadeID2, Anette BoklundID2, Louise LohseID1, Bertel Strandbygaard1, Morten RasmussenID1, Thomas Yssing MichaelsenID3, Sten MortensenID4, Anders FomsgaardID1, Graham J. BelshamID2, Anette Bøtner2 1 Department of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen S, Denmark, 2 Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark, 3 Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark, 4 Danish Veterinary and Food Administration, Glostrup, Denmark a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * grbe@sund.ku.dk (GJB); aneb@sund.ku.dk (AB) OPEN ACCESS Citation: Rasmussen TB, Fonager J, Jørgensen CS, Lassaunière R, Hammer AS, Quaade ML, et al. (2021) Infection, recovery and re-infection of farmed mink with SARS-CoV-2. PLoS Pathog 17(11): e1010068. https://doi.org/10.1371/journal. ppat.1010068 Editor: Kanta Subbarao, The Peter Doherty Institute and Melbourne University, AUSTRALIA Editor: Kanta Subbarao, The Peter Doherty Institute and Melbourne University, AUSTRALIA Copyright: © 2021 Rasmussen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene as well as the A22920T mutation, result- ing in the Y453F substitution within this protein, seen previously in mink. The infected mink recovered and after free-testing of 300 mink (a level giving 93% confidence of detecting a 1% prevalence), the animals remained seropositive. During further follow-up studies, after a period of more than 2 months without any virus detection, over 75% of tested animals again scored positive for SARS-CoV-2 RNA. Whole genome sequencing showed that the viruses circulating during this re-infection were most closely related to those identified in the first out- break on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies in serum samples after the second round of infection than at free-testing or during recovery from initial infection, consistent with a boosted immune response. Thus, it was concluded that following recovery from an initial infection, seropositive mink were readily re-infected by SARS-CoV-2. PLOS PATHOGENS PLOS PATHOGENS PLOS PATHOGENS * grbe@sund.ku.dk (GJB); aneb@sund.ku.dk (AB) Author summary Early on, in the course of SARS-CoV-2 infections among mink in Denmark, we identified a farm, with about 15,000 mink, that had virus-infected animals. At this time, we found that a very high proportion of the mink had been infected and made antibodies against the virus. In contrast to the three previously infected farms, the mink were allowed to recover (the mink had shown few signs of disease and only low mortality) and our testing demonstrated the absence of circulating virus. Continued screening, in the following weeks, supported the absence of infection in the mink but the maintenance of antibodies against the virus. However, less than 3 months after the initial infection, we again Data Availability Statement: All sequences have been submitted to the GISAID database and accession numbers are listed in the Supporting Information, S1 and S2 Tables. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist Competing interests: The authors have declared that no competing interests exist 1 / 13 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 identified the presence of virus in some dead mink from this farm and in many live mink. The viruses responsible for this second wave of infection were slightly different from those found in the first wave but were closer to each other than to the SARS-CoV-2s found on other mink farms. The antibody levels in mink during this second wave of infec- tion were much higher than observed after the initial infection. We concluded that the ini- tial round of infection in mink was insufficient to confer protection against re-infection. Introduction The SARS-CoV-2 has caused a pandemic with about 200 million cases reported and it has con- tributed to the deaths of over 5 million people [1]. Farmed mink (Neovison vison) are also highly susceptible to infection by SARS-CoV-2 [2, 3]. As in humans, the infection in mink can cause respiratory distress and, in some cases, mortality. However, often the proportion of infected mink that show clinical disease is low. Cases of SARS-CoV-2 infection in farmed mink were initially observed in the Netherlands (NL), in April 2020 [3], and then indepen- dently in Denmark (DK) in June 2020 (note, different clades of the virus were involved, [2]). Outbreaks continued and about 70 farms in the NL became infected [4], while 290 farms out of about 1200 mink farms in DK were found positive for the virus [5]. All farmed mink (>15,000,000) in DK have now been culled [6]. Similarly, the termination of mink farming in the NL was brought forward by 3 years from the previous plan of 1st January 2024 [4]. The routes of transmission of the virus between mink farms are not fully understood [5] but it has become apparent that spread of the virus can occur not only from humans to mink but also from mink to humans [2, 7]. After the initial cases of SARS-CoV-2 infection in mink in DK, on Farms 1–3 in Northern Jutland [2], a screening program was established to test dead mink from all Danish mink farms for the presence of SARS-CoV-2, every 3rd week [6]. Infection of mink on Farm 4 was identified through this Early Warning (EW) program but, in contrast to Farms 1–3, the mink on this farm were not culled and the seropositive animals apparently cleared the infection. This allowed an evaluation of the duration and efficacy of the immune response in mink to protect against re-infection. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 Infection of mink on Farm 4 Farm 4 (with about 2400 adult mink and 12600 kits in 24 open-sided houses, S1 Fig), was located near Hjørring (in Northern Jutland). It had been classified as having “Aleutian disease- free status” since 2015. This farm was tested for the presence of the SARS-CoV-2 as part of the EW screening program. On 20th July 2020, 5 dead mink from this farm were tested for the virus and all were RT-qPCR negative. However, on 11th August, a further 5 dead mink were tested and all were positive in this assay (Table 1). In follow-up testing, on 13th August, 23 of 30 live mink tested (16 adults and 14 kits) were positive. A further 7 (of 10) dead mink also tested positive. All live mink tested (30 kits and 30 adults) were also strongly seropositive on 19th August, but a reduced proportion of the mink (13 of these 60 mink tested) were positive for SARS-CoV-2 RNA. However, throat swab samples from 21 dead mink were all positive for viral RNA. Furthermore, on 31st August, 7 out of 24 dead mink also tested positive by RT- qPCR. The mink on the farm were not culled but closely followed and, from 15th September onwards, no virus was detected by RT-qPCR among the mink. For “free-testing”, on 30th PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 2 / 13 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 Table 1. Summary of laboratory analysis of mink sampling from Farm 4. ELISA RT-qPCR Sample origin Sera (positive/tested) % Throat swabs (positive/tested) % Date of sample collection Dead mink (EW) n.d. 0/5 0 20-07-2020 Dead mink (EW) n.d. 5/5 100 11-08-20201 Live adult mink n.d. 11/16 69 13-08-2020 Live mink kits n.d. 12/14 86 13-08-2020 Dead mink n.d. 7/10 70 13-08-2020 Live adult mink 30/30 100 4/30 13 19-08-2020 Live mink kits 30/30 100 9/30 30 19-08-2020 Dead mink n.d. 21/21 100 19-08-2020 Dead mink n.d. 7/24 29 31-08-2020 Dead mink n.d. 0/31 0 15-09-2020 Dead mink n.d. 0/25 0 28-09-2020 Live mink n.d. 0/60 0 30-09-2020 Live adult mink2 29/29 100 0/29 0 05-10-2020 Live mink kits2 30/30 100 0/30 0 05-10-2020 Dead mink (EW) n.d. 1/2 50 02-11-2020 Dead mink (EW) n.d. 1/2 50 04-11-2020 Live mink 30/30 100 23/30 77 06-11-2020 Dead mink n.d. 3/5 60 06-11-2020 Table 1. Summary of laboratory analysis of mink sampling from Farm 4. https://doi.org/10.1371/journal.ppat.1010068.t001 Infection of mink on Farm 4 n.d.: not done 300 animals were tested in pools of 5, i.e. in 60 assays two pools of 5 samples tested 1: Samples were received at SSI on this date. 2: Samples were collected from the same animals as 19-08-2020 https://doi.org/10.1371/journal.ppat.1010068.t001 September, a larger sampling was performed involving some 300 animals, from across each of the different animal houses. All the samples were tested (in 60 pools of 5 samples) with nega- tive results (Table 1). At least 12 samples were collected from each of the 24 houses, from adults and kits in similar proportions. This testing was designed to detect, with 95% confidence, a 1% prevalence of SARS-CoV-2 RNA positive animals. Hence, the infection had apparently disap- peared among the mink on this farm at that time. Surveillance of the farm continued and, in early October, 59 of 60 live mink tested in mid- August were again all found negative by RT-qPCR but all these mink remained seropositive (Table 1). Thus, no animals tested positive by RT-qPCR in September and October but all tested animals had antibodies against SARS-CoV-2. However, unexpectedly, 1 of 2 pools of 5 dead mink tested, as part of the continuing EW program, on both 2nd and 4th November were found to be positive by RT-qPCR (Table 1). Consequently, during the culling process that was initiated, a further 30 animals were tested on 6th November and 23 (77%) were found SARS-CoV-2 RNA positive and 100% of these animals were found to be seropositive. In addi- tion, 3 out of 5 additional dead mink were found positive by RT-qPCR (Ct values for 15 of 23 samples were below 30). During the initial infection, the farmer noticed reduced feed intake with some cases of diarrhea among the mink, and a few mink later displayed respiratory dis- tress, however, no respiratory signs were displayed before culling during the reinfection. Fig 1 shows a timeline for the percentages of the mink tested that were seropositive and RT-qPCR positive on Farm 4 during the period August-November 2020. 3 / 13 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 Fig 1. Timeline for infection of mink on Farm 4. Assessment of anti-SARS-CoV-2 antibodies in mink sera by ELISA Titration, using ELISA, of the anti-SARS-CoV-2 antibodies (that recognize the receptor bind- ing domain (RBD)) in seropositive serum samples collected from August onwards showed that much higher levels of these antibodies were present in the mink in November, following the second round of infection than in August or October (Fig 2A). However, the proportion of seropositive animals among the tested mink had been high throughout. In August (at initial testing), the median antibody titre observed (from 16 animals tested) was 800 (range from 100 to 3200), with just a single animal having the highest titre. In early October (at free-testing), the titres in 15 sera tested were higher (ranging from 800 to 12800, with 4 of the sera having titres of 6400, the median titre was 3200). However, in November, following re-infection as judged by the reappearance of RT-qPCR positive animals, 16 of 22 sera tested, had titres 6400 and 12 had titres of 25600, see Fig 2A). There were significant differences in the levels of antibodies detected by ELISA in the serum samples collected at different times (P < 0.001, Kruskal-Wallis test), with higher levels at the time of “free-testing” compared to initial sam- pling and much higher levels following reinfection compared to the earlier testing. Infection of mink on Farm 4 The percentage of live and dead mink assayed by RT-qPCR that tested positive is shown throughout the period of August to November 2020, together with the proportion of live mink that tested positive by ELISA for anti-CoV-2 antibodies. The numbers of animals tested on each date are shown in Table 1. Dotted lines have been used to connect the data points for clarity but should not be used to infer intermediate percentage levels. Fig 1. Timeline for infection of mink on Farm 4. The percentage of live and dead mink assayed by RT-qPCR that tested positive is shown throughout the period of August to November 2020, together with the proportion of live mink that tested positive by ELISA for anti-CoV-2 antibodies. The numbers of animals tested on each date are shown in Table 1. Dotted lines have been used to connect the data points for clarity but should not be used to infer intermediate percentage levels. Fig 1. Timeline for infection of mink on Farm 4. The percentage of live and dead mink assayed by RT-qPCR that tested positive is shown throughout the period of August to November 2020, together with the proportion of live mink that tested positive by ELISA for anti-CoV-2 antibodies. The numbers of animals tested on each date are shown in Table 1. Dotted lines have been used to connect the data points for clarity but should not be used to infer intermediate percentage levels. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 Assessment of neutralizing antibodies Anti-SARS-CoV-2 antibody titres were measured by ELISA. Selected sera from mink collected at the time of initial diagnosis (blue circles), at free-testing (grey circles) and following re-infection (red circles), on 19-08-20, 05-10-20 and 06-11-20 respectively, that scored positive when assayed undiluted were titrated and assayed again by ELISA. The reciprocals of the highest dilution yielding a positive signal are plotted (on a log2 scale). Mean (+/- SEM) values are indicated by horizontal black lines. Panel B. The same serum samples were also assayed in virus neutralization assays and the calculated antibody titres are plotted (on a log2 scale) using the same colour scheme. The statistical significance of the differences between anti-CoV-2 antibody levels was determined using the Kruskal-Wallis test (see Materials and Methods). https://doi.org/10.1371/journal.ppat.1010068.g002 https://doi.org/10.1371/journal.ppat.1010068.g002 ELISA positive sera tested had neutralization activity but the levels of these antibodies were greatly elevated after the re-infection (sera collected in November). This difference in levels of neutralizing antibodies between the time of “free testing” and after reinfection was statistically significant (P = 0.0009), while there was no significant difference in levels of neutralizing anti- bodies between the time of initial testing and the time of free testing (P = 0.38). There was a high degree of correspondence between the levels of antibodies detected in the two different types of assay (for all samples, the Spearman correlation co-efficient r = 0.793, P <0.0001). Assessment of neutralizing antibodies To assess whether the anti-SARS-CoV-2 antibodies in the mink that were detected by ELISA could neutralize virus infectivity, the same serum samples were also tested in virus neutraliza- tion assays using a human SARS-CoV-2 isolate that had the same amino acid changes in the spike protein as the viruses identified initially on Farm 4 (as used previously [8]). The results (Fig 2B) showed a similar profile of antibody levels as observed in the ELISA. All of these PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 4 / 13 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 Fig 2. Assessment of anti-SARS CoV-2 antibody levels in mink sera. Panel A. Anti-SARS-CoV-2 antibody titres were measured by ELISA. Selected sera from mink collected at the time of initial diagnosis (blue circles), at free-testing (grey circles) and following re-infection (red circles), on 19-08-20, 05-10-20 and 06-11-20 respectively, that scored positive when assayed undiluted were titrated and assayed again by ELISA. The reciprocals of the highest dilution yielding a positive signal are plotted (on a log2 scale). Mean (+/- SEM) values are indicated by horizontal black lines. Panel B. The same serum samples were also assayed in virus neutralization assays and the calculated antibody titres are plotted (on a log2 scale) using the same colour scheme. The statistical significance of the differences between anti-CoV-2 antibody levels was determined using the Kruskal-Wallis test (see Materials and Methods). https://doi.org/10.1371/journal.ppat.1010068.g002 Fig 2. Assessment of anti-SARS CoV-2 antibody levels in mink sera. Panel A. Anti-SARS-CoV-2 antibody titres were Fig 2. Assessment of anti-SARS CoV-2 antibody levels in mink sera. Panel A. Anti-SARS-CoV-2 antibody titres were measured by ELISA. Selected sera from mink collected at the time of initial diagnosis (blue circles), at free-testing (grey circles) and following re-infection (red circles), on 19-08-20, 05-10-20 and 06-11-20 respectively, that scored positive when assayed undiluted were titrated and assayed again by ELISA. The reciprocals of the highest dilution yielding a positive signal are plotted (on a log2 scale). Mean (+/- SEM) values are indicated by horizontal black lines. Panel B. The same serum samples were also assayed in virus neutralization assays and the calculated antibody titres are plotted (on a log2 scale) using the same colour scheme. The statistical significance of the differences between anti-CoV-2 antibody levels was determined using the Kruskal-Wallis test (see Materials and Methods). https://doi.org/10.1371/journal.ppat.1010068.g002 Fig 2. Assessment of anti-SARS CoV-2 antibody levels in mink sera. Panel A. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 Whole genome sequencing of viruses on Farm 4 The complete genome sequences of the viruses, from multiple samples, from infected mink in August and in November were determined. The viruses on Farm 4 in August were very closely related to the viruses that were previously identified on Farms 1, 2 and 3 [2], they were from clade 20B / lineage B.1.1.298 (Fig 3A and 3B and Table 2) and appeared part of the same trans- mission chain. In particular, they each had the mutation A22920T in the spike protein coding sequence, resulting in the amino acid substitution Y453F. However, in addition to this change, the spike protein gene in the viruses on Farm 4 had a deletion of 6 nt (Δ21766–21771). This affected 3 codons, changing GCT.ATA.CAT.GTC.TCT to GCT.ATC.TCT, the encoded amino acid sequence is changed from A-I-H69-V70-S to A-I-S thus residues H69 and V70 in the N-terminal domain (NTD) of the spike protein are lost. This deletion had not been identified previously in mink or in humans in combination with the Y453F substitution (see Table 2) but the deletion of these residues is shared with the SARS CoV-2 variant of concern (VOC) 202012/01 [9]. Two other deletions in the ORF1a coding sequence (Δ517–519 and Δ6510– 6512) and two other amino acid substitutions (P3395S in ORF1a and S2430I in ORF1b) were also observed in some of the viruses present in the mink during this initial infection in August. The viruses present on Farm 4 in November were most closely related to those seen previously PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 5 / 13 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 Fig 3. Phylogenetic trees showing the relationships between the full genome sequences of SARS-CoV-2 samples from Danish mink farms with the lineage B.1.1.298 variants. Panel A. All known SARS-CoV2 lineage B.1.1.298 genome sequences (436 in total) from Danish mink along with the Wuhan reference sequence (NC_045512.2) were included in this analysis. Sequences from the re-infection on Farm 4 (collected in November) are indicated by open red triangles while samples collected in August are indicated by blue triangles. Sequences from Farm 1 are indicated with green squares and the Wuhan reference strain with a black filled circle. The GISAID accession IDs are listed in S1 Table. Panel B. The Maximum Likelihood phylogenetic relationships between the viruses collected on Farm 4 and those from Farms 1–3 are shown. The GISAID accession IDs are listed in S2 Table. Fig 3. Whole genome sequencing of viruses on Farm 4 Phylogenetic trees showing the relationships between the full genome sequences of SARS-CoV-2 samples from Danish mink farms with the lineage B.1.1.298 variants. Panel A. All known SARS-CoV2 lineage B.1.1.298 genome sequences (436 in total) from Danish mink along with the Wuhan reference sequence (NC_045512.2) were included in this analysis. Sequences from the re-infection on Farm 4 (collected in November) are indicated by open red triangles while samples collected in August are indicated by blue triangles. Sequences from Farm 1 are indicated with green squares and the Wuhan reference strain with a black filled circle. The GISAID accession IDs are listed in S1 Table. Panel B. The Maximum Likelihood phylogenetic relationships between the viruses collected on Farm 4 and those from Farms 1–3 are shown. The GISAID accession IDs are listed in S2 Table. https://doi.org/10.1371/journal.ppat.1010068.g003 on Farm 4, over 2 months earlier (Fig 3A and 3B). It should be noted that, by November 2020, over 200 farms in DK had been identified as having infected mink [5] and a number of differ- ent variants had been observed in the animals [6]. The viruses on Farms 1–3 were closely related to each other and also to the viruses present in August on Farm 4, but the latter viruses had some additional changes (e.g. the deletion of residues H69 and V70 in the spike protein, see Table 2), which were also found in most of the later outbreaks in farmed mink in DK. Thus, viruses in farms infected after Farm 4 (identified on August 11th) were nearly all derived from those first detected on Farm 4. As indicated above, the November viruses (following re- infection) from Farm 4 had the A22920T mutation and the deletions in the S and ORF1a cod- ing sequences. However, they also had additional changes across the genome, both within and outside of the S gene, compared to the viruses from Farm 4 in August (Table 2). It is notewor- thy that the Farm 4 sequences in November had changes at nt 10448 (encoding the substitu- tion P3395S in ORF1a) and 20756 (encoding S2430I in ORF1b) that had only been seen in a subset of the August sequences from Farm 4 (samples Farm4_18_13-08-2020 and Farm4_19_13-08-2020, see Fig 3B). These changes strongly suggest that the infection in on Farm 4, over 2 months earlier (Fig 3A and 3B). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 Whole genome sequencing of viruses on Farm 4 It should be noted that, by November 2020, over 200 farms in DK had been identified as having infected mink [5] and a number of differ- ent variants had been observed in the animals [6]. The viruses on Farms 1–3 were closely related to each other and also to the viruses present in August on Farm 4, but the latter viruses had some additional changes (e.g. the deletion of residues H69 and V70 in the spike protein, see Table 2), which were also found in most of the later outbreaks in farmed mink in DK. Thus, viruses in farms infected after Farm 4 (identified on August 11th) were nearly all derived from those first detected on Farm 4. As indicated above, the November viruses (following re- infection) from Farm 4 had the A22920T mutation and the deletions in the S and ORF1a cod- ing sequences. However, they also had additional changes across the genome, both within and outside of the S gene, compared to the viruses from Farm 4 in August (Table 2). It is notewor- thy that the Farm 4 sequences in November had changes at nt 10448 (encoding the substitu- tion P3395S in ORF1a) and 20756 (encoding S2430I in ORF1b) that had only been seen in a subset of the August sequences from Farm 4 (samples Farm4_18_13-08-2020 and Farm4_19_13-08-2020, see Fig 3B). These changes strongly suggest that the infection in November was not due to an entirely new introduction of virus into the farm from elsewhere (Fig 3A). Furthermore, the viruses on Farm 4 in November also all shared changes at nt 3792 (resulting in A1176V), 5167, 10887 (resulting in G3541E), 21727 and 23815 (these latter two silent changes are in the S gene) that were not present in any of the Farm 4 sequences in August (Table 2). The presence of these additional sequence changes indicates that the virus had been replicating between August and November. Farm4_19_13-08-2020, see Fig 3B). These changes strongly suggest that the infection in November was not due to an entirely new introduction of virus into the farm from elsewhere (Fig 3A). Furthermore, the viruses on Farm 4 in November also all shared changes at nt 3792 (resulting in A1176V), 5167, 10887 (resulting in G3541E), 21727 and 23815 (these latter two silent changes are in the S gene) that were not present in any of the Farm 4 sequences in August (Table 2). Whole genome sequencing of viruses on Farm 4 Shared additional changes that occurred in viruses on Farm 4 between August and November 2020 are indicated with colour codes, encoded amino acid changes, where applicable, are also shown. The Accession IDs for all the indicated sequences are listed in S2 Table. Table 2. Sequence changes within SARS-CoV-2 in mink on Farm 4. Location 5’- UTR ORF1a ORF1b S ORF3a N Nt 241 3037 5144 10448 11776 14408 15656 20756 Δ21766– 21771 22920 23403 25936 28854 other Virus Wuhan C C C C C C C G - A A C C EPI_ISL_455326 20B T T C C C T C G - A G C C Farm 1 T T C C C T T G - T/A G T C Farm 2 T T C C C T T G - T G T C Farm 3 T T C C C T T G - T G T C Aug 2020 Farm4_5 T T T C T T T G + T G T T Farm4_6 T T T C T T T G + T G T T G488A Farm4_8 T T T C T T T G + T G T T Δ21984–21995 Farm4_18 T T T T T T T T + T G T T Farm4_19 T T T T T T T T + T G T T Farm4_21 T T T C T T T G + T G T T A652C (K129N)1 Farm4_35 T T T C T T T G + T G T T Δ27982–28030 Farm4_37 T T T C T T T G + T G T T T1873C,G2035T(L590F) Nov 2020 Farm4_1 T T T T T T T T + T G T T C1913T (R550C), C3792T ðA1176VÞ , C5167T , G10887A ðG3541EÞ , C21727T , T23815C Farm4_14 T T T T T T T T + T G T T A3303G, C3792T ðA1176VÞ , C5167T , G10887A ðG3541EÞ , C21727T , T23815C Farm4_15 T T T T T T T T + T G T T A3303G, C3792T ðA1176VÞ , C5167T , G10887A ðG3541EÞ , C21727T , T23815C AA change - - - P3395S - P314L T730I S2430I ΔH69-V70 Y453F D614G H182Y S194L 1: Note the same additional sequence change was also present in 4 other samples (Farm4_16_13-08-2020, Farm4_20_13-08-2020, Farm4_22_13-08-2020 and Farm4_4_19-08-2020). N.B. Whole genome sequencing of viruses on Farm 4 All the mink viruses, together with the EPI_ISL455326 clade 20B representative sequence, shown here were from clade 20B and had the changes G28881A, G28882A and G28883C compared to the Wuhan strain. In addition, the mink viruses from Farm 4 also lacked nt 517–519 and nt 6510–6512. Nucleotide changes from the Wuhan reference sequence are highlighted in yellow. Shared additional changes that occurred in viruses on Farm 4 between August and November 2020 are indicated with colour codes, encoded amino acid changes, where applicable, are also shown. The Accession IDs for all the indicated sequences are listed in S2 Table. Table 2. Sequence changes within SARS-CoV-2 in mink on Farm 4. 2. Sequence changes within SARS-CoV-2 in mink on Farm 4 1: Note the same additional sequence change was also present in 4 other samples (Farm4_16_13-08-2020, Farm4_20_13-08-2020, Farm4_22_13-08-2020 and Farm4_4_19-08-2020). N.B. All the mink viruses, together with the EPI_ISL455326 clade 20B representative sequence, shown here were from clade 20B and had the changes G28881A, G28882A and G28883C compared to the Wuhan strain. In addition, the mink viruses from Farm 4 also lacked nt 517–519 and nt 6510–6512. Nucleotide changes from the Wuhan reference sequence are highlighted in yellow. Shared additional changes that occurred in viruses on Farm 4 between August and November 2020 are indicated with colour codes, encoded amino acid changes, where applicable, are also shown. The Accession IDs for all the indicated sequences are listed in S2 Table. 1: Note the same additional sequence change was also present in 4 other samples (Farm4_16_13-08-2020, Farm4_20_13-08-2020, Farm4_22_13-08-2020 and Farm4_4_19-08-2020). N.B. All the mink viruses, together with the EPI_ISL455326 clade 20B representative sequence, shown here were from clade 20B and had the changes G28881A, G28882A and G28883C compared to the Wuhan strain. In addition, the mink viruses from Farm 4 also lacked nt 517–519 and nt 6510–6512. Nucleotide changes from the Wuhan reference sequence are highlighted in yellow. Shared additional changes that occurred in viruses on Farm 4 between August and November 2020 are indicated with colour codes, encoded amino acid changes, where applicable, are also shown. The Accession IDs for all the indicated sequences are listed in S2 Table. Whole genome sequencing of viruses on Farm 4 above, two of the early Farm 4 viruses (Farm4_18_13-08-2020 and Farm4_19_13-08-2020) shared additional changes at nt 10448 and 20756 (see Table 2) and the November viruses formed their own distinct branch from these (Fig 3B), due to the presence of the further sequence changes (Table 2). Whole genome sequencing of viruses on Farm 4 The presence of these additional sequence changes indicates that the virus had been replicating between August and November. Phylogenetic analysis clearly showed that all viruses from Farm 4 were very closely related to each other, including the viruses from both August and November (Fig 3B). As described PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 6 / 13 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 Table 2. Sequence changes within SARS-CoV-2 in mink on Farm 4. Whole genome sequencing of viruses on Farm 4 Location 5’- UTR ORF1a ORF1b S ORF3a N Nt 241 3037 5144 10448 11776 14408 15656 20756 Δ21766– 21771 22920 23403 25936 28854 other Virus Wuhan C C C C C C C G - A A C C EPI_ISL_455326 20B T T C C C T C G - A G C C Farm 1 T T C C C T T G - T/A G T C Farm 2 T T C C C T T G - T G T C Farm 3 T T C C C T T G - T G T C Aug 2020 Farm4_5 T T T C T T T G + T G T T Farm4_6 T T T C T T T G + T G T T G488A Farm4_8 T T T C T T T G + T G T T Δ21984–21995 Farm4_18 T T T T T T T T + T G T T Farm4_19 T T T T T T T T + T G T T Farm4_21 T T T C T T T G + T G T T A652C (K129N)1 Farm4_35 T T T C T T T G + T G T T Δ27982–28030 Farm4_37 T T T C T T T G + T G T T T1873C,G2035T(L590F) Nov 2020 Farm4_1 T T T T T T T T + T G T T C1913T (R550C), C3792T ðA1176VÞ , C5167T , G10887A ðG3541EÞ , C21727T , T23815C Farm4_14 T T T T T T T T + T G T T A3303G, C3792T ðA1176VÞ , C5167T , G10887A ðG3541EÞ , C21727T , T23815C Farm4_15 T T T T T T T T + T G T T A3303G, C3792T ðA1176VÞ , C5167T , G10887A ðG3541EÞ , C21727T , T23815C AA change - - - P3395S - P314L T730I S2430I ΔH69-V70 Y453F D614G H182Y S194L 1: Note the same additional sequence change was also present in 4 other samples (Farm4_16_13-08-2020, Farm4_20_13-08-2020, Farm4_22_13-08-2020 and Farm4_4_19-08-2020). N.B. All the mink viruses, together with the EPI_ISL455326 clade 20B representative sequence, shown here were from clade 20B and had the changes G28881A, G28882A and G28883C compared to the Wuhan strain. In addition, the mink viruses from Farm 4 also lacked nt 517–519 and nt 6510–6512. Nucleotide changes from the Wuhan reference sequence are highlighted in yellow. Discussion SARS-CoV-2 can readily infect humans and mink. In addition, certain other species, e.g. cats, dogs and ferrets, can also be infected following direct inoculation under experimental condi- tions [10, 11]. Furthermore, some cases of transmission from infected people to their cats and PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 7 / 13 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 dogs have occurred but it does not seem to happen more generally. Both cellular and humoral immune responses occur within SARS-CoV-2-infected people and animals [12, 13] and it is common for both humans and animals to be both seropositive and RT-qPCR positive simulta- neously (see [2, 14]). However, as people and animals recover, the levels of virus subside but antibody levels persist, or increase, at least for some time. Farm 4 was the first Danish mink farm, where the animals were allowed to recover follow- ing SARS-CoV-2 infection and were then tested with the purpose of documenting freedom from the virus about 2 months after the initial infection. Thus, Farm 4 gave a unique opportu- nity to follow the maintenance of anti-SARS-CoV-2 antibodies over an extended period and the resistance of the animals to reinfection. As also observed on other mink farms in DK [5], very widespread infection of the mink on Farm 4 by SARS-CoV-2 occurred in the first wave of infection, with 100% of the tested animals being seropositive. During the following period of over 2 months, the animals were repeatedly screened and shown to be negative by RT-qPCR, while 100% of the tested mink remained seropositive. However, in November, it was found that the mink had become infected again. Surprisingly, a high proportion (>75%) of the ani- mals tested had been re-infected by SARS-CoV-2 (Table 1 and Fig 1). The virus responsible for the second round of infection was most closely related to the virus found almost 3 months ear- lier on this farm (Fig 3A and 3B), but with distinctive differences (see Table 2) from the viruses responsible for the initial infections observed in mink on Farms 1–3 [2]. The virus acquired sequence changes during the period between the infections recognized in August and Novem- ber (Table 2), indicative of continued replication, rather than simply having been preserved in an infectious form. Discussion Since the viruses present on Farm 4 in November were most closely related to viruses present on the same farm in August, it seems most likely that re-infection of the mink from within the farm had occurred. It cannot be established, however, whether the virus had continued to replicate in a small number of mink on the farm, but with very restricted spread, or if it had replicated in an alternative host, linked to the farm, during this time and had then been re-introduced into the seropositive mink. It has been demonstrated, in both DK and the NL, that transmission between humans and mink can occur in both directions [2, 6]. Transmission to, and from, other host species is theoretically possible (but not described previ- ously; some cats were found to be infected on mink farms in DK and in the NL but they do not seem to spread the virus). It has been found that there was a cluster of occurrences of SARS-- CoV-2 with the ΔH69/N70 and Y453F changes (as in Farm 4) in the local human population in August. Furthermore, a virus containing these changes plus the additional mutations (i.e. C3792T, C5167T, G10887A, C21727T and T23815C, see Table 2), which were present in the mink viruses from Farm 4 in November, was found in one person in early November. It seems likely that these human cases were infections derived from mink. A high proportion of the sequence changes observed in mink (Table 2), which occurred in the viruses from Farm 4 between August and November (and also between the clade 20B viruses and the Wuhan virus, see [2]), involved C to T changes (in cDNA) that correspond to C to U changes in the viral RNA. Several of these nt changes are synonymous, i.e., they do not result in amino acid sequence changes. It has been suggested that such changes reflect host immune pressure via RNA editing systems (e.g. by APOBEC) rather than selection for increased transmissibility in particular hosts [15–17]. However, this process of RNA editing is not relevant to the key mutation in the S gene (A22920T), which seems to be an adaptation that occurred during the initial infection of mink [2], or to the generation of deletions. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 Discussion The loss of residues H69 and V70 in the spike protein, seen in mink for the first time on Farm 4, and in certain variants from people, has been reported to double the infectivity of pseudo- viruses displaying the mutant spike protein compared to the wild type particles [18]. This dele- tion appears to emerge in SARS-CoV-2 following other changes in the spike protein [19]. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 8 / 13 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 The sampling of the mink on Farm 4 tested, at most, 300 animals on any particular date, out of a population of about 15,000 animals. It is clearly possible that a small number of infected mink were missed although the repeated follow-up screening makes this unlikely. The level of seropositivity among tested animals, prior to the second round of infection, remained very high (100%). Thus, it is not clear why so many animals (77% of 30 animals tested) were susceptible to a second round of infection. It has been considered whether the seropositivity detected in kits in August may be a consequence of maternally derived antibodies that could potentially decline more rapidly than antibodies generated from the infection in each animal. However, it seems difficult to reconcile this with the fact that >80% of throat swabs from mink kits tested clearly positive by RT-qPCR in August, which indicated a high level of infection amongst the kits in the first wave also. The measurements of antibody responses were made using an ELISA that targets the recep- tor binding domain (RBD) of the spike protein. Antibodies to the SARS-CoV-2 spike protein were present in up to 100% of the infected mink. The antibody titres, measured in this assay, increased to very high levels during the period of re-infection (Fig 2A). In studies on human sera, samples testing clearly positive (10 x cut-off) in this ELISA all had neutralizing antibodies [20]. Indeed, assessment of the same samples of mink sera as tested by ELISA in virus neutrali- zation tests indicated a high correspondence between these two types of assay. Discussion Thus, the ELISA positive mink sera neutralized the virus and, furthermore, the sera collected in Novem- ber, after reinfection, had significantly higher levels of anti-SARS-CoV-2 antibodies as mea- sured in each assay (Fig 2A and 2B), these results are consistent with boosting of the immune response due to a second round of infection. Due to the relatively low number of samples tested, the calculated proportions of virus or antibody positive animals may not directly correspond to prevalence of infection on the farm. However, during the course of the SARS-CoV-2 infections in mink in Denmark, the collection of samples from 62 Danish mink farms, where no clinical signs were present, a prevalence of virus of 100% was found on 45% of the farms, and on only 11 farms (18%) was a prevalence of virus below 50% observed [5]. On 40 farms, where blood samples were collected at the first sampling date, i.e. shortly after suspicion of infection was raised, a seroprevalence of 100% was found in 22 farms (55%), while on 12 farms (30%), a seroprevalence below 50% was observed [5]. This indicates that when animals were entirely randomly selected (due to the complete absence of clinical signs) for sampling, high prevalence of virus as well as of seropositive ani- mals was observed. In Denmark, mink were usually kept in long rows of open houses, i.e. in contiguous wire netting cages (S1 Fig). Cages were typically side-by-side, with the possibility of neighbouring mink having nose-to-nose contact and of air exchange between cages. Samples of air, fur and straw from mink farms have previously been found PCR-positive for SARS-CoV-2 [3, 5, 21], indicating that transmission indirectly through feed, bedding and airborne dust might occur within farms. It appears that the virus responsible for the infections in November was antigenically identi- cal to the virus in August since there were no non-synonymous changes within the spike pro- tein gene during this time, although some silent sequence changes (i.e. C21727T and T23815C) had occurred in it, as well as changes elsewhere within the virus genome. The most plausible conclusion is that infection of farmed mink with SARS-CoV-2 does not induce long-term protection against the virus. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 Sampling strategy At the time of first detection of infection on Farm 4, the practice was that suspected farms were visited by an official veterinarian, who took throat swabs from animals with clinical signs of SARS-CoV-2, if present, or alternatively from randomly selected animals. The numbers of samples taken varied during the epidemic. Here we describe only the samples taken in Farm 4. At first suspicion, following positive detection in the EW samples, 30 samples were taken from live animals, this approach provided 95%-confidence of detecting an infection prevalence of 0.1 with a test sensitivity of 99% and a specificity of 100%. When the samples were found posi- tive, staff from University of Copenhagen (UCPH) collected further throat swab samples as well as blood samples for serology. UCPH staff collected samples randomly from each house (S1 Fig) and randomly from the periphery and centres of the rows [5]. UCPH collected 60 samples from live animals, providing 95%-confidence of detecting a disease prevalence of 0.05 with a test sensitivity of 99% and a specificity of 100%. As increased mortality was one of the signs observed most often on infected farms [3, 5], throat swab samples from 5–21 recently deceased animals (within the previous 14 days) were collected when indicated (Table 1). Ethics statement No experimental procedures were performed on animals in this study. Only routine diagnostic samples (blood samples and throat swabs) were collected from the live, farmed, animals with no harm to them. Therefore, no prior ethical approval was required. Discussion This should be compared with the situation in rhesus macaques where primary infection did protect against reinfection at about 1 month post-initial infection [13, 22] and in humans where protection from reinfection may last at least eight months [12, 23]. However, some cases of re-infection have been reported in health care workers in Brazil [24], although this occurred in people who only developed a weak PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 9 / 13 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 immune response during the initial infection. Furthermore, only about 50% of people aged over 65 years in DK, who had been infected with SARS-CoV-2, were found to be protected against re-infection [25]. On a mink farm, a large number of animals live in close proximity to each other and, potentially, once the infection occurs in some animals then there can be a rapid increase in virus production and a strong challenge to neighbouring animals. Perhaps this is sufficient to overcome the immune response. It is notable that greatly enhanced levels of anti-SARS-CoV-2 antibodies were detected in the mink following the second round of infec- tion (Fig 2A and 2B), but this was also observed following challenge of previously infected rhe- sus macaques which did not become re-infected [13, 22]. Interestingly, two doses of a spike protein-based subunit vaccine did confer protection in mink against virus challenge at 5 weeks following the second vaccination [26] but it is not known how long this resistance to infection was maintained. Similarly, in ferrets, two doses of the adenovirus-vectored S gene vaccine pro- vided good protection against the virus [27]; however, it is noteworthy that virus challenge after a single dose of this vaccine resulted in an increase in neutralizing antibody titres whereas no such increase was observed following challenge after two doses of the vaccine. In our study, the mink did exhibit a boost in antibody levels following the re-infection, suggesting the immune response to the initial infection was inadequate to block virus replication. Currently, there are no “correlates of protection” that can be used to evaluate the immune responses in mink. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 Laboratory analyses Blood and throat-swab samples were collected from mink (adults and kits) as indicated in Table 1. The presence of SARS-CoV-2 RNA was determined by RT-qPCR as described previ- ously [2]. The SARS-CoV-2 Ab ELISA (Beijing Wantai Biological Pharmacy Enterprise, Bei- jing, China) was used as described previously [2], according to the manufacturer’s protocol 10 / 13 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 (the assay has sensitivity of 97% and specificity of 100%). Undiluted sera were used to identify seropositive animals and selected sera were then diluted (2-fold steps) and re-assayed in the ELISA. Antibody titres are presented as the reciprocal of the highest dilution of the serum giv- ing a positive result. Neutralizing antibody titres were determined as described previously [8]. For documentation of freedom from circulating virus (free-testing), 300 randomly selected mink were tested (in 60 pools of 5 samples) by RT-qPCR. This testing strategy was designed to detect, with 95% confidence, a 1% prevalence of SARS-CoV-2 RNA positive animals in 300 individual samples, leading to 93% confidence when pooling of 5 samples was taken into account. Selected SARS-CoV-2 positive RNA samples were sequenced as described [2] and SARS-- CoV-2 sequences were aligned using MAFFT [28]. Phylogenetic analysis was performed using MEGA version X, using the Maximum Likelihood method and General Time Reversible model with elimination of all sites with less than 95% coverage [29]. The GISAID accession IDs are listed in S1 and S2 Tables. The statistical significance of the differences between anti-CoV-2 antibody levels was deter- mined using the Kruskal-Wallis test in R [30]. Supporting information S1 Fig. Layout of mink houses on Farm 4. The locations from which samples were collected are indicated in dark blue (for August and October sampling) or red (November sampling). The open houses (in light blue unless sampled) in both the old and new parts of the farm are numbered and the number of mink sampled from each house are shown (in parenthesis). (TIF) S1 Table. List of accession numbers for SARS-CoV-2 sequences obtained from Danish mink farms. The names of virus samples, as used in the phylogenetic analysis (Fig 3A), together with their date of collection plus their GISAID accession ID numbers are shown. (XLSX) S2 Table. List of accession numbers for SARS-CoV-2 sequences obtained from mink on Farms 1–4. The names of virus samples, as used in the phylogenetic analysis (Fig 3B), together with their site and date of collection plus their GISAID accession ID numbers are shown. (XLSX) Acknowledgments We greatly appreciate the collaborative attitude of the farmer. Furthermore, we appreciate the work done by technicians and official veterinarians at the Danish Veterinary and Food Administration (FVST) who collected many of the samples. The Danish COVID-19 Genome Consortium (https://www.covid19genomics.dk) is acknowledged for sequencing. Writing – original draft: Graham J. Belsham. Writing – review & editing: Thomas Bruun Rasmussen, Jannik Fonager, Charlotte Sværke Jørgensen, Ria Lassaunière, Anne Sofie Hammer, Michelle Lauge Quaade, Anette Boklund, Louise Lohse, Bertel Strandbygaard, Morten Rasmussen, Thomas Yssing Michaelsen, Sten Mortensen, Anders Fomsgaard, Graham J. Belsham, Anette Bøtner. Author Contributions Conceptualization: Thomas Bruun Rasmussen, Sten Mortensen, Anders Fomsgaard, Graham J. Belsham, Anette Bøtner. Data curation: Thomas Bruun Rasmussen, Graham J. Belsham, Anette Bøtner. Formal analysis: Thomas Bruun Rasmussen, Jannik Fonager, Anette Boklund, Louise Lohse, Morten Rasmussen. 11 / 13 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1010068 November 15, 2021 PLOS PATHOGENS Re-infection of mink with SARS-CoV-2 Investigation: Thomas Bruun Rasmussen, Jannik Fonager, Charlotte Sværke Jørgensen, Ria Lassaunière, Anne Sofie Hammer, Michelle Lauge Quaade, Anette Boklund, Louise Lohse, Bertel Strandbygaard. Methodology: Jannik Fonager, Ria Lassaunière, Anette Boklund, Morten Rasmussen, Thomas Yssing Michaelsen. Supervision: Thomas Bruun Rasmussen, Louise Lohse, Anders Fomsgaard, Anette Bøtner. Validation: Thomas Bruun Rasmussen, Jannik Fonager, Ria Lassaunière, Anette Boklund, Sten Mortensen, Anders Fomsgaard, Graham J. Belsham, Anette Bøtner. Visualization: Thomas Bruun Rasmussen, Jannik Fonager, Anette Boklund, Graham J. Belsham. Writing – original draft: Graham J. Belsham. References 1. 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W4390758395.txt	https://www.mdpi.com/2673-4931/27/1/15/pdf?version=1704962957	en		Growth of Inner Carbon Nanotubes inside Cobaltocene-Filled Single-Walled Carbon Nanotubes	null	2,023	cc-by	1,200	Proceeding Paper Growth of Inner Carbon Nanotubes inside Cobaltocene-Filled Single-Walled Carbon Nanotubes † Marianna V. Kharlamova 1,2 1 2 † Centre for Advanced Materials Application (CEMEA), Slovak Academy of Sciences, Dúbravská Cesta 5807/9, 845 11 Bratislava, Slovakia; mv.kharlamova@gmail.com Moscow Institute of Physics and Technology, 9 Institutskiy per., 141700 Dolgoprudny, Russia Presented at the 6th International Electronic Conference on Atmospheric Sciences, 15–30 October 2023; Available online: https://ecas2023.sciforum.net/. Abstract: In this work, the single-walled carbon nanotubes (SWCNTs) were filled with cobaltocene. The growth properties of individual chirality nanotubes were studied with Raman spectroscopy. It was shown that the larger nanotubes grow slower. The growth of inner nanotubes becomes faster with increasing annealing temperature. These results are of high importance as they stimulate research on carbon nanotubes, and bring ideas from laboratories into factories. Keywords: Raman spectroscopy; growth properties; cobaltocene 1. Introduction Filling of single-walled carbon nanotubes (SWCNTs) is important for applications [1–4]. Metallocenes are promising filler material as they modify the chemical and physical properties of SWCNTs. Metallocenes inside SWCNTs represent a unique system for the growth of inner carbon nanotubes. The electronic properties of filled SWCNTs are also modified [5,6]. In this work, I filled the SWCNTs with cobaltocene, and I investigated the growth of inner carbon nanotubes. Raman spectroscopy allowed me to trace the process of growth with high precision. The growth of inner SWCNTs with chiralities of (13,1), (12, 3), and (11, 1) was detected. The chirality-specific growth curves of SWCNTs were obtained at different temperatures to compare kinetics. Citation: Kharlamova, M.V. Growth of Inner Carbon Nanotubes inside Cobaltocene-Filled Single-Walled Carbon Nanotubes. Environ. Sci. Proc. 2023, 27, 15. https://doi.org/ 10.3390/ecas2023-16351 Academic Editor: Anthony Lupo Published: 27 November 2023 Copyright: © 2023 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/). 2. Experiments I performed the filling of SWCNTs with cobaltocene in gas phase. The filling was performed in glass ampoule at low temperature (~59 ◦ C). The filled SWCNTs were annealed at different temperatures to investigate kinetics. This system is very interesting for investigation of these properties as it gives a clean environment for highly precise study of growth of SWCNTs. 3. Results Figure 1 shows the growth curves of inner nanotubes inside cobaltocene-filled SWCNTs at temperatures of 540 ◦ C (a), 560 ◦ C (b), 580 ◦ C (c), 600 ◦ C (d), 620 ◦ C (e) and 640 ◦ C (f). With these lots of data, it is visible that the larger nanotubes grow slower. The growth of inner nanotubes becomes faster with increasing annealing temperature. This is because the kinetics of growth of inner SWCNTs depends on their chirality. Here, I present the chiralityspecific kinetics, which is important knowledge for applications. For example, this will allow decreasing the growth temperature of SWCNTs to make the preparation processes easier, simpler, and low cost. It should be noted that the growth kinetics of SWCNTs in this system should be investigated with different conditions, i.e., precursor types, which lead to new results, and important information on synthesis protocols is implemented in industry. The kinetics experiment is time-consuming, but the established fundamental dependencies Environ. Sci. Proc. 2023, 27, 15. https://doi.org/10.3390/ecas2023-16351 https://www.mdpi.com/journal/environsciproc n. Sci. Proc. 2023, 27, x FOR PEER REVIEW 2 of 3 Environ. Sci. Proc. 2023, 27, 15 2 of 3 types, which lead to new results, and important information on synthesis protocols is implemented in industry. The kinetics experiment is time-consuming, but the established and trends lead to trends quick modernization of preparationof processes. I expect fundamental dependencies and lead to quick modernization preparation pro- that this data will bethis involved in factory process developments cesses. I expect that data will be involved in factory processsoon. developments soon. Figure 1. The growth inner curves nanotubes inside cobaltocene-filled SWCNTs at temperatures Figurecurves 1. The of growth of inner nanotubes inside cobaltocene-filled SWCNTs at temperatures of 540 °C (a), 560 °C (b), 580 °C (c), 600 °C (d), 620 °C (e) and 640 °C (f) [2]. by the ◦ ◦ ◦ ◦ ◦ of 540 C (a), 560 C (b), 580 C (c), 600 C (d), 620 C (e) andCopyright 640 ◦ C (f)2021 [2]. Copyright 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the the terms and conditions of the Creative Commons Attribution (CC BY) license. terms and conditions of the Creative Commons Attribution (CC BY) license. 4. Conclusions 4. Conclusions In this work, the growth of properties individualofchirality nanotubes studied. In this work,properties the growth individual chiralitywere nanotubes were studied. It It was shown was that shown the larger grow slower. The growth inner nanotubes bethatnanotubes the larger nanotubes grow slower. Theofgrowth of inner nanotubes becomes comes faster with annealing temperature. The high of this of data fasterincreasing with increasing annealing temperature. Theimportance high importance thisisdata is caused caused by theby necessity of a driving force force for researchers to study. WithWith thesethese materials the necessity of a driving for researchers to study. materials obtained, obtained, research on carbon nanotubes is going faster. research on carbon nanotubes is going faster. Funding: TheseFunding: studies were partly performed during the implementation of the project of BuildingThese studies were partly performed during the implementation the project Building-up up Centre for advanced materials application of the Slovak Academy of Sciences, ITMS project Centre for advanced materials application of the Slovak Academy of Sciences,code ITMS project code 313021T081 supported by Research & Innovation Programme funded by the ERDF. 313021T081 supported by ResearchOperational & Innovation Operational Programme funded by the ERDF. Institutional Review Board Statement: NotStatement: applicable. Not applicable. Institutional Review Board Informed Consent Statement: Not applicable. Environ. Sci. Proc. 2023, 27, 15 3 of 3 Data Availability Statement: The data are available on request of Marianna V. Kharlamova. Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. 2. 3. 4. 5. 6. Kharlamova, M.V.; Kramberger, C. Applications of Filled Single-Walled Carbon Nanotubes: Progress, Challenges, and Perspectives. Nanomaterials 2021, 11, 2863. [CrossRef] [PubMed] Kharlamova, M.V.; Kramberger, C. Metal Cluster Size-Dependent Activation Energies of Growth of Single-Chirality Single-Walled Carbon Nanotubes inside Metallocene-Filled Single-Walled Carbon Nanotubes. Nanomaterials 2021, 11, 2649. [CrossRef] [PubMed] Kharlamova, M.V.; Kramberger, C. Spectroscopy of filled single-walled carbon nanotubes. Nanomaterials 2022, 12, 42. [CrossRef] [PubMed] Kharlamova, M.V. Advances in tailoring the electronic properties of single-walled carbon nanotubes. Prog. Mater. Sci. 2016, 77, 125–211. [CrossRef] Kharlamova, M.V. Electronic properties of pristine and modified single-walled carbon nanotubes. Phys. Uspekhi 2013, 56, 1047–1073. [CrossRef] Kharlamova, M.V.; Kramberger, C. Metallocene-filled single-walled carbon nanotube hybrids. Nanomaterials 2023, 13, 77. [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/W2479491462	http://wrap.warwick.ac.uk/114077/1/WRAP-disabililty-act-vaccine-damage-payments-act-scare-Millward-2017.pdf	English	null	A Disability Act? The Vaccine Damage Payments Act 1979 and the British Government’s Response to the Pertussis Vaccine Scare	Social history of medicine	2,016	cc-by	14,359	Centre for History in Public Health, Faculty of Public Health and Policy, London School of Hygiene and Tropical Medicine, 15–17 Tavistock Place, London WC1H 9SH, UK. E-mail: gareth.millward@lshtm.ac.uk or gareth@ garethmillward.com Gareth Millward is a Research Fellow at the Centre for History in Public Health at the London School of Hygiene and Tropical Medicine, currently working on a Wellcome Trust funded project entitled Placing the Public in Public Health: Public Health in Britain, 1948–2010. His PhD, completed in 2013, focused on British disability policy from 1965 to 1995. A Disability Act? The Vaccine Damage Payments Act 1979 and the British Government’s Response to the Pertussis Vaccine Scare Gareth Millward at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from Summary. The Vaccine Damage Payments Act 1979 provided a lump-sum social security benefit to children who had become severely disabled as a result of vaccination. It came in the wake of a scare over the safety of the whooping cough (pertussis) vaccine. Yet very little has been written about it. Existing literature focuses more on the public health and medical aspects of both the Act and the scare. This article uses material from the archives of disability organisations and official documents to show that this Act should be seen as part of the history of post-war British disability policy. By framing it thus, we can learn more about why the government responded in the specific way that it did, as well as shed new light on public attitudes towards vaccination and disability. Keywords: vaccination; disability; policy; social security; public health In the mid-1970s, a group of British parents claimed that their children had become dis- abled as a result of government-recommended vaccinations. Although their complaints covered a range of diseases, it was the whooping cough—pertussis—vaccine that cap- tured the public imagination. Sections of the medical community backed the parents’ po- sition, and the vaccination rate for pertussis plummeted. The confusion was such that when the government was advised by its own expert bodies that a major publicity cam- paign was necessary to avoid a whooping cough epidemic, it declined to do so until it had received the results of epidemiological studies into the safety of the vaccination pro- gramme. In an attempt to restore confidence, the Labour government forced through legislation that would provide payments of £10,000 to those who could show that their children had been damaged. But this was too late to avoid a pertussis epidemic in the winter of 1978/79. The Department of Health and Social Security (DHSS) engaged in a two-pronged de- fence of the vaccination programme. First, its advisory bodies the Committee on the Safety of Medicines and the Joint Committee on Vaccination and Immunisation (JCVI) re- viewed the evidence on the safety and efficacy of the pertussis vaccine. Second, to re- store public trust it passed the Vaccine Damage Payments Act 1979 to provide social security payments to families of damaged children. © The Author 2016. Published by Oxford University Press on behalf of the Society for the Social History of Medicine 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. doi:10.1093/shm/hkv140 Social History of Medicine Vol. 0, No. 0 pp. 1–19 Social History of Medicine Advance Access published August 4, 2016 Social History of Medicine Vol. 0, No. 0 pp. 1–19 *Centre for History in Public Health, Faculty of Public Health and Policy, London School of Hygiene and Tropical Medicine, 15–17 Tavistock Place, London WC1H 9SH, UK. E-mail: gareth.millward@lshtm.ac.uk or gareth@ garethmillward.com Gareth Millward is a Research Fellow at the Centre for History in Public Health at the London School of Hygiene and Tropical Medicine, currently working on a Wellcome Trust funded project entitled Placing the Public in Public Health: Public Health in Britain, 1948–2010. His PhD, completed in 2013, focused on British disability policy from 1965 to 1995. © The Author 2016. Published by Oxford University Press on behalf of the Society for the Social History of Medicine 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. doi:10.1093/shm/hkv140 3Porter and Porter, ‘The Politics of Prevention’; Nadja Durbach, Bodily Matters: The Anti-Vaccination Movement in England, 1853–1907 (Durham, NC: Duke University Press, 2004); Nelson Marie Clark and John Rogers, ‘The right to die? Anti-vaccination activ- ity and the 1874 smallpox epidemic in Stockholm’, Social History of Medicine, 1992, 5, 369–88. paigners and how war veterans were ‘rehabilitated’. See: Julie Anderson, War, Disability and Rehabilitation in Britain (Manchester: Manchester University Press, 2011); Michael Mantin, ‘Educational Experiences of Deaf Children in Wales: The Cambrian Institution for the Deaf and Dumb, 1847–1914’ (unpublished PhD thesis, Swansea University, 2012); Jameel Hampton, ‘Discovering Disability: The General Classes of Disabled People and the Classic Welfare State, 1948– 1964’, Historian, 2013, 75. 2Anne Borsay, Disability and Social Policy (Basingstoke: Palgrave Macmillan, 2005). Again, recent studies have investigated the lives of children, disability cam- paigners and how war veterans were ‘rehabilitated’. See: Julie Anderson, War, Disability and Rehabilitation in Britain (Manchester: Manchester University Press, 2011); Michael Mantin, ‘Educational Experiences of Deaf Children in Wales: The Cambrian Institution for the Deaf and Dumb, 1847–1914’ (unpublished PhD thesis, Swansea University, 2012); Jameel Hampton, ‘Discovering Disability: The General Classes of Disabled People and the Classic Welfare State, 1948– 1964’, Historian, 2013, 75. A Disability Act? The Vaccine Damage Payments Act 1979 and the British Government’s Response to the Pertussis Vaccine Scare The former has received attention from historians and researchers of public health. The latter, however, has been largely Gareth Millward 2 ignored, or presented as part of the medical establishment’s response to the ‘pertussis vaccine scare’. As this article demonstrates, such analyses overlook the crucial influence of contemporary political factors. In particular, developments in disability policy and the position of disabled people fuelled and, in turn, provided some of the tools for respond- ing to the crisis. at London School of Hygien http://shm.oxfordjournals.org/ Downloaded from at London School of Hy http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from 1Dorothy Porter and Roy Porter, ‘The Politics of Prevention: Anti-vaccinationism and Public Health in Nineteenth-century England’, Medical History, 1988, 32, 231–52. Other investigations in smallpox and the Vaccination Acts include E. P. Hennock, ‘Vaccination Policy Against Smallpox, 1835–1914: A Comparison of England with Prussia and Imperial Germany’, Social History of Medicine, 1998, 11, 49–71; Ann Clark, ‘Compliance with Infant Smallpox Vaccination Legislation in Nineteenth-century Rural England: Hollingbourne, 1876–88’, Social History of Medicine, 2004, 17, 175–98; Michael Bennett, ‘Jenner’s Ladies: Women and Vaccination against Smallpox in Early Nineteenth-Century Britain’, History, 2008, 93, 497– 513. 1Dorothy Porter and Roy Porter, ‘The Politics of Prevention: Anti-vaccinationism and Public Health in Nineteenth-century England’, Medical History, 1988, 32, 231–52. Other investigations in smallpox and the Vaccination Acts include E. P. Hennock, ‘Vaccination Policy Against Smallpox, 1835–1914: A Comparison of England with Prussia and Imperial Germany’, Social History of Medicine, 1998, 11, 49–71; Ann Clark, ‘Compliance with Infant Smallpox Vaccination Legislation in Nineteenth-century Rural England: Hollingbourne, 1876–88’, Social History of Medicine, 2004, 17, 175–98; Michael Bennett, ‘Jenner’s Ladies: Women and Vaccination against Smallpox in Early Nineteenth-Century Britain’, History, 2008, 93, 497– 513. 5Notable exceptions concern Bacillus Calmette-Gue´rin (BCG), Heaptitis B policy, and a growing interest in polio. Linda Bryder, ‘“We shall not find salvation in in- oculation”’, Social Science & Medicine, 1999, 49, 1157–67; Jennifer Stanton, ‘What Shapes Vaccine Policy?’, Social History of Medicine, 1994, 7, 427–46; Gareth Williams, Paralysed with Fear: The Story of Polio (Basingstoke: Palgrave Macmillan, 2013); Ulrike Lindner and Stuart S. Blume, ‘Vaccine Innovation and Adoption: Polio Vaccines in the UK, the Netherlands and West Germany, 1955–1965’, Medical History, 2006, 50, 425–46. Disability, Vaccination and Social History Disability, Vaccination and Social History The current historiography on the whooping cough crisis of the 1970s and early 1980s has focused on the public health ramifications. This has meant that the primary source materials and analytical focus have been predominantly medical or concerned with the minutiae of public health policy. Yet the events are better explained through the prevail- ing political and social context. This is by no means a new approach to the history of medicine. Porter and Porter, for example, have shown that opposition to compulsory smallpox vaccination in the nineteenth century was tied into a number of cultural atti- tudes towards poverty and pauperism, as well as scepticism over the truth claims of the emerging fields of epidemiology and public health.1 On the disability side, Borsay has shown how disabled people were institutionalised during the modern period; but at the same time, many participated in public and private pursuits. This has provided a richer view of how health status and concepts such as disability and capacity were understood in modern British society.2 By highlighting the disability aspects of the Vaccine Damage Payments Act 1979, we can better understand reactions to both public health and dis- ability issues in the post-war era. The Act was explicitly framed using disability legislation, telling us much about the legal framework derived from a definition of disability that had evolved from earlier decades. The catalyst was a medical scandal, born in the wake of the thalidomide crisis and played out in the political discourse of the period. This is, there- fore, an opportunity to see public anxieties over acute and chronic health concerns in action. Work has been done on public and organised opposition to smallpox vaccination in the nineteenth century, and has included debates over class, religion, scientific consensus and the rights of individuals versus the collective.3 Many of these themes continued into A Disability Act? The Vaccine Damage Payments Act 1979 3 the twentieth century, as has been shown with other emerging immunisation techniques and how they were accepted by their target population.4 However, immunisation in post- war Britain is much less studied from a historical perspective.5 For whooping cough in par- ticular, we are left with mainly medical and epidemiological analyses rather than political or social history. These locate the controversy almost exclusively within public health prac- tice and policy. 6G. Amirthalingam, S. Gupta and H. Campbell, ‘Pertussis Immunisation and Control in England and Wales, 1957 to 2012: A Historical Review’, Euro Surveillance, 2013, 18; Maria A. Riolo, Aaron A. King and Pejman Rohani, ‘Can Vaccine Legacy Explain the British Pertussis Resurgence?’, Vaccine, 2013, 31, 5903–8. 4See: Anne Hardy, ‘Straight Back to Barbarism: Anti-ty- phoid Inoculation and the Great War, 1914’, Bulletin of the History of Medicine, 2000, 74, 265–90; Jane Lewis, ‘The Prevention of Diphtheria in Canada and Britain 1914–1945’, Journal of Social History, 1986, 20, 163–76. 8Jeffrey P. Baker, ‘The Pertussis Vaccine Controversy in Great Britain, 1974–1986’, Vaccine, 2003, 21, 4003– 10; Department of Health and Social Security, Committee on Safety of Medicines, and Joint Committee on Vaccination and Immunisation, Whooping Cough (London: HMSO, 1981). Disability, Vaccination and Social History This ignores much of the contemporary political climate, including the growing disability movement, sweeping reforms to social security benefits for disabled people, the legacy of the recent thalidomide scandal and a deepening financial crisis. As a result, the scare has been somewhat dehistoricised and placed in the context of proceed- ing. Broadly, this has created two types of analysis. The first uses the pertussis vaccination and incidence data from the 1970s and 1980s to show that the epidemics of 1979 and 1982 were much larger than at any point before or since the introduction of a routine diphtheria-tetanus-pertussis (DTP) vaccine in 1957.6 The scare is therefore presented as a cautionary tale of the risks of allowing fear of vaccine safety to grow amongst the public, as well as evidence for the efficacy of mass vaccination programmes. The second type of analysis draws parallels with the later MMR controversy of the late 1990s and early 2000s.7 This is problematic, because both scares were produced in very specific historical conditions. Instead, they are thought equivalent because the central concern of these studies is how to measure the effects of declining vaccination rates. Both these forms of analysis gloss over the political and social context of the period, and take as granted the hindsight that the pertussis vaccination was declared safe in the early 1980s.8 Further, they tend to disregard the historical importance of the scare in its own right in favour of wider practical questions about public health and vaccination. For public health practi- tioners, the scandal represents ‘bad science’, to borrow a term, in a world that is more prone to focus on the ‘lesson of history’ for concrete action rather than to understand the motives of policy actors within their own specific historical context.9 at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from 7Chris T. British Pertussis Resurgence?’, Vaccine, 2013, 31, 5903–8. 7 7Chris T. Bauch and Samit Bhattacharyya, ‘Evolutionary Game Theory and Social Learning Can Determine How Vaccine Scares Unfold’, PLoS Computational Biology, 2012, 8, 1–12; Rachel Casiday, ‘Risk Communication in the British Pertussis and MMR Vaccine Controversies’, in Peter Bennett, Kenneth Calman, Sarah Curtis and Denis Fischbacher-Smith, eds, Risk Communication and Public Health (Oxford: Oxford University Press, 2010), 129–46; Rachel Casiday, ‘Risk and Trust in Vaccine Decision Making’, Durham Anthropology Journal, 2005, 13. (London: Harper Perennial, 2009). For histories of so- cial welfare provision, see: Bernard Harris, The Origins of the British Welfare State: Society, State and Social Welfare in England and Wales, 1800–1945 (Basingstoke: Palgrave Macmillan, 2004); Howard Glennerster, British Social Policy, 1945 to the Present, 3rd edn (Oxford: Blackwell, 2007); David Gladstone, ‘Renegotiating the Boundaries: Risk and Responsibility in Personal Welfare since 1945’, in Helen Fawcett and Rodney Lowe, eds, Welfare Policy in Britain: The Road from 1945 (Basingstoke: Macmillan, 1999), 34–51. 11Mark Drakeford and Ian Butler, ‘Everyday Tragedies: Justice, Scandal and Young People in Contemporary Britain’, The Howard Journal of Criminal Justice, 2007, 46, 219–35. 12John W. Kingdon, Agendas, Alternatives and Public Policies, 2nd edn (New York: Longman, 1995); Gareth Millward, ‘Invalud Definitions, Invalid Responses: Disability and the Welfare State, 1965– 1995’ (unpublished PhD thesis, London School of Hygiene & Tropical Medicine, 2014). 10Mark Drakeford and Ian Butler, Scandal, Social Policy and Social Welfare (Bristol: Policy Press, 2005). (London: Harper Perennial, 2009). For histories of so- cial welfare provision, see: Bernard Harris, The Origins of the British Welfare State: Society, State and Social Welfare in England and Wales, 1800–1945 (Basingstoke: Palgrave Macmillan, 2004); Howard Glennerster, British Social Policy, 1945 to the Present, 3rd edn (Oxford: Blackwell, 2007); David Gladstone, ‘Renegotiating the Boundaries: Risk and Responsibility in Personal Welfare since 1945’, in Helen Fawcett and Rodney Lowe, eds, Welfare Policy in Britain: The Road from 1945 (Basingstoke: Macmillan, 1999), 34–51. 13The DHSS had created the post of Minister for the Disabled in 1974, while governments since the mid- 1960s had dedicated much administrative time to the question of disability benefits and access to ser- vices. See Gareth Millward, ‘Social Security Policy and the Early Disability Movement—Expertise, Disability, and the Government, 1965–77’, Twentieth Century British History, 2015, 26, 274–97. 14For a historiographical review, see: Julie Anderson and Ana Carden-Coyne, ‘Enabling the Past: New Perspectives in the History of Disability’, European Review of History, 2007, 14, 447–57, and other arti- cles in this special edition; Anne Borsay, ‘History and Disability Studies: Evolving Perspectives’, in Nick Watson, Alan Roulstone and Carol Thomas, Routledge Handbook of Disability Studies (Abingdon: Routledge, 2012), 324–35; Catherine J. Kudlick, ‘Disability History: Why We Need Another “Other”’, The American Historical Review, 2003, 108, 763–93. 15Rodney Lowe, The Welfare State in Britain since 1945 (Basingstoke: Palgrave Macmillan, 2005); Glennerster, British Social Policy; Derek Fraser, The Evolution of the British Welfare State (Basingstoke: Palgrave Macmillan, 2009). Disability, Vaccination and Social History Bauch and Samit Bhattacharyya, ‘Evolutionary Game Theory and Social Learning Can Determine How Vaccine Scares Unfold’, PLoS Computational Biology, 2012, 8, 1–12; Rachel Casiday, ‘Risk Communication in the British Pertussis and MMR Vaccine Controversies’, in Peter Bennett, Kenneth Calman, Sarah Curtis and Denis Fischbacher-Smith, eds, Risk Communication and Public Health (Oxford: Oxford University Press, 2010), 129–46; Rachel Casiday, ‘Risk and Trust in Vaccine Decision Making’, Durham Anthropology Journal, 2005, 13. 8Jeffrey P. Baker, ‘The Pertussis Vaccine Controversy in Great Britain, 1974–1986’, Vaccine, 2003, 21, 4003– 10; Department of Health and Social Security, Committee on Safety of Medicines, and Joint Committee on Vaccination and Immunisation, Whooping Cough (London: HMSO, 1981). 9Virginia Berridge, Public Health in History (Maidenhead: Open University Press, October 2011), 213–16; Virginia Berridge, ‘History Matters?’, Medical History, 2008, 52, 311–26; Ben Goldacre, Bad Science 4 Gareth Millward 4 4 Gareth Millward 4 For these reasons, it is important for historians to look beyond the medical sphere. As Drakeford and Butler have shown, ‘scandals’ such as this are manufactured to a certain extent.10 The mere existence of morally offensive action is not enough; it needs to be ar- ticulated through public discourse.11 This is true not just of a scandal, but also of the spe- cific responses that are chosen by policy makers. Kingdon has noted that policy action requires the confluence of a perceived problem, the political will to act, and the technical capacity to respond.12 That is to say, it was neither inevitable that the knowledge of vac- cine damage would turn into the scandal that it did; nor that the Vaccine Damage Payments Act would be one of the policy results. The pertussis scare occurred at a crucial time in disability politics in which both Labour and Conservative governments had en- acted a range of policies aimed at improving the lives of disabled people. Disability was being seen as a social issue as well as (if not instead of) a medical one; and it had become a branch of policy with its own machinery for creating solutions to policy problems.13 Further, a voluntary organization in the form of the Association of Parents of Vaccine Damaged Children was able to convince medical, state and private institutions that their favoured solution to the problem—statutory compensation—was the morally correct course of action. Disability, Vaccination and Social History at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from The Vaccine Damage Payments Act has received relatively little scrutiny of this type partly because it is, historically speaking, a recent event. The traditional ‘thirty-year rule’ at The National Archives means that much of the material upon which this article is based has only been publicly available for a few years. Moreover, disability histories are them- selves relatively new.14 Histories of welfare provision have tended to focus on the wider ‘rediscovery of poverty’ and the politics of ‘consensus’ in the 1960s and 1970s which provided a fertile environment for extending the welfare state to groups excluded from the post-war settlement.15 Recent work has shed light on the lives of disabled people 13The DHSS had created the post of Minister for the Disabled in 1974, while governments since the mid- 1960s had dedicated much administrative time to the question of disability benefits and access to ser- vices. See Gareth Millward, ‘Social Security Policy and the Early Disability Movement—Expertise, Disability, and the Government, 1965–77’, Twentieth Century British History, 2015, 26, 274–97. 14For a historiographical review, see: Julie Anderson and Ana Carden-Coyne, ‘Enabling the Past: New Perspectives in the History of Disability’, European Review of History, 2007, 14, 447–57, and other arti- cles in this special edition; Anne Borsay, ‘History and Disability Studies: Evolving Perspectives’, in Nick Watson, Alan Roulstone and Carol Thomas, Routledge Handbook of Disability Studies (Abingdon: Routledge, 2012), 324–35; Catherine J. Kudlick, ‘Disability History: Why We Need Another “Other”’, The American Historical Review, 2003, 108, 763–93. 15Rodney Lowe, The Welfare State in Britain since 1945 (Basingstoke: Palgrave Macmillan, 2005); Glennerster, British Social Policy; Derek Fraser, The Evolution of the British Welfare State (Basingstoke: Palgrave Macmillan, 2009). A Disability Act? The Vaccine Damage Payments Act 1979 and the various institutions which governed their lives.16 This has begun to include dis- cussions of the social construction of impairment and the differing experiences of dis- abled people.17 This journal has also shown a growing interest in the implications for the social history of medicine.18 But this relatively new endeavour has only just begun to in- vestigate the period after 1970. 17Helen Bolderson, Social Security, Disability and Rehabilitation (London: Jessica Kingsley, 1991); Borsay, Disability and Social Policy; Julie Anderson, War, Disability and Rehabilitation; Hampton, ‘Discovering Disability’. Reassessment’, Social History of Medicine, 2013, 26, 56–73; Laura L. Phillips, ‘Gendered Dis/ability: Perspectives from the Treatment of Psychiatric Casualties in Russia’s early Twentieth-century Wars’, Social History of Medicine, 2007, 20, 333–50; Beth Linker, ‘Feet for Fighting: Locating Disability and Social Medicine in First World War America’, Social History of Medicine, 2007, 20, 91–109; and passim. 19For thalidomide see: Jack Ashley, Acts of Defiance (London: Reinhardt, 1992); The Sunday Times, Suffer the Children: The Story of Thalidomide (New York: Viking Press, 1979); Mary Wilkinson, Defying Disability (London: Jessica Kingsley, 2009), 35–56; David Mason, Thalidomide (London: Allen and Unwin, 1976); Louise Medus, Laughing and Loving— A Thalidomide Survivor’s Story (Pembroke Dock: Accent Press, 2009). For vaccine damage see: Rosemary Fox, Helen’s Story (London: John Blake, 2006); Wilkinson, Defying Disability, 35–56; Ashley, Acts of Defiance. 20Jameel Hampton, ‘Disabled People and the Classic Welfare State’ (Unpublished PhD thesis, University of Bristol, 2011). 16See in particular: Anne Borsay and Pamela Dale (eds), Disabled Children: Contested Caring, 1850–1979 (London: Pickering & Chatto, 2012); David M. Turner and Kevin Stagg (eds), Social Histories of Disability and Deformity (Abingdon: Routledge, 2006); Nick Watson (ed.), Disability: Major Themes in Health and Social Welfare (Abingdon: Routledge, 2008); Sonali Shah and Mark Priestley, Disability and Social Change: Private Lives and Public Bodies (Bristol: Policy Press, 2011). 18Ben Curtis and Stephen Thompson, ‘“A Plentiful Crop of Cripples Made by all this Progress”: Disability, Artificial Limbs and Working-class Mutualism in the South Wales Coalfield, 1890– 1948’, Social History of Medicine, 2014, 27, 708–27; Alistair Ritch, ‘English Poor Law Institutional Care for Older People: Identifying the “Aged and Infirm” and the “Sick” in Birmingham Workhouse, 1852–1912’, Social History of Medicine, 2014, 27, 64–85; Heli Leppa¨la¨, ‘Duty to Entitlement: Work and Citizenship in the Finnish Post-war Disability Policy, early 1940s to 1970’, Social History of Medicine, 2014, 27, 144– 64; Gwen A. Parsons, ‘The Construction of Shell Shock in New Zealand, 1919–1939: A 21Claire Sewell, ‘“If one member of the family is dis- abled the family as a whole is disabled”: Thalidomide Children and the Emergence of the Family Carer in Britain, c. 1957–1978’, Family and Community History (2015), 18, 37–52. 16See in particular: Anne Borsay and Pamela Dale (eds), Disabled Children: Contested Caring, 1850–1979 (London: Pickering & Chatto, 2012); David M. Turner and Kevin Stagg (eds), Social Histories of Disability and Deformity (Abingdon: Routledge, 2006); Nick Watson (ed.), Disability: Major Themes in Health and Social Welfare (Abingdon: Routledge, 2008); Sonali Shah and Mark Priestley, Disability and Social Change: Private Lives and Public Bodies (Bristol: Policy Press, 2011). Disability, Vaccination and Social History As such, there is very little on the two big disability com- pensation crises of the decade—thalidomide and vaccine damage. Memoirs and biographical material of the major players have been produced in which the campaigners narrate their side of the story, yet there is almost nothing on how these affected the posi- tion of ‘disability’ as a social and legal concept.19 Jameel Hampton has discussed the quandary confronted by the Disablement Income Group (DIG) when faced with the claims by the thalidomide parents, but by ending his study of British disability policy in 1975 he does not tackle vaccine damage.20 Similarly, Claire Sewell’s work on the parents of disabled children is analysed in the wake of the thalidomide crisis, but does this to draw wider conclusions about parenting, childhood and the concept of ‘the carer’.21 Other than Jeffrey Baker’s overview of the pertussis vaccine controversy and its effects on American anti-vaccination campaigns, there is no investigation into the primary material surrounding the Vaccine Damage Payments Act and its significance in British disability policy.22 at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from This article argues that by placing the Act in this wider political and social discussion, historians can learn more about why the government responded as it did to the whoop- ing cough scare. Campaigners successfully used the tactics of other voluntary organiza- tions to press their case to the public. They were able to draw on both the successes of 18Ben Curtis and Stephen Thompson, ‘“A Plentiful Crop of Cripples Made by all this Progress”: Disability, Artificial Limbs and Working-class Mutualism in the South Wales Coalfield, 1890– 1948’, Social History of Medicine, 2014, 27, 708–27; Alistair Ritch, ‘English Poor Law Institutional Care for Older People: Identifying the “Aged and Infirm” and the “Sick” in Birmingham Workhouse, 1852–1912’, Social History of Medicine, 2014, 27, 64–85; Heli Leppa¨la¨, ‘Duty to Entitlement: Work and Citizenship in the Finnish Post-war Disability Policy, early 1940s to 1970’, Social History of Medicine, 2014, 27, 144– 64; Gwen A. Parsons, ‘The Construction of Shell Shock in New Zealand, 1919–1939: A 21Claire Sewell, ‘“If one member of the family is dis- abled the family as a whole is disabled”: Thalidomide Children and the Emergence of the Family Carer in Britain, c. 1957–1978’, Family and Community History (2015), 18, 37–52. y 22Baker, ‘The Pertussis Vaccine Controversy’. 23See The National Archives, Kew (hereafter TNA) MH 154/1053, ‘“Society should compensate for brain damage”’, Birmingham Post, 26 June 1973 [page numbers omitted]. 24Mary McCormack, ‘The Hazards of Health’, The Guardian, 3 August 1973, 11. See also Fox, Helen’s Story. 25 23See The National Archives, Kew (hereafter TNA) MH 154/1053, ‘“Society should compensate for brain damage”’, Birmingham Post, 26 June 1973 [page numbers omitted]. 24Mary McCormack, ‘The Hazards of Health’, The Guardian, 3 August 1973, 11. See also Fox, Helen’s Story. 25Hampton, ‘Disabled People and the Classic Welfare State’; Campbell and Oliver, Disability Politics. 26Since 1994, the Spastics Society has been called Scope. See: ibid.; Pat Thane, ‘Voluntary Action in Britain since Beveridge’, in Melanie Oppenheimer and Nicholas Deakin, eds, Beveridge and Voluntary Disability, Vaccination and Social History 22Baker, ‘The Pertussis Vaccine Controversy’. Gareth Millward 6 the poverty lobby over the 1960s and recent health scandals which remained fresh in the memory. Importantly, the specific framework of the Act drew heavily on existing disabil- ity policies and definitions. It is only by moving beyond the existing medical narratives that we can access this history. To go further, even though the Act has not been part of the traditional narrative of disability policy, we can understand much about government attitudes towards disability through the provisions contained within the Act. at London School of Hygien http://shm.oxfordjournals.org/ Downloaded from at London School of Hy http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from Action in Britain and the Wider British World (Manchester: Manchester University Press, 2011), 121–34. 27Disablement Income Group, Creating a National Disability Income (London: DIG, 1972); Hampton, ‘Disabled People and the Classic Welfare State’; Millward, ‘Social Security Policy and the Early Disability Movement’. 28Disability Alliance, Poverty and Disability (London: Disability Alliance, 1975); Howard Glennerster, ‘Peter Townsend’, Oxford Dictionary of National Biography (Oxford: Oxford University Press, 2013). Action in Britain and the Wider British World (Manchester: Manchester University Press, 2011), 121–34. 27Disablement Income Group, Creating a National Disability Income (London: DIG, 1972); Hampton, ‘Disabled People and the Classic Welfare State’; Millward, ‘Social Security Policy and the Early Disability Movement’. 28 25Hampton, ‘Disabled People and the Classic Welfare State’; Campbell and Oliver, Disability Politics. 26Since 1994, the Spastics Society has been called Scope. See: ibid.; Pat Thane, ‘Voluntary Action in Britain since Beveridge’, in Melanie Oppenheimer and Nicholas Deakin, eds, Beveridge and Voluntary 28Disability Alliance, Poverty and Disability (London: Disability Alliance, 1975); Howard Glennerster, ‘Peter Townsend’, Oxford Dictionary of National Biography (Oxford: Oxford University Press, 2013). 25Hampton, ‘Disabled People and the Classic Welfare State’; Campbell and Oliver, Disability Politics. 26Since 1994, the Spastics Society has been called Scope. See: ibid.; Pat Thane, ‘Voluntary Action in Britain since Beveridge’, in Melanie Oppenheimer and Nicholas Deakin, eds, Beveridge and Voluntary The Association of Parents of Vaccine Damaged Children The Association of Parents of Vaccine Damaged Children The Association of Parents of Vaccine Damaged Children (hereafter the Association) was formed in 1973 by two mothers who blamed their children’s brain damage on the polio- myelitis vaccine. A piece in the Birmingham Post in June 1973 on the subject of vaccine damage included a call from Rosemary Fox and Renee Lennon to establish a new soci- ety.23 By the time their story was published in a Guardian article in August, this society was calling itself the Association.24 Fox and the Association became the public face of the campaign to provide compensation for victims of vaccine damage. In many ways, the Association drew on the tactics and successes of earlier disability or- ganisations. Unlike others in the ‘poverty lobby’ or ‘welfare rights’ sphere, it was largely a single-issue group. DIG became the first pan-impairment disability organisation to lobby central government in 1965.25 Other voluntary organisations at this time concerned themselves with specific impairments or groups of conditions—notable examples being the Spastics Society and MENCAP—or were charities providing care for disabled people—such as Leonard Cheshire.26 DIG’s concern was wide-ranging, and included a complete reformulation of the social security system with regard to disabled people. It established a campaign for a National Disability Income, an ideal social security benefit that would compensate disabled people for the lost earnings and additional costs associ- ated with living with single or multiple impairments.27 In 1974, the Disability Alliance would promote a similar campaign led by prominent sociologist and poverty campaigner Professor Peter Townsend.28 The Association, however, focused solely on the issue of compensation for vaccine damaged children. This demand for special treatment was politically problematic. Both DIG and the Disability Alliance argued against the system, which had developed after the Second World War and gave preferential treatment to certain categories of disabled peo- ple. Claimants with National Insurance records or those injured in industrial accidents or the armed forces were entitled to higher levels of benefit; while married women qualified A Disability Act? The Vaccine Damage Payments Act 1979 7 for no benefits at all. Moreover, the system was designed to provide temporary cover for sickness and unemployment rather than the specific effects of chronic illness. This approach remained most prominent throughout the 1970s,29 but it was already coming under scrutiny from more radical opponents. The Association of Parents of Vaccine Damaged Children Social security was seen by the Union of the Physically Impaired Against Segregation as a symptom of disability, not the root cause of why disabled people were discriminated against. Drawing on feminist and black critiques of sexism and racism, it directly challenged the dominant ‘medical model’—whereby most political and cultural institutions defined disabled people by medical diagnoses, or what was ‘wrong’ with their bodies and minds. A new social model was proposed in which people were said to be disabled by society.30 For example—a person is not dis- abled because they cannot climb stairs; they are disabled because buildings are designed for an assumed level of capacity in which everyone can climb stairs. Thus, the focus of disability policy should not be on manipulating the individual to walk (necessarily), but should instead look to install escalators and lifts in public buildings so that everyone has access to core services.31 While these groups did not gain significant public attention until the 1980s, it must be noted that the Association’s focus on the specific medical problems of their members’ children went against many of the political developments of the decade. at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from Until the financial crises of the Callaghan years restricted government expenditure, the campaigns for disability benefits were largely successful. DIG and the Disability Alliance became frustrated at the slow rate of progress, but after the passing of the Social Security Benefits Act 1975, most of the groups that DIG had campaigned for were now covered by at least some sort of benefit.32 The outgoing Wilson government in 1970 at- tempted to create a limited invalidity pension, but the National Superannuation and National Insurance Bill was lost to the general election. Such was the political consensus on the matter, however, that the new Heath administration quickly established Invalidity Benefit and Attendance Allowance to help unemployed disabled people and the cost of caring for a disabled relative respectively. When it returned to power, Labour created benefits for housewives, the costs of transport and a non-contributory version of Invalidity Benefit in 1975. 33Parliamentary Debates (Commons), 767, 24 July 1969, 2158; ibid., 846, 14 November 1972, 96; ibid., 881, 21 November 1974, 1558. 29Campbell and Oliver, Disability Politics, 55; Millward, ‘Social Security Policy and the Early Disability Movement’. 30This was first properly articulated in Oliver, Politics of Disablement. However, its roots are deeper. See Paul Hunt, Stigma (London: G. Chapman, 1966); The Disability Archive, University of Leeds: Union of the Physically Imapaired Against Segregation and The Disability Alliance, Fundamental Principles of Disability <http://disability-studies.leeds.ac.uk/files/library/UPIAS- fundamental-principles.pdf>, accessed 13 August 2015; Victor Finkelstein, Attitudes and Disabled People: Issues for Discussion (New York: International 32Millward, ‘Social Security Policy and the Early Disability Movement’. 29Campbell and Oliver, Disability Politics, 55; Millward, ‘Social Security Policy and the Early Disability Movement’. 30 Exchange of Information in Rehabilitation, 1980); Tom Shakespeare, Disability Rights and Wrongs (Abingdon: Routledge, 2006). 31Victor Finkelstein’s story about the only non-wheel- chair user in a world of wheelchair users is a good il- lustrative example. See: Victor Finkelstein, ‘Phase 2: Discovering the Person in “Disability” and “Rehabilitation”’, Magic Carpet, 1975, 27, 31–8. 32Millward, ‘Social Security Policy and the Early Disability Movement’. 33Parliamentary Debates (Commons), 767, 24 July 1969, 2158; ibid., 846, 14 November 1972, 96; 31Victor Finkelstein’s story about the only non-wheel- chair user in a world of wheelchair users is a good il- lustrative example. See: Victor Finkelstein, ‘Phase 2: Discovering the Person in “Disability” and “Rehabilitation”’, Magic Carpet, 1975, 27, 31–8. Exchange of Information in Rehabilitation, 1980); Tom Shakespeare, Disability Rights and Wrongs (Abingdon: Routledge, 2006). The Association of Parents of Vaccine Damaged Children Campaigners had managed to secure statements from succes- sive Secretaries of State for Social Services that, once the economy recovered, the system would be reformed and improved upon.33 For the Association, this meant that it was widely accepted that monetary payments were an important facet of social policy for Gareth Millward 8 8 disabled people; and that there was a growing set of precedents upon which a compen- sation scheme could be built. The result of this growing movement was a wider consideration of the needs of dis- abled people, and parliamentarians were beginning to specialise in this area of policy. This gave the Association the opportunity to build alliances with sympathetic figures in Westminster. In 1969, Alfred Morris introduced the Chronically Sick and Disabled Persons Bill. When it received Royal Assent the following year, it was the first such Act of its kind in the world, giving powers to local authorities to provide services for disabled people.34 The result of this growing movement was a wider consideration of the needs of dis- abled people, and parliamentarians were beginning to specialise in this area of policy. This gave the Association the opportunity to build alliances with sympathetic figures in Westminster. In 1969, Alfred Morris introduced the Chronically Sick and Disabled Persons Bill. When it received Royal Assent the following year, it was the first such Act of its kind in the world, giving powers to local authorities to provide services for disabled people.34 While it never had the powers of compulsion that Morris and the Bill’s supporters had hoped for, it meant that after the February 1974 General Election Morris became the world’s first Minister for the Disabled.35 He had been helped by the creation of the All Party Group on Disablement, founded by Jack Ashley (Labour, Stoke-on-Trent South) and John Astor (Conservative, Newbury).36 Ashley was central to the Association’s activities in Westminster. He was also disabled, deaf as a result of an infection he had contracted after surgery on his ears. In 1974 he became Parliamentary Private Secretary to Barbara Castle (Secretary of State for Social Services), which gave the Association an opportunity to speak directly to the head of the DHSS.37 Even when Ashley left his post, his relationship with Morris and new Secretary of State David Ennals ensured that the issue remained on the agenda. 39Millward, ‘Invalid Definitions, Invalid Responses’. 40Fox, Helen’s Story, 88. 41Parliamentary Debates (Commons) 847, 29 November 1972, 446. 42See: Hampton, ‘Disabled People and the Classic Welfare State’; Derek Kinrade, Alf Morris (London: National Information Forum, 2007), 207–18; Ashley, Acts of Defiance; The Sunday Times, Suffer the Children; Medus, Laughing and Loving. 43Colin Pearson, Royal Commission on Civil Liability and Compensation for Personal Injury, vol. 1 (Cmnd. 7054–I) (London: HMSO, 1978), specifically quotes thalidomide cases as one of the reasons for its appointment. 36He was, for instance, the Labour vice president of DIG. See Ashley, Acts of Defiance; Beth Capper, A Celebration of the Work of the APPDG (London: RADAR, 2008), 12. 38See correspondence with Ashley in TNA: BN 13/360; and also TNA: BN 124/20, Meeting on the Vaccine Damage Payments Scheme, 16 June 1981. 37Fox, Helen’s Story, 57–61. 35Alfred Morris and Arthur Butler, No Feet to Drag: Report on the Disabled (London: Macmillan, 1972); Derek Kinrade, Alf Morris: People’s Parliamentarian: Scenes from the Life of Lord Morris of Manchester (London: National Information Forum, 2007), esp. 155–82. 34Eda Topliss and Bryan Gould, A Charter for the Disabled (Oxford: B. Blackwell & M. Robertson, 1981). 35Alfred Morris and Arthur Butler, No Feet to Drag: Report on the Disabled (London: Macmillan, 1972); Derek Kinrade, Alf Morris: People’s Parliamentarian: Scenes from the Life of Lord Morris of Manchester (London: National Information Forum, 2007), esp. 155–82. 34Eda Topliss and Bryan Gould, A Charter for the Disabled (Oxford: B. Blackwell & M. Robertson, 1981). 44A. Robens, Safety and Health at Work, Report of the Committee (Cmnd. 5034) (London: HMSO, 1972). 45Sewell, ‘Thalidomide Children and the Emergence of the Family Carer’. 46Anon., ‘Help for Victims of Immunizations’, Br Med J., 1973, 1, 758. 47Virginia Berridge, Martin Gorsky and Alex Mold, Public Health in History (Maidenhead: Open University Press, 2011), 195–210. 48Parliamentary Debates (Commons) 766, 20 June 1968, 1294–5; ibid., 795, 11 February 1970, 1356– 7; ibid., 914, 8 July 1976, 1979; ibid., 957, 7 November 1978, 110–2W; Parliamentary Debates (Lords) 317, 7 April 1971, 376–7. 49See in particular Fox’s autobiography in which she details the campaign from her perspective. Fox, Helen’s Story. 49See in particular Fox’s autobiography in which she details the campaign from her perspective. Fox, Helen’s Story. 48Parliamentary Debates (Commons) 766, 20 June 1968, 1294–5; ibid., 795, 11 February 1970, 1356– 7; ibid., 914, 8 July 1976, 1979; ibid., 957, 7 November 1978, 110–2W; Parliamentary Debates (Lords) 317, 7 April 1971, 376–7. The Association of Parents of Vaccine Damaged Children This is evidenced not only by the correspondence between Ashley and the DHSS during the 1970s but the involvement of the three men in meetings with Conservative ministers over the Vaccine Damage Payments Scheme in the early 1980s.38 at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from Most crucially of all, disability was seen as a social category and a matter of policy.39 Yet the rhetoric had been based around equal treatment based on need, not on cause of impairment. The Association was trying to argue that it was a ‘special case’.40 The recent thalidomide crisis gave campaigners an analogous medical scandal that could be ex- ploited. The Sunday Times campaign for full compensation from the drug’s manufac- turers had only just concluded, pursued vigorously by Jack Ashley in parliament. In its wake, the Heath government created the Family Fund to provide payments to ‘congeni- tally disabled children’.41 The experience had made the public and medical establishment wary about the dangers that could be posed by drugs and treatments presumed to be safe.42 As a result, The Royal Commission on Civil Liability and Compensation for Personal Injury added medical negligence to its remit.43 Chaired by Lord Pearson, it was A Disability Act? The Vaccine Damage Payments Act 1979 9 9 primarily concerned with the current system of accident compensation, including indus- trial injuries following the earlier Robens Report into Health and Safety.44 Thalidomide had also made the parents of disabled children more visible, even if, as Sewell argues, it had not resulted in a fundamental shift in public attitudes or their legal status.45 By simul- taneously claiming that the Family Fund did not provide adequate coverage, and appeal- ing to the potential for another thalidomide-like scandal, the Association could make its specific claims for compensation. at London School of Hygien http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from The Vaccine Damage Campaign The Association’s breakthrough in 1973 came as a result of Fox’s campaigning and the recent publication of an article in the British Medical Journal (BMJ) which argued: The Vaccine Damage Campaign The Association’s breakthrough in 1973 came as a result of Fox’s campaigning and the recent publication of an article in the British Medical Journal (BMJ) which argued: The moral justification for compensation . . . is based on the social contract. National immunization programmes not only aim to protect the individual but also to protect society. . . . If individuals are asked to accept a risk (even a very small one) partly for the benefit of society then it seems equitable that society should compensate the victims of occasional unlucky mishaps.46 ‘Protect[ing] society’ had taken on a dual meaning. In the short-term, vaccination policy had been focused on preventing infectious disease; but it had also come to mean protec- tion from disability. While early public health interventions had focused more on infec- tious diseases, the relative increase of chronic disease had seen a shift in priorities.47 Vaccination was still seen as an important tool against infectious diseases such as diph- theria and tuberculosis, but immunisations against poliomyelitis and rubella were driven by concerns over the public and private costs of disabled children surviving into adult- hood. The campaign against rubella, particularly aimed at women of child-bearing age, was regularly cited throughout the 1970s as part of the government’s disability policy.48 Similarly, the request for monetary ‘compensation’ was entirely consistent with demands from welfare rights organisations and the recent success of the parents of children affected by thalidomide. Fox’s daughter, Helen, had received the polio vaccine in the early 1960s and soon af- terwards showed signs of brain damage. She began to have fits and, by the time of the campaign, was eleven years old with a diagnosed mental age of three. Believing that the vaccine had caused this behaviour, Fox and the other parents were angered by the atti- tude of the profession, with many doctors refusing to acknowledge that vaccination could lead to damage at all. They began to collect detailed medical information from Association members to make their case to the medical authorities.49 This had been a core tactic among welfare rights organisations. The Vaccine Damage Campaign The Association’s breakthrough in 1973 came as a result of Fox’s campaigning and the recent publication of an article in the British Medical Journal (BMJ) which argued: The Child Poverty Action Group and DIG Gareth Millward 10 had collected information from members and those it sought to help in order to be able to provide illustrative examples of the difficulties suffered by those who could not access help from the welfare authorities.50 The majority view of the medical establishment was that vaccines were safe and effec- tive measures of disease prevention. A large trial of 36,000 subjects in 1957 conducted by the Medical Research Council had shown the pertussis vaccine to be safe and effec- tive, with no cases of brain damage.51 But the parents were not ‘fobbed off’ by every- one.52 In particular, Professor Gordon Stewart and Dr John Wilson offered their support to the campaign. A letter to The Guardian by Drs J. V. T. Gosling and J. H. Moseley al- leged that the pertussis vaccine was not effective enough to be worth administering. They also made reference to some cases of brain damage that might be linked to its use.53 This was pressed further in 1974 by Wilson and colleagues at Great Ormond Street Hospital, who alleged a link between brain-damaged children and the whooping cough vaccine.54 ‘People seem to worry’, Fox told The Guardian, ‘doctors in particular, that they may get themselves involved in another highly publicised thalidomide episode’.55 at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from It would also not have been the first high-profile instance of damage to children en masse from vaccination. The American Cutter Incident, in which thousands of children were injected with live polio virus as the result of a faulty batch of the new Salk vaccine, had occurred less than 20 years previously.56 This led the Association to make a tactical decision and focus its efforts on pertussis. 55McCormack, ‘The Hazards of Health’, 11. 56This created ripples across the developed world. See: Per Axelsson, ‘The Cutter Incident and the Development of a Swedish Polio Vaccine, 1952– 1957’, Dynamis, 2012, 32, 311–28; Alison Day, ‘“An American Tragedy”. The Cutter Incident and its Implications for the Salk Polio Vaccine in New Zealand 1955–1960’, Health & History: Journal of the Australian & New Zealand Society for the History of Medicine, 2009, 11, 42–61. 57As of January 1977, Fox claimed to have 281 cases of damage, of which 182 were blamed on pertussis. Hugh Herbert, ‘Parents Gain Ground in Medical Fight’, The Guardian, 6 January 1977, 6. See also: Fox, Helen’s Story; DHSS et al., Whooping Cough. 59G. Stewart, ‘Vaccination against Whooping-cough’, Lancet, 1977, 1, 234–7; ‘Rush for Cough Vaccine “could do harm”, The Guardian, 19 December 1977, 2; Baker, ‘The Pertussis Vaccine Controversy’. 50Whiteley and Winyard, Pressure for the Poor: The Poverty Lobby and Policy Making (London: Methuen, 1987); Matthew Hilton, James McKay, Nicholas Crowson and Jean-Franc¸ois Mouhot, The Politics of Expertise: How NGOs Shaped Modern Britain (Oxford: Oxford University Press, 2013), esp. 133–5; Matthew Hilton, Nick Crowson, Jean-Franc¸ois Mouhot and James McKay, A Historical Guide to NGOs in Britain: Charities, Civil Society and the Voluntary Sector since 1945 (Basingstoke: Palgrave Macmillan, 2012), 122–5. 52This had been a core complaint in the interview with the Guardian. Mary McCormack, ‘The Hazards of Health’, The Guardian, 3 August 1973, 11. 51‘Vaccination against Whooping-cough’, Br Med J., 1956, 2, 454. 54M. Kulenkampff, J. S. Schwartzman and J. Wilson, ‘Neurological Complications of Pertussis Inoculation’, Archives of Disease in Childhood, 1974, 49, 46–9. 58William Breckon, ‘A Vaccinating Question’, The Guardian, 29 August 1973, 9. 53J. V. T. Gosling and J. H. Moseley, ‘To Jab or Not to Jab?’, The Guardian, 20 August 1973, 9. 50Whiteley and Winyard, Pressure for the Poor: The Poverty Lobby and Policy Making (London: Methuen, 1987); Matthew Hilton, James McKay, Nicholas Crowson and Jean-Franc¸ois Mouhot, The Politics of Expertise: How NGOs Shaped Modern Britain (Oxford: Oxford University Press, 2013), esp. 133–5; Matthew Hilton, Nick Crowson, Jean-Franc¸ois Mouhot and James McKay, A Historical Guide to NGOs in Britain: Charities, Civil Society and the Voluntary Sector since 1945 (Basingstoke: Palgrave Macmillan, 2012), 122–5. Medicine, 2009, 11, 42–61. 63‘The Departments’ referred to the Department of Health and Social Security (and its predecessors in England and Wales) and the Scottish and Northern Ireland Offices which had devolved responsibility for health. Parliamentary Commissioner for Administration, Sixth Report, Whooping Cough Vaccination (London: HMSO, 1977), HC571 (1976– 77), 3. ibid., 924, 17 January 1977, 73–4W and passim. For questions from Adley see: ibid., 918, 2 November 1976, 547–8W; ibid., 923, 20 December 1976, 240– 59; ibid., 925, 7 February 1977, 575–7W and passim.` The Vaccine Damage Campaign The Association’s breakthrough in 1973 came as a result of Fox’s campaigning and the recent publication of an article in the British Medical Journal (BMJ) which argued: Not only did many of its members (around two-thirds) blame the vaccine for their children’s injuries, there was a growing literature that suggested that there was hard evidence for their case.57 Professor George Dick, a member of the JCVI, had noted in 1973 that he had evidence for around 80 cases of damage from the vaccine per year.58 By 1977, Gordon Stewart’s claims that it was safer to catch pertussis than receive the inoculation reflected how much attention had been brought to the subject in the media and the medical community.59 Although there was never consensus that the vaccine was dangerous, enough doubt had been sown for there to be a genuine debate. A Disability Act? The Vaccine Damage Payments Act 1979 1 11 Parliamentarians became interested in the crisis, and the Association’s rising profile saw both the Heath and Wilson governments of the mid-1970s forced to refute allega- tions of medical negligence or a cover up.60 A succession of Early Day Motions, signed by dozens of MPs, suggest at least “soft” support for the campaign’s broad goal of provid- ing compensation for accident victims.61 Parliamentary questions from many MPs, but particularly Jack Ashley and Robert Adley (Conservative, Bristol North East until February 1974, then Christchurch and Lymington), pressed the government to release more infor- mation and to conduct further enquiries and tests into vaccine safety.62 In 1977, Ashley referred a complaint to the Parliamentary Commissioner, Sir Idwal Pugh, on behalf of the Association. Fox and Ashley argued that the health services had at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygien http://shm.oxfordjournals.org/ Downloaded from failed to make available to parents all the information they should have taken into account before they agreed to have their children vaccinated against whooping cough (pertussis). Mrs Fox added that she felt medical practitioners and health visi- tors were generally ill-informed about the conditions which made it inadvisable to give pertussis vaccine in the first place (contra-indications). . . . She considered that the Departments . . . had a responsibility also to see that everyone involved had ad- equate information and guidance on the subject.63 Pugh’s report was significant in that it brought specific cases to the attention of Parliament and the media. 62For questions from Astley see: ibid., 867, 17 January 1974, 172–5W; ibid., 914, 1 July 1976, 277–8W; 64Peter Hillmore, ‘MP Picks 4 Vaccine Victims for Fight’, The Guardian, 25 January 1976, 6. 61Fox, Helen’s Story, 67–70. Early Day Motion 70 (1974–75), for example, had been signed by ‘more than 50’ MPs by 3 December 1974: ‘That this House is concerned at the lack of statistics concerning vac- cine-damaged children: believes that their case for compensation is at least as just as those children suf- fering as a result of the thalidomide tragedy; and de- mands an immediate investigation into the problem’, Parliamentary Debates (Commons) 882, 3 December 1974, 1514–26. ibid., 924, 17 January 1977, 73–4W and passim. For questions from Adley see: ibid., 918, 2 November 1976, 547–8W; ibid., 923, 20 December 1976, 240– 59; ibid., 925, 7 February 1977, 575–7W and passim.` 60See, e.g., correspondence between Fox and the Heath government in Kew, London: The National Archives, TNA: MH 154/1053; and with the Wilson/ Callaghan governments in TNA: PIN 35/549. The Government Response The government resisted early calls for a compensation scheme for the children, despite showing political sympathy with the Association’s cause. Eventually, however, multiple pressure points forced action. Not only had the campaign for compensation gathered pace, it was becoming increasingly clear that the dramatic decline in vaccination rates was likely to lead to an epidemic in 1978 or 1979. at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from Ennals made an announcement to the House of Commons in February 1977. The JCVI had urged him to begin a publicity campaign, but he opted to wait until he had harder evidence of the vaccine’s safety, on the advice of the Committee for the Safety of Medicines.66 In his speech, he expressed sympathy for the parents, but argued that any action on the matter would have to wait for a detailed report from the JCVI on the scien- tific evidence, and from the Pearson Report on the legal position of any compensatory scheme.67 Pearson had been used as a delaying tactic throughout the Association’s cam- paign. Castle, in her first meeting with Fox in May 1974, had suggested referring vaccine damage to the Royal Commission.68 It also allowed the government to stall (and eventu- ally fight off) a case in the European Commission of Human Rights that was being pursued by the Association.69 A series of coinciding factors forced the government into action in the summer of 1977. The entire vaccination programme was in crisis. Vaccination rates for whooping cough had declined 59 per cent between 1971 and 1975.70 Pearson was due to publish towards the end of the year, and would most likely recommend a compensation scheme. Consumer in- formation group Which? had also made its support for compensation public.71 Further, Ennals clearly believed that the whole episode was inflicting significant political damage on the Labour Party. Ashley and the Association had successfully argued their case, and the government’s lack of action was ‘undermining our reputation as a caring government, and many of our supporters do not understand why we are resisting a claim which they see as obviously just’. Since the DHSS had already accepted, privately, that the compensation prin- ciple was sound, ‘political considerations favour an early announcement ... The Vaccine Damage Campaign The Association’s breakthrough in 1973 came as a result of Fox’s campaigning and the recent publication of an article in the British Medical Journal (BMJ) which argued: Ashley had chosen the medical stories of four children that, he argued, showed clear signs of contra-indications that were ignored by doctors at the time, leading directly to vaccine damage.64 ‘T’ was apparently ‘normal and healthy’ until he received his second dose of DTP and began having fits. The doctor gave the child a third dose regardless of the symptoms, and now he was considered ‘severely brain-dam- aged and ineducable’. ‘M’ had an epileptic mother, but has ‘begun to deteriorate after immunisation at fourteen months old and by eighteen months had become totally unre- sponsive’. ‘K’ had been born prematurely, and after receiving her vaccinations had be- come prone to ‘bouts of screaming’. The final case, ‘R’, had developed a ‘curious jerking of his left arm’ soon after being vaccinated for the first time, but the issue was dismissed as unimportant. After the second dose, the child began to have convulsions. The Commissioner did not agree entirely with the Association’s assertions of cause and ef- fect, and believed that there was enough information about the benefits and risks of Gareth Millward 12 vaccination in the broad public health sense. Still, he believed that information on ad- verse reactions was poor, and argued that parents and doctors should be given better ad- vice on how to spot contra-indications.65 69Fox, Helen’s Story, 88–90. See also TNA: MH 154/ 1057. 70TNA: CAB 129/195/14, Merlyn Rees, Vaccine Damage, 29 April 1977, 1. 71Hugh Herbert, ‘Which? Backs Vaccine Injury Compensation’, The Guardian, 13 January 1977, 7. 72TNA: CAB 129/195/16, David Ennals, Payment for Vaccine Damaged Children, 2 May 1977, 1. 73‘Medicine: Doubts over Vaccine Damage’, The Times, 26 February 1977, 14; Hugh Herbert, ‘“No cause for alarm” as Whooping Cough Spreads’, The Guardian, 26 April 1978. 65Parliamentary Commissioner for Administration, Sixth Report, 17–18, 22. 66 68‘Bringing the Law up to Scratch’, The Guardian, 7 August 1974. 69Fox, Helen’s Story, 88–90. See also TNA: MH 154/ 1057. 70TNA: CAB 129/195/14, Merlyn Rees, Vaccine Damage, 29 April 1977, 1. 66‘Vaccination Campaign Shelved’, The Guardian, 22 November 1977, 4; ‘Minister Defers Campaign to Encourage Vaccination’, The Times, 22 November 1974, 2; ‘Vaccine Campaign Launched Soon’, The Guardian, 8 February 1978, 5. 67Parliamentary Debates (Commons) 925, 8 February 1977, 1227–39. 65Parliamentary Commissioner for Administration, Sixth Report, 17–18, 22. 66‘Vaccination Campaign Shelved’, The Guardian, 22 November 1977, 4; ‘Minister Defers Campaign to Encourage Vaccination’, The Times, 22 November 1974, 2; ‘Vaccine Campaign Launched Soon’, The Guardian, 8 February 1978, 5. 67Parliamentary Debates (Commons) 925, 8 February 1977, 1227–39. 68‘Bringing the Law up to Scratch’, The Guardian, 7 August 1974. 69Fox, Helen’s Story, 88–90. See also TNA: MH 154/ 1057. 71Hugh Herbert, ‘Which? Backs Vaccine Injury Compensation’, The Guardian, 13 January 1977, 7. p y 72TNA: CAB 129/195/16, David Ennals, Payment for Vaccine Damaged Children, 2 May 1977, 1. A Disability Act? The Vaccine Damage Payments Act 1979 13 The Cabinet resolved to accept the general principle of compensation for victims of vaccine damage in order to restore faith in the vaccination programme. Partially, this was to ‘mollify’ Ashley and the Association.74 But it was also designed to play to the wider public. The belief was that by accepting the compensation principle, it would allay the fears of parents by showing that if something went wrong the state would protect them. It was also seen as a sign of strength and confidence. The government was explicitly stat- ing that it was sure that there were so few cases that it was willing to compensate par- ents even if they could not definitively prove that vaccines were the sole cause of their child’s disability.75 On the other hand, it was possible that such action would bring atten- tion to those rare cases, and give parents cause for concern.76 On balance, the govern- ment chose to acquiesce to the principle of the Association’s demands, and produce a solution that was financially affordable and would not open the government up to com- peting claims for no-fault compensation from other interest groups.77 at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygien http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medic http://shm.oxfordjournals.org/ Downloaded from The government engineered a public exchange of correspondence between Lord Pearson and James Callaghan, orchestrated by Ennals and Lord Chancellor Frederick Elwyn-Jones.78 On 6 June 1977, Callaghan wrote: My ministerial colleagues and I are greatly concerned by the small, but tragic, number of cases in which vaccination against serious childhood diseases may have caused damage to the children concerned. . . . It would therefore go far to relieve the anxi- eties and concern of myself and colleagues, and to restore public confidence, if you were able to assure me that the Commission will be dealing specifically with the problem of vaccine damage and to give an indication of your thinking at this stage.79 To which Pearson responded: I can readily give you the assurance you seek. .. . 76TNA: CAB 129/195/14, Vaccine Damage, 1. Similar views were expressed in a Guardian editorial: ‘Either Way, the Children still Die’, The Guardian, 12 February 1977. 80TNA: BN 120/10, Lord Pearson to James Callaghan, 9 June 1977. This exchange was reinforced by a state- ment by Ennals in the House of Commons: Parliamentary Debates (Commons) 933, 14 June 1977, 240–1. 77TNA: CAB 128/61/18, Cabinet minutes, 5 May 1977, 8–11. 78See TNA: BN 120/10, esp. Lord Elwyn-Jones to James Callaghan, 20 May 1977; Cabinet Office, Meeting between Lord Chancellor, Secretary of State for Social Services and Chief Secretary, Treasury, held 19 May 1977. 79 75TNA: CAB 129/195/14, Vaccine Damage; CAB 129/ 195/16, Payment for Vaccine Damaged Children; Parliamentary Debates (Commons) 925, 8 February 1977, 1227–39. 79TNA: BN 120/10, James Callaghan to Lord Pearson, 6 June 1977. 80 78See TNA: BN 120/10, esp. Lord Elwyn-Jones to James Callaghan, 20 May 1977; Cabinet Office, Meeting between Lord Chancellor, Secretary of State for Social Services and Chief Secretary, Treasury, held 19 May 1977. 79TNA: BN 120/10, James Callaghan to Lord Pearson, 6 June 1977. 80TNA: BN 120/10, Lord Pearson to James Callaghan, 9 June 1977. This exchange was reinforced by a state- ment by Ennals in the House of Commons: Parliamentary Debates (Commons) 933, 14 June 1977, 240–1. The Government Response rather than waiting for many months, during which the pressure will build up and the vaccination pro- gramme ... further damaged’.72 To give the government confidence, it was also becoming increasingly clear that respected medical evidence supported the pertussis vaccine.73 A Disability Act? The Vaccine Damage Payments Act 1979 74TNA: CAB 128/61/18, CM(77) 18th Conclusions, Cabinet minutes, 5 May 1977 10.30am, 10. A Disability Act? The Vaccine Damage Payments Act 1979 We see it as a particular part of a very difficult field with which our Report will have to deal, but we have all reached the conclusion that some kind of financial assistance should be made available for very seri- ous injury resulting from vaccination recommended by a public health authority.80 The Association saw Pearson as a victory. While Pugh’s report had been seen as too tame, Pearson reaffirmed many of the core arguments, especially the one made in the BMJ in 1973—that is, that if the government was going to encourage all children to be vaccinated on public health grounds, it should also compensate those rare cases of 74TNA: CAB 128/61/18, CM(77) 18th Conclusions, Cabinet minutes, 5 May 1977 10.30am, 10. 77TNA: CAB 128/61/18, Cabinet minutes, 5 May 1977, 8–11. Gareth Millward 14 damage that followed.81 Although the report only briefly covered vaccination policy (six pages out of 545), it acknowledged that almost every expert voice on the matter agreed that there was a moral case for compensation. ‘Nobody argued in the contrary sense.’82 The Government was forced to produce legislation quickly as the 1979 General Election was looming.83 Callaghan had already ordered that the scheme should be planned in the background to ensure it could be brought to the House as quickly after Pearson’s publication as possible.84 This meant that the drafting was essentially com- plete, and cross-party support for the Bill ensured that the passage through the Houses of Parliament became a formality.85 The resulting Act showed some very clear choices on the part of the DHSS which cannot be explained outside of the realm of disability policy. As the full title states, this was: at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from An Act to provide for payments to be made out of public funds in cases where severe disablement occurs as a result of vaccination against certain diseases or of contact with a person who has been vaccinated against any of those diseases.86 ‘Severe disablement’ is, obviously, a reference to disability; the definition was based on long-standing medico-legal practice that had only recently been reaffirmed with the 1975 expansion of disability benefits. Thus, a person qualified for payment if they ‘[suf- fered] disablement to the extent of 80 per cent. 85TNA: BN 13/360; The Bill got its second reading on 5 February and received Royal Assent on 22 March, days before the no-confidence vote. Parliamentary Debates (Commons) 962, 5 February 1979; ibid., 964, 22 March 1979, 1760. 84TNA: CAB 128/61/18, Cabinet minutes, 5 May 1977, 10–11. 86Vaccine Damage Payments Act 1979. 87Ibid., c. 1, para. 4. In Northern Ireland it references the Social Security (Northern Ireland) Act 1975. 88Disability Alliance, Poverty and Disability; DIG, Creating a National Disability Income. 89Definition of ‘disablement’, used in all subsequent Acts until the Disability Discrimination Act 1995. Disabled Persons (Employment) Act 1944, c. 1. 90Social Security Act 1975, Schedule 8. 91Paul Abberley, ‘Counting Us Out: A Discussion of the OPCS Disability Surveys’, Disability, Handicap & Society, 1992, 7, 139–155; Glenn T. Fujiura and Violet Rutkowski-Kmitta, ‘Counting Disability’, in Albrecht et al., eds, Handbook of Disability Studies, 69–96. 83An election had to be called by October as it had been five years since the previous one, but it would be brought forward by a vote of no confidence in March 1979. Parliamentary Debates (Commons) 965, 28 March 1979, 461–590. 81For allegations that the Pugh report was too tame see: Gillian Linscott, ‘“Frail” Vaccine Report Disappoints Parents’, The Guardian, 28 October 1972, 2. y , 91Paul Abberley, ‘Counting Us Out: A Discussion of the OPCS Disability Surveys’, Disability, Handicap & Society, 1992, 7, 139–155; Glenn T. Fujiura and Violet Rutkowski-Kmitta, ‘Counting Disability’, in Albrecht et al., eds, Handbook of Disability Studies, 69–96. 81For allegations that the Pugh report was too tame see: Gillian Linscott, ‘“Frail” Vaccine Report Disappoints Parents’, The Guardian, 28 October 1972, 2. 82Pearson, Royal Commission, para. 1397, 296. A Disability Act? The Vaccine Damage Payments Act 1979 or more, assessed as for the purposes of section 57 of the Social Security Act 1975’.87 The idea of ‘percentage of disablement’ came from the Industrial Injuries and War Pensions schemes from before the Second World War. The Disability Alliance favoured this system as a way of determining pay- ments based on need for all disabled people, though in this respect they differed from DIG’s wider National Disability Income Scheme.88 The concept is, however, rooted in older medical definitions of disability. Percentage of disablement was designed to deter- mine the effects on an adult being able to find employment ‘of a kind which apart from that injury, disease or deformity would be suited to his age, experience and qualifica- tions’.89 The degree of disablement was measured against ‘a person of the same age and sex whose physical and mental condition is normal’ by a medical practitioner, who would provide a written assessment for the social security authorities.90 Transposing this concept onto children was not unheard of, despite the potential difficulties in applying such measures of disablement.91 By choosing 80 per cent—a generally accepted level of A Disability Act? The Vaccine Damage Payments Act 1979 15 ‘severe disablement’ that would also be used when Severe Disablement Allowance was introduced in 1984—the government also made a decision that only the ‘most in need’ would receive benefit.92 This was a traditional tactic in restricting access to new benefits, with the DHSS and the Treasury often wary of opening the door to an avalanche of claims, and a seemingly exponential rise in public expenditure over time.93 It remains clear that the Vaccine Damage Payments Act 1979 could not have operated without the legal framework of disability that had been established over decades of legis- lation. Indeed, the restrictions on access were defended by reference to the supposed im- provements in disability policy over the past decade. The Act was originally intended as an interim measure—the Labour government specifically brought it to Parliament with the caveat that once final recommendations on vaccines safety from JCVI were available (and Person had been fully digested) that there would be follow-up legislation to create a more comprehensive Act.94 This was in part a defence of the relatively low sum of money available: £10,000 was not considered enough by campaigners to truly cover the costs of caring for a severely disabled child over its lifetime. 94JCVI published its recommendations in 1981, along with data on the effectiveness of the vaccine. See DHSS et al., Whooping Cough; Anon., ‘Efficacy of Pertussis Vaccination in England’, Br Med J., 1982, 96TNA: BN 124/20, Norman Fowler to Fox, 4 March 1983; Margaret Thatcher to Fox, (?)20 April 1982; TNA: PIN 35/549, Gerard Vaughan to Fox, 22 September 1980; Parliamentary Debates (Commons) 977, 22 January, 163W. p 95Parliamentary Debates (Commons) 962, 5 February 1979, 32–86. A Disability Act? The Vaccine Damage Payments Act 1979 Yet this was also countered by refer- ence to recent improvements in the general state of disability benefits.95 After the gen- eral election, Margaret Thatcher’s Conservative government used the defence that it planned to improve life for all disabled people and was not willing to give more special treatment to a group that already benefited hugely over other equally disabled people.96 Once again, the Act and its implementation were rooted in disability policy, legally and politically. at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hy http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 rnals.org/ 97TNA: CAB 129/195/14, Vaccine Damage, Annex. 93Millward, ‘Invalid Definitions, Invalid Responses’. See also Stone’s work on how disability schemes tend to grow as pressure builds from within: Deborah A. Stone, The Disabled State (Philadelphia: Temple University Press, 1984); Deborah A. Stone, ‘Physicians as Gatekeepers’, Public Policy, 1979, 27, 227–54. 92For the Severe Disablement Allowance levels of disable- ment see: Health and Social Security Act 1984, c. 11. 285, 357–39; R. Alderslade, Department of Health and Social Security, and Middlesex Hospital Medical School, National Childhood Encephalopathy Study (London: Middlesex Hospital Medical School, 1981). 285, 357–39; R. Alderslade, Department of Health and Social Security, and Middlesex Hospital Medical School, National Childhood Encephalopathy Study (London: Middlesex Hospital Medical School, 1981). 977, 22 January, 163W. 92For the Severe Disablement Allowance levels of disable- ment see: Health and Social Security Act 1984, c. 11. 93Millward, ‘Invalid Definitions, Invalid Responses’. See also Stone’s work on how disability schemes tend to grow as pressure builds from within: Deborah A. Stone, The Disabled State (Philadelphia: Temple University Press, 1984); Deborah A. Stone, ‘Physicians as Gatekeepers’, Public Policy, 1979, 27, 227–54. 94JCVI published its recommendations in 1981, along with data on the effectiveness of the vaccine. See DHSS et al., Whooping Cough; Anon., ‘Efficacy of Pertussis Vaccination in England’, Br Med J., 1982, 92For the Severe Disablement Allowance levels of disable- ment see: Health and Social Security Act 1984, c. 11. The Significance of the Act By looking at the historical and political context of the Vaccine Damage Payments Act, we can see more clearly why this specific piece of legislation was passed. Winning the medical argument through the Medical Research Council and JCVI evidence was not enough. A political statement needed to be made that accorded with public opinion and concern over the vaccination programme in general. The provisions contained within the Act were relatively cheap, a welcome relief in the economic circumstances. Initial esti- mates predicted only around 300 to 500 initial claims, followed by 14 to 70 claims per year thereafter. On the basis of £25,000 lump-sum payments, this would have cost around £10 million, and then £350,000 to £1,750,000 per annum.97 In the end, only £10,000 was awarded to each of 349 children in 1979 and 255 in 1980, before the claim Gareth Millward 16 rate fell significantly.98 It was also seen, to quote Ennals, as ‘one essentially of political judgement’. Vaccination was not compulsory in Britain, and it would be possible to argue that these cases affected the tiniest of minorities.99 Still, if the Association’s campaign had continued, Ennals believed that ‘the vaccination programme can be got going again’. It was ‘vital’ that it were, ‘because of the possibility of an outbreak of poliomyelitis this summer—an event for which many people would lay responsibility at the Government’s door’.100 The Act, and pronouncements leading to it, were part of a spe- cific political response to a particular public threat. rate fell significantly.98 It was also seen, to quote Ennals, as ‘one essentially of political judgement’. Vaccination was not compulsory in Britain, and it would be possible to argue that these cases affected the tiniest of minorities.99 Still, if the Association’s campaign had continued, Ennals believed that ‘the vaccination programme can be got going again’. It was ‘vital’ that it were, ‘because of the possibility of an outbreak of poliomyelitis this summer—an event for which many people would lay responsibility at the Government’s door’.100 The Act, and pronouncements leading to it, were part of a spe- cific political response to a particular public threat. at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from The Association’s success was due to a number of factors, many of which are seen as typical of the campaigning landscape at the time. 100TNA: CAB 129/195/16, Payment for vaccine dam- aged children, 1. 101See: Whiteley and Winyard, Pressure for the Poor. 102CAB 128/61/18, Cabinet minutes 5 May 1977, 8. 103See Ennals’ arguments in CAB 12/195.16, Payment for vaccine damaged children. 104Drakeford and Butler, ‘Everyday Tragedies’. 105DHSS et al., Whooping Cough; Baker, ‘The Pertussis Vaccine Controversy’. 98While the value of the award would be scaled up at points over the proceeding decade to combat the ef- fects of inflation, the number of claims remained rel- atively low. A total of 74 successful claims were made in 1981; 42 in 1983; 29 in 1984; 26 in 1985; 15 in 1986 and 10 up to December 1987. See Parliamentary Debates (Commons) 124, 18 December 1987, 931W. 99TNA: CAB 128/61/18, Cabinet minutes 5 May 1977, 8–11. 111The Disability Alliance’s files in the Peter Townsend Collection at the University of Essex cover this pe- riod in great detail. See especially: Peter Townsend Collection, University of Essex, Colchester: 79.03, ‘A note about the Pearson Commission Report’, Peter Townsend 16 June 1978; 77.02, Disability Alliance Steering Group minutes, 29 June 1978, 1. The Significance of the Act The use of more professionalised re- search, exploitation of media coverage and the creation of key allies in positions of power greatly aided the parents’ cause.101 The relationship between Rosemary Fox and Jack Ashley MP was central to this. Ashley had experience in precisely this sort of campaign for medical compensation following thalidomide. Cabinet spoke about the Association and Ashley in the same breath, referring to ‘Mr Ashley’s campaign’.102 Recent develop- ments in disability policy cannot be ignored in explaining how these issues came to be recognised by the public as worthy of discussion. Social security payments were seen at this time as logical responses to social injustices; and disabled people were seen as wor- thy recipients of new benefit schemes. There were also a number of parliamentarians able to articulate these points. In many ways, the Association was unlike DIG and the Disability Alliance for its dogged focus on one specific benefit for a special medical case. However, it clearly benefited from many of the successes of those organisations, drawing on their tactics and building on their political arguments. For it should be noted that The Association won the moral argument, and won it early. The only scientific debate to be won was to prove that vaccine damage existed. It man- aged to provide hundreds of potential examples, enough to spread doubt among the public and to win support for a compensation scheme. Pugh’s report, coupled with news- paper coverage of the medical doubts of Dick, Stewart and Wilson made vaccine damage a fact. It took the government three years to publicly announce that it accepted the thrust of the Association’s argument, as a direct result of the public pressure Fox and her allies had generated.103 This cannot be separated from the wider medical scare, but it reem- phasises Drakeford and Butler’s claims that scandals have to be articulated and pur- sued.104 No person or body ever provided enough evidence to overturn the initial MRC trials; and two years later the completed review by the JCVI reaffirmed the medical estab- lishment’s position.105 Without the wider focus on the political pressures of the period, we cannot explain why this particular medical debate became a full-blown ‘scandal’. A Disability Act? 108For examples of the Interdepartmental Group on Disablement and of cabinet sub-committee discus- sions Morris led, see TNA: CAB 134/3845; CAB 134/ 4036; CAB 134/4235; MH 154/848; and passim. Evelyn Adelaide Sharp chaired the Sharp Report, published as Mobility of Physically Disabled People (London: HMSO, 1974). The Silver Jubilee Committee was chaired by Peter Large and its re- port published as ‘Can Disabled People Go Where You Go?’: Report by the Silver Jubilee Committee on Improving Access for Disabled People (London: HMSO, 1979). See also: Committee on Restrictions Against Disabled People, Report (London: HMSO, 1982). 106See Kingdon’s work on policy making and agenda setting: Kingdon, Agendas, Alternatives and Public Policies. 110Only 843 payments had been made in total from the Vaccine Damage Payments scheme up to December 1987. By contrast, over one million peo- ple were claiming invalidity benefit at any one time in 1987/88. See Parliamentary Debates (Commons) 124, 18 December 1987, 931W. There were an av- erage of 1,067,000 claimants at any one time of in- validity benefit, the main National Insurance unemployment benefit for disabled people, in the fi- nancial year 1987/88. Department of Work and Pensions, Benefit Expenditure Tables, 2013 <http:// statistics.dwp.gov.uk/asd/asd4/expenditure_tables_ Budget 2013.xls>, accessed 12 June 2013. 110Only 843 payments had been made in total from the Vaccine Damage Payments scheme up to December 1987. By contrast, over one million peo- ple were claiming invalidity benefit at any one time in 1987/88. See Parliamentary Debates (Commons) 124, 18 December 1987, 931W. There were an av- erage of 1,067,000 claimants at any one time of in- validity benefit, the main National Insurance unemployment benefit for disabled people, in the fi- nancial year 1987/88. Department of Work and Pensions, Benefit Expenditure Tables, 2013 <http:// statistics.dwp.gov.uk/asd/asd4/expenditure_tables_ Budget 2013.xls>, accessed 12 June 2013. 109Campbell and Oliver, Disability Politics; Michael Oliver and Colin Barnes, The New Politics of Disablement (Basingstoke: Palgrave Macmillan, 2012). 107TNA: BN 59/75. 108 The Significance of the Act The Vaccine Damage Payments Act 1979 17 By establishing that there was a policy problem and generating the political will to rec- tify it, a solution needed to be found.106 Developments in disability benefits over the de- cade provided the bureaucratic tools for this. Since the late 1960s, the DHSS had been planning for a number of disability benefits. Even after the Social Security Benefits Act 1975, it had continued to be involved in developing a scheme for disabled drivers to pur- chase cars.107 Alfred Morris had established an Interdepartmental Group on Disablement within government, and the Sharp Report, Silver Jubilee Committee and Committee on Restrictions Against Disabled People were considering social rights issues with regard to access to businesses and services.108 In short, the British government had created the tools necessary for dealing with disability issues, both in the form of a bureaucratic appa- ratus for investigating policy solutions and the legal precedents of previous schemes. As we have also seen, concerns surrounding thalidomide had led to the introduction of medical issues in the Pearson Report. Thus, even if the decline in public support for vacci- nation is seen as a medical issue, the response cannot be explained outside the disability and social security politics of the late 1970s. at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from And yet, it must be stressed that most of the other disability organisations and cam- paigners of the period wrote very little about vaccine damage compensation. Disability studies activists, where they have written historical analyses of the 1970s, have focused on the battles they themselves fought. 112Reports of declining vaccination rates and increased risk of epidemic were published across the winter of 1977/78. See for example: ‘Whooping Cough Vaccinations Fall’, The Guardian, 23 November 1977, 4; ‘Whooping Cough Could Sweep Britain’, The Guardian, 10 December 1977, 24; ‘The Omens so Far Are Bad—These Could be the First Ripples of a Whooping Cough Wave’, The Guardian, 25 January 1978, 9. The Significance of the Act Since many of them were involved in DPOs such as the Union of the Physically Impaired Against Segregation and the British Council of Organisations of Disabled People, such legislation was neither part of their remit, nor was it a core constituent of the wider struggle for disabled people’s civil rights.109 Moreover, while the scheme is still running, very few disabled people received payments as a pro- portion of all the disabled people in the United Kingdom.110 Even at the time, organisa- tions such as the Disability Alliance had covered Pearson in great detail and submitted evidence; but it had focused on industrial injuries compensation, not mentioning vaccina- tion at all.111 Vaccine damage, then, occupies an interesting analytical hinterland, being 109Campbell and Oliver, Disability Politics; Michael Oliver and Colin Barnes, The New Politics of Disablement (Basingstoke: Palgrave Macmillan, 2012). 109Campbell and Oliver, Disability Politics; Michael Oliver and Colin Barnes, The New Politics of Disablement (Basingstoke: Palgrave Macmillan, 2012). Gareth Millward 18 both rooted in the disability politics and policy of the 1970s, while seemingly ignored as a disability issue by many of its contemporaries. This again stresses the need to look beyond solely medical readings of the pertussis vaccine scare. It also should make historians aware of the need to acknowledge that medical definitions of disability—while rejected by social model advocates as politically illegitimate—offer a useful lens for understanding and framing the decisions and attitudes of institutions in the past. at London School of Hygiene http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from 117Baker, ‘The Pertussis Vaccine Controversy’, esp. 4006. 113Dr Tony Smith, ‘Clearing the Doubts over Whooping Cough’, The Times, 18 August 1978, 12. 114Melanie Phillips, ‘Row over Cause of Whooping Cough Outbreak’, The Guardian, 9 August 1978, 3. g 116‘Whooping Cough Vaccine “almost run out”’, The Times, 16 December 1977, 2. 115Ashley, Acts of Defiance. See also Parliamentary Debates (Commons) 962, 5 February 1979, 33; Parliamentary Debates (Lords) 551, 12 January 1994, 125. Fox and the Association were also keen to emphasise that they were not anti-vaccination, but were looking for more informed choice and compensation for those injured:‘Mary McCormack, ‘The Hazards of Health’, 11; ‘Bringing the Law up to Scratch’, The Guardian, 7 August 1974. 113Dr Tony Smith, ‘Clearing the Doubts over Whooping Cough’, The Times, 18 August 1978, 12. 114Melanie Phillips, ‘Row over Cause of Whooping Cough Outbreak’, The Guardian, 9 August 1978, 3. Conclusions The Association’s success quickly turned sour. Between 1974 and 1977, it won the moral argument for compensation, and legislation soon followed. After 1977, however, the fo- cus shifted back towards public health. JCVI had warned of a potential whooping cough epidemic for 1978 or 1979, and when it hit, the Association and Jack Ashley took much of the blame.112 Dr Tony Smith in the The Times argued that while the press should have been more responsible in providing a balanced review of the evidence, it was the atten- tion drawn by the Association that had caused the controversy.113 The JCVI and Office of Population Censuses and Surveys went further, arguing that the Association was to blame for scaring parents.114 Ashley was forced to refute at the time and many years later that he opposed vaccination, making it very clear that he and his colleagues sup- ported the national programme and believed declining vaccination rates were worri- some.115 Such was the volatility of public opinion on the matter that in the summer of 1978 the government reported a shortage of whooping cough vaccine owing to the rush from parents who had previously opted out.116 In some ways, this marked a new medical scandal—not surrounding the failure of government protection against vaccine damage, but of its failure to protect against infectious disease. As Baker has shown, the targets of opprobrium were not a complacent medical establishment, but those who undermined the vaccination programme through scaremongering.117 The Vaccine Damage Payments Act was just a part of the government’s response to the pertussis vaccine scare. Ostensibly, it was designed to restore faith in the vaccination programme, allowing the state to resume its protective policies against infectious disease. But the specific form of this legislation and the way in which the campaigns for compen- sation were run owed a lot to the context of disability and social security developments over the course of the 1970s. The government had to act in such a way that took note of the economic conditions of the time, as well as public attitudes towards medical risk, vac- cination and notions of the role of the state in protecting and providing for disabled A Disability Act? The Vaccine Damage Payments Act 1979 19 people. Yet there is a paradox here for historians. Conclusions Despite being seen as largely a matter of medical and public health policy, the responses to the pertussis scare were rooted in the context of disability and social security policy of the 1970s. It is clear that medical his- torians need to pay closer attention to the disability issues; and by the same token, disability historians can learn much from re-examining the Act using the skills they have developed over the past 20 years. This will begin to provide a wider view of the pertussis vaccine scare as a social phenomenon, and not just one of crisis within the public health profession. at London School of Hygiene & Tropical Medicine on October 28, 2016 http://shm.oxfordjournals.org/ Downloaded from at London School of Hygien http://shm.oxfordjournals.org/ Downloaded from at London School of Hygiene & Tropical Medicine http://shm.oxfordjournals.org/ loaded from Acknowledgements The author would like to thank Alex Mold, Harriet Palfreyman, Martin Moore and Emily Andrews for their notes and feedback on earlier drafts of this article. Thanks also to the examiners of the PhD, Pat Thane and the late Anne Borsay, from which some of this work is based.
https://openalex.org/W2236998591	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-016-2698-5	English	null	Overweight and obesity among women: analysis of demographic and health survey data from 32 Sub-Saharan African Countries	BMC public health	2,015	cc-by	7,842	© 2016 Neupane et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Overweight and obesity are risk factors for many chronic diseases globally. However, the extent of the problem in low-income countries like Sub-Saharan Africa is unclear. We assessed the magnitude and disparity of both phenomena by place of residence, level of education and wealth quintile using cross-sectional data from 32 countries. Methods: Demographic and Health Survey (DHS) data collected in 32 Sub-Saharan African countries between January 2005 and December 2013 were used. A total of 250651 women (aged 15–49 years) were analyzed. Trained personnel using a standardized procedure measured body weight and height. Body mass index (BMI) was calculated by dividing body weight by height squared. Prevalence of overweight (25.0–29.9 kg/m2) and obesity (≥30.0 kg/m2) were estimated for each country. Analysis of the relationships of overweight and obesity with place of residence, education and wealth index were carried out using logistic regression. Results: The pooled prevalence of overweight for the region was 15.9 % (95 % CI, 15.7–16.0) with the lowest in Madagascar 5.6 % (95 % CI, 5.1–6.1) and the highest in Swaziland 27.7 % (95 % CI, 26.4–29.0). Similarly, the prevalence of obesity was also lowest in Madagascar 1.1 % (95 % CI, 0.9–1.4) and highest in Swaziland 23.0 (95 % CI, 21.8–24.2). The women in urban residence and those who were classified as rich, with respect to the quintile of the wealth index, had higher likelihood of overweight and obesity. In the pooled results, high education was significantly associated with overweight and obesity. Conclusions: The prevalence of overweight and obesity varied highly between the countries and wealth index (rich vs. poor) was found to be the strongest predictor in most of the countries. Interventions that will address the socio-cultural barriers to maintaining healthy body size can contribute to curbing the overweight and obesity epidemic in Africa. Keywords: Overweight, Obesity, Women, BMI, DHS, Sub-Saharan Africa Keywords: Overweight, Obesity, Women, BMI, DHS, Sub-Saharan Africa are attributable to overweight and obesity [4]. The World Health Organization (WHO) defines a person to be overweight if his or her body mass index (BMI) is >25, and obese if BMI is ≥30 [5]. Overweight and obesity among women: analysis of demographic and health survey data from 32 Sub-Saharan African Countries Subas Neupane1, Prakash K.C.1 and David Teye Doku2* Subas Neupane1, Prakash K.C.1 and David Teye Doku2* * Correspondence: dokudavid@gmail.com 2Department of Population and Health, University of Cape Coast, Private Mail Bag, University Post Office, Cape Coast, Ghana Full list of author information is available at the end of the article Neupane et al. BMC Public Health (2016) 16:30 DOI 10.1186/s12889-016-2698-5 Neupane et al. BMC Public Health (2016) 16:30 DOI 10.1186/s12889-016-2698-5 Background Obesity has become a major public health problem both in developed and developing countries [1]. Overweight and obesity are the fifth leading risk for global deaths [2]. At least 2.8 million adults die each year as a result of being overweight or obese [3]. In addition, 44 % of the diabetes burden, 23 % of the ischemic heart disease bur- den and between 7 and 41 % of certain cancer burdens According to a recent research reports, the burden of obesity is increasing in developing countries and also no significant reduction has been seen in its burden in de- veloped countries over the past few decades [1]. For ex- ample, in West Africa, the prevalence of obesity rapidly increased during the last two decades of the 20th century and it continues to increase in the 21st cen- tury. A recent review indicates that the prevalence * Correspondence: dokudavid@gmail.com 2Department of Population and Health, University of Cape Coast, Private Mail Bag, University Post Office, Cape Coast, Ghana Full list of author information is available at the end of the article Neupane et al. BMC Public Health (2016) 16:30 Page 2 of 9 Page 2 of 9 information on age and height were included. DHS surveys are available to investigators through the World Wide Web (http://www.dhsprogram.com). All 32 countries, DHS survey followed the same standard procedures. Detail descriptions of DHS sampling procedures, validation of questionnaire, and data collection methods are published elsewhere (http:// www.dhsprogram.com). Briefly, the DHS used a strati- fied two-stage random sampling approach, consisting of a selection of census enumeration areas based on a probability, followed by a random selection of house- hold from a complete listing of a household within the selected enumeration areas. In this study, all to- gether 366885 women from 32 countries responded to the surveys with the response rates varying from 86.2 to 100.0 %. However, the present analysis is based on all women who had information on weight and height (N = 250651). Women granted written in- formed consent before interviewing them. Ethical ap- proval was given by ICF International (Calverton, MD, USA) institutional review board and by individ- ual review boards within every participating country. of obesity in the region between 2000 and 2004 was roughly 10 % [6, 7]. Measurements of variables Over all, some few studies on obesity in Africa have been conducted [13–15]. While these studies have made various contributions to understanding the phenomenon in the region, their focuses differ from the present stud- ies. For instance, Steyn and Mchiza [13] focused on un- derstanding whether changes in nutrition diet and obesity have taken place over the past three decades in Sub-Saharan Africa. Ziraba et al. [14] on the other hand, explored the phenomenon in urban Africa. Another challenge with these previous findings is that they used different data sets, which have varying representativeness and methodological procedures. Therefore, the findings are not comparable across countries. To address these constraints, this study used nationally representative data from 32 Sub-Saharan African countries with similar method of data collection to explore the prevalence of overweight and obesity in the region. Furthermore, the study explored disparity in the phenomenon by place of residence, level of education and wealth quintile. In all DHS survey, trained personnel measured the height and weight using a standardized procedure. Weight was measured using solar-powered scales with accuracy of 0.1 kg and height was measured using stan- dardized measuring boards with accuracy to 0.1 cm. Body mass index (BMI) was calculated by dividing body weight (kg) by squared height (m2). Overweight and obesity were defined as recommended by World Health Organization [5]: overweight 25.0–29.9 kg/m2 and obes- ity ≥30.0 kg/m2. As the prevalence of obesity was low (<1 %) in some countries, the categories of overweight and obesity were combined together in the logistic re- gression analyses. Only the respondents who had infor- mation on BMI were included in the analyses. The participants’ place of residence was designated as rural and urban according to country specific definitions. The wealth index was calculated using easy-to-collect data on a household’s ownership of selected assets (e.g. televisions, bicycles, cars, materials used for housing construction and types of water access and sanitation fa- cilities). The wealth index was then generated as a com- posite variable by demographic and health survey (DHS) staff using principal components analysis. Continuous scale of relative wealth was then categorized into five (poorest, poorer, middle, richer, and richest) according to the quintile of the sample. Wealth index was not available from the DHS data from Chad. Maternal edu- cation was assessed from self-report of the completed educational level (no education, primary, secondary, or higher). Background Overweight and obesity is as a result of energy imbal- ance that consumes more calories than what is equiva- lently expended in physical activities. Westernization and Urbanization are said to be among the main reasons for this energy imbalance in the African region and they are viewed from two perspectives [7]. First, urbanization and westernization lead to decreased physical activity. Sec- ond, urbanization and westernization result in increased food supply, which include access to high caloric fast foods and sugar sweetened beverages (fatty foods) [8]. Previous studies have found socioeconomic differences in obesity and overweight [9], and in developing countries the direc- tion is not consistent [9]. While some studies found the likelihood of being obese or overweight to the detriment of those with higher socio-economic status (SES) [10, 11], others reported higher chances of being overweight or obese among those with lower SES [12]. Height, which is a component of BMI is itself socioeconomically patterned because past socioeconomic status and nutrition during the developmental stages of life (childhood and adoles- cence) affect height. Statistics number of outcome events to reflect their influence on the correlation. Analyses of the relationships between overweight and obesity and place of residence, education and wealth index were carried out. Odds ratios (ORs) and their 95 % confidence intervals (CIs) for overweight and obesity (<25.0 = 0, ≥25.0 = 1) were estimated in multivari- ate logistic regressions model including the place of residence (urban = 0, rural =1), maternal education (secondary or higher =0, no or primary education =1) and wealth index (richer or richest =0, poorest to Sample weights were applied to the data to remove the bias due to unequal selection probabilities. Descriptive figures of the study participants are reported in percent- ages and the prevalence of overweight and obesity with their 95 % confidence intervals (CIs) are reported separ- ately for each country. The pooled prevalence for the re- gion was also estimated. Data sources and procedures Thirty-two nationally representative cross-sectional data from the most recent Demographic and Health survey (DHS) conducted between January 1, 2005, and Decem- ber 31, 2013 in Sub-Saharan Africa were used. The DHS survey data were collected at about 5-year intervals across low and middle-income countries. DHS collect data on health and welfare by interviewing women of re- productive age (15–49 years), their children, and their households. In this analysis only women who had Neupane et al. BMC Public Health (2016) 16:30 Page 3 of 9 Page 3 of 9 a Number of women of reproductive age (15–49 years) group who had information on BMI NA data not available Statistics Scatterplots were used to visualize the relationship between no education and over- weight and obesity with the size of a marker relative to the Table 1 Characteristics of women in Demographic Health Surveys across countries Country Na Mean age (years) Urban residents (%) Lowest house-hold wealth quintile (%) Highest house-hold wealth quintile (%) No education (%) Mean value of total children ever born Benin, 2006 16071 28.93 45.6 17.1 23.8 60.0 2.77 Burkina Faso, 2010 8503 28.72 26.7 18.2 24.4 74.0 3.27 Burundi, 2010 4616 27.74 10.5 20.0 20.0 45.4 2.74 Cameroon, 2011 7872 27.82 54.0 15.8 24.3 19.0 2.73 Chad 3801 28.26 18.5 NA NA 77.1 4.42 Comoros 5155 27.68 33.4 16.3 22.0 31.2 2.19 Congo (Brazzaville) 2011-12 5480 28.48 66.8 17.7 20.3 5.7 2.54 Congo Republic, 2007 4763 28.52 45.0 18.7 21.9 22.1 3.08 Cote d’Ivoire, 2011-12 4694 28.20 49.6 18.0 23.6 54.4 2.70 Ethiopia, 2011 16065 27.70 23.3 18.2 25.1 51.0 2.91 Gabon, 2012 5408 28.68 88.4 14.8 21.9 4.5 2.35 Ghana, 2008 4822 28.99 48.5 15.8 23.1 21.2 2.33 Guinean, 2005 4756 28.16 35.0 17.5 22.5 66.4 2.95 Kenya, 2008 8295 28.44 25.3 16.5 26.2 8.8 2.68 Lesotho, 2009 3903 28.19 32.6 14.6 26.0 1.2 1.82 Liberia, 2007 6951 29.43 42.1 17.7 22.0 42.7 3.11 Madagascar, 2008-09 8389 28.84 16.9 17.9 24.2 19.2 2.88 Malawi, 2010 7554 27.95 19.6 16.9 24.4 15.2 3.06 Mali, 2006 5261 28.77 24.2 19.9 22.9 75.0 3.35 Mozambique, 2011 13619 28.58 34.5 19.0 23.4 31.3 2.90 Namibia, 2006-07 9446 28.31 47.7 16.9 22.9 6.7 1.92 Niger, 2006 5149 28.83 17.6 17.7 21.3 80.7 4.22 Nigeria, 2013 38263 28.81 42.1 18.2 22.8 37.7 3.07 Rwanda, 2010 6948 28.23 15.2 18.1 21.9 15.3 2.38 Sao Tome, 2008-09 2246 29.02 63.3 17.5 24.1 5.9 2.73 Senegal, 2010-11 5768 27.97 49.1 16.3 24.1 57.4 2.73 Sierra Leon, 2008 3487 29.26 34.7 18.4 21.4 67.8 3.08 Swaziland, 2006-07 4853 27.73 26.1 15.8 24.9 8.0 2.28 Tanzania, 2010 10002 28.58 28.4 16.7 23.6 18.9 2.89 Uganda, 2011 2666 27.94 20.6 17.3 25.7 12.2 3.43 Zambia, 2007 7037 28.08 42.1 17.5 25.0 10.4 3.04 Zimbabwe, 2010-11 8806 28.17 37.7 17.1 24.4 2.3 2.11 a Number of women of reproductive age (15–49 years) group who had information on BMI Neupane et al. Results Data from a total of 250651 women from 32 countries in Sub-Saharan Africa were analysed in this study. Table 1 shows the country level characteristics of the study participants. The mean age of the studied women varied from 27.68 years (Comoros) to 29.43 years (Liberia). The overall mean age of the study participants was 28.46 years. Only 10.5 % of women were urban resi- dents in Burundi which is the least among countries studied whereas Congo (Brazzaville) had the highest number of urban residents (66.8 %). On the average, 36.6 % of the women resided in urban areas. According to wealth index, Lesotho had the fewest poorest (14.6 %) and the highest number of poorest women were found in Burundi (20 %). Similarly, Burundi also had the fewer richest (20 %) people compared to Kenya (26.2 %). The largest number of women who had no education (80.7%) were found in Niger compared to only 1.2 % in Lesotho. Similarly, women in Lesotho also had the lowest mean number of children (1.82) compared to 4.42 % in Chad. The pooled prevalence of overweight for the region was 15.9 % (95 % CI, 15.7–16.0) with the lowest prevalence in Madagascar 5.6 % (95 % CI, 5.1–6.1) and the highest in Swaziland 27.7 % (95 % CI, 26.4–29.0) Table 2. Other countries such as Burkina Faso, Burundi, Chad and Ethiopia had also lower prevalence (<10 %) of over- weight. Moreover, Cameroon, Comoros, Gabon, Sao Tome, and Zimbabwe were among those with high prevalence (>20 %) of overweight among women. Simi- larly, the lowest and the highest prevalence of obesity were also found in the same countries; lowest in Madagascar 1.1 % (95 % CI, 0.9–1.4) and the highest in Swaziland 23.0 (95 % CI, 21.8–24.2). Many countries such as Burkina Faso, Burundi, Chad, Congo Repub- lic, Guinean, Malawi, Niger, Rwanda, Tanzania and Zambia have obesity prevalence of less than 5 %. Figures 1, 2 and 3 show clear substantial heterogeneity in prevalence estimates of overweight and obesity be- tween countries of Sub-Saharan Africa. Table 3 presents the odds ratio and 95 % CI for over- weight and obesity among women in urban vs. rural, high education vs. low and vs. poor . The place of residence, education and wealth index strongly predicted overweight and obesity among Sub-Saharan African women. Statistics BMC Public Health (2016) 16:30 Page 4 of 9 Table 2 Prevalence of overweight and obesity with their 95 % confidence intervals (CIs) among women by countries Country Prevalence of overweight (95 % CI) Prevalence of obesity (95 % CI) Benin, 2006 19.4 (18.8–20.0) 6.4 (6.0–6.8) Burkina Faso, 2010 8.0 (7.5–8.6) 2.8 (2.4–3.1) Burundi, 2010 8.3 (7.5–9.1) 2.3 (1.9–2.8) Cameroon, 2011 22.0 (21.0–22.9) 10.2 (9.5–10.9) Chad 8.9 (8.0–9.8) 2.5 (2.0–3.0) Comoros 25.3 (24.1–26.5) 13.1 (12.2–14.1) Congo (Brazzaville) 2011–12 14.6 (13.7–15.5) 5.7 (5.1–6.4) Congo Republic, 2007 10.4 (9.6–11.3) 2.6 (2.1–3.0) Cote d’Ivoire, 2011–12 17.6 (16.5–18.7) 5.9 (5.3–6.6) Ethiopia, 2011 6.2 (5.8–6.6) 1.7 (1.5–2.0) Gabon, 2012 23.6 (22.5–24.7) 14.9 (13.9–15.8) Ghana, 2008 19.8 (18.6–20.9) 8.5 (7.7–9.3) Guinean, 2005 14.5 (13.5–15.6) 4.4 (3.8–5.0) Kenya, 2008 17.9 (17.0–18.7) 7.5 (6.9–8.0) Lesotho, 2009 24.5 (23.1–25.8) 15.8 (14.7–16.9) Liberia, 2007 15.4 (14.6–16.3) 5.6 (5.1–6.2) Madagascar, 2008–09 5.6 (5.1–6.1) 1.1 (0.9-1.4) Malawi, 2010 13.5 (12.7–14.3) 3.2 (2.8–3.6) Mali, 2006 13.5 (12.6–14.4) 5.8 (5.2–6.5) Mozambique, 2011 15.1 (14.5–15.7) 5.3 (4.9–5.6) Namibia, 2006–07 16.3 (15.6–17.0) 11.6 (11.0–12.3) Niger, 2006 15.2 (14.3–16.2) 4.9 (4.3–5.5) Nigeria, 2013 17.8 (17.3–18.1) 7.6 (7.3–7.8) Rwanda, 2010 15.6 (14.7–16.4) 2.4 (2.0–2.7) Sao Tome, 2008–09 23.0 (21.3–24.7) 11.2 (10.0–12.5) Senegal, 2010–11 13.3 (12.4–14.2) 5.1 (4.5–5.7) Sierra Leon, 2008 21.3 (20.0–22.7) 9.9 (8.9–10.9) Swaziland, 2006–07 27.7 (26.4–29.0) 23.0 (21.8–24.2) Tanzania, 2010 15.5 (14.8–16.2) 6.2 (5.7–6.7) Uganda, 2011 15.3 (14.0–16.7) 4.6 (3.8–5.4) Zambia, 2007 15.0 (14.1–15.8) 4.6 (4.1–5.1) Zimbabwe, 2010–11 20.7 (19.9–21.6) 9.9 (9.3–10.6) Pooled prevalence for the region 15.9 (15.7–16.0) 6.7 (6.6–6.8) middle =1). All the analyses were performed using the SPSS statistical software package version 21 and Stata version 13. Table 2 Prevalence of overweight and obesity with their 95 % confidence intervals (CIs) among women by countries Results Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe NER MLI TCD BFA GIN SLE BEN CIV SEN ETH BDI LBR NGA COM CMR SWZ LSO GAB STP ZWE GHA KEN NAM COG TZA RWA UGA ZMB MWI COD MDG 0 20 40 60 80 No education 5 10 15 20 25 30 Overweight Fitted values No Education : Fig. 1 Scatterplot of overweight prevalence of women in each country by no education (weighted by total sample size of the country). Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe Fig. 1 Scatterplot of overweight prevalence of women in each country by no education (weighted by total sample size of the country). Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe Fig. 1 Scatterplot of overweight prevalence of women in each country by no education (weighted by total sample size of the country). Results Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe NER MLI BFA TCD GIN SLE BEN SEN CIV ETH BDI LBR NGA COM MOZ SWE CMR GHA TZA LSO GAB NAM STP ZWE KEN COG UGA ZMB MWI RWA COD MDG 0 20 40 60 80 No education 0 5 10 15 20 25 Obesity Fitted values No education Fig. 2 Scatterplot of obesity prevalence of women in each country by no education (weighted by total sample size of the country). Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe NER MLI BFA TCD GIN SLE BEN SEN CIV ETH BDI LBR NGA COM MOZ SWE CMR GHA TZA LSO GAB NAM STP ZWE KEN COG UGA ZMB MWI RWA COD MDG 0 20 40 60 80 No education 0 5 10 15 20 25 Obesity Fitted values No education Fig. 2 Scatterplot of obesity prevalence of women in each country by no education (weighted by total sample size of the country). Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe Fig. 2 Scatterplot of obesity prevalence of women in each country by no education (weighted by total sample size of the country). Results Signifi- cantly, the largest variation of overweight and obesity was between urban and rural (OR 7.94, 95 % CI, 6.95–9.07), high vs. low education (OR 3.71, 95 % CI, 3.27–4.21) and rich vs. poor (OR 7.86, 95 % CI, 6.66–9.28) in Ethiopia. Likewise, the least variation of overweight and obesity was by urban vs. rural (OR 1.24, 95 % CI, 1.10–1.40) in Gabon, high vs. low education (OR 0.65, 95 % CI, 0.58–0.73) and rich vs. poor (OR 1.31, 95 % CI, 1.17–1.47) in Comoros. Urban residents compared to rural had higher odds of overweight and obesity. Also, in many countries women with high education had higher odds of overweight and obesity except in Benin, Comoros, Sao Tome and Senegal. In almost all studied countries the overweight and obesity was more common among rich women compared to poor with an average odds of 2.45 (95 % CI 2.40–2.50). Neupane et al. BMC Public Health (2016) 16:30 Page 5 of 9 NER MLI TCD BFA GIN SLE BEN CIV SEN ETH BDI LBR NGA COM CMR SWZ LSO GAB STP ZWE GHA KEN NAM COG TZA RWA UGA ZMB MWI COD MDG 0 20 40 60 80 No education 5 10 15 20 25 30 Overweight Fitted values No Education : Fig. 1 Scatterplot of overweight prevalence of women in each country by no education (weighted by total sample size of the country). Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe NER MLI BFA TCD GIN SLE BEN SEN CIV ETH BDI LBR NGA COM MOZ SWE CMR GHA TZA LSO GAB NAM STP ZWE KEN COG UGA ZMB MWI RWA COD MDG 0 20 40 60 80 No education 0 5 10 15 20 25 Obesity Fitted values No education Fig. 2 Scatterplot of obesity prevalence of women in each country by no education (weighted by total sample size of the country). Discussion Twenty-seven of the studied countries had overweight prevalence higher than 10 % and 7 countries had more than 10 % prevalence of obesity. Urban residents, women with high education and rich women as measured by wealth index quintile had higher odds of overweight and obesity in general. These findings put these countries at the risk of high burden of obesity related morbidity and mortality in the future. Twenty-seven of the studied countries had overweight prevalence higher than 10 % and 7 countries had more than 10 % prevalence of obesity. Urban residents, women with high education and rich women as measured by wealth index quintile had higher odds of overweight and obesity in general. These findings put these countries at the risk of high burden of obesity related morbidity and mortality in the future. A number of previous studies have reported the emer- gence of obesity and overweight in developing countries [1, 10, 16, 17]. Although these earlier studies provide ag- gregate picture of the epidemic in developing countries, they lack in-depth analysis of the phenomenon in spe- cific regions, alongside socio-demographic dimensions. Furthermore, with the exception of few studies [1, 10], the previous studies used data collected from different sources using varied methodologies and therefore it was difficult for comparison across the diverse regions within the developing countries. An additional constraint of a number of the previous studies is that the BMI was esti- mated from self-reported weight and height and therefore it may be biased by the social desirability regarding weight in the respective countries and communities within these countries. To build on these previous studies and to ad- dress the aforementioned gaps, the present study used a large sample of the data which were collected using simi- lar methods among women aged 15 to 49 years in 32 Afri- can countries to explore the prevalence of overweight and obesity in the region and to investigate the socio- demographic disparities in the two outcomes across the region. There exist some systematic reviews and meta-analyses [1, 6, 9, 12] therefore we did not perform a systematic re- view of the scientific literature. However we search the re- cent literature published in English, employing no date restrictions. Search terms included BMI, overweight, obes- ity, developing countries, Sub-Saharan Africa etc. We identified 20 relevant primary research articles based on low- and middle-income countries. Results Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO Lesotho, LBR Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe Neupane et al. BMC Public Health (2016) 16:30 Page 6 of 9 NER MLI BFA TCD GIN SLE BEN CIV SEN NGA LBR BDI ETH MOZ COD MDG TZA COM CMR GHA SWZ GAB LSO STP ZWE NAM KEN COG ZMB UGA MWI RWA 0 20 40 60 80 No education 10 20 30 40 50 Overweight and obesity Fitted values No education Fig. 3 Scatterplot of overweight obesity prevalence of women in each country by no education (weighted by total sample size of the country). Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO = Lesotho, LBR = Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe Fig. 3 Scatterplot of overweight obesity prevalence of women in each country by no education (weighted by total sample size of the country). Abbreviation: BEN Benin, BFA Burkina Faso, BDI Burundi, CMR Cameron, TCD Chad, COM Comoros, COG Congo (Brazzaville), COD Congo Republic, CIV Cote d’Ivorie, ETH Ethiopia, GAB Gabon, GHA Ghana, GIN Guinean, KEN Kenya, LSO = Lesotho, LBR = Liberia, MDG Madagascar, MWI Malawi, MLI Mali, MOZ Mozambique, NAM Namibia, NER Niger, NGA Nigeria, RWA Rwanda, STP Sao Tome, SEN Senegal, SLE Sierra Leon, SWZ Swaziland, TZA Tanzania, UGA Uganda, ZMB Zambia, ZWE Zimbabwe Discussion Only very few of them were based on national data. Considerable differences in overweight and obesity across Sub-Saharan Africa were found. This ranged from low overweight and obesity of 6.7 % in Madagascar to 50.8 % in Swaziland. Unlike in previous studies [10, 18] where the characteristics of overweight and obesity have been associated with the level of development in the countries, it seems that the prevalence in the present study does not reflect the developmental level of coun- tries. The emerging prevalence of overweight and Our study found high variation in overweight and obesity problem across Sub-Saharan African countries. Neupane et al. BMC Public Health (2016) 16:30 Page 7 of 9 Page 7 of 9 Table 3 Odds ratios and their 95 % confidence intervals (CIs) for overweight and obesity among women due to place of residence, education and wealth index across countries Table 3 Odds ratios and their 95 % confidence intervals (CIs) for overweight and obesity among women due to place of residence, education and wealth index across countries education and wealth index across countries Country Percentagesa OR (95 % CI) Residence, urban vs. rural Education, high vs. low Wealth index, rich vs. Discussion poor Benin, 2006 27.3 1.66 (1.55–1.79) 0.96 (0.88–1.05) 2.10 (1.95–2.26) Burkina Faso, 2010 11.2 4.27 (3.70–4.92) 2.55 (2.16–3.02) 4.46 (3.80–5.22) Burundi, 2010 8.3 5.75 (4.72–6.99) 3.11 (2.54–3.82) 4.01 (3.24–4.97) Cameroon, 2011 32.6 2.25 (2.04–2.48) 1.66 (1.51–1.82) 2.51 (2.28–2.76) Chad 7.9 5.16 (4.05–6.57) 3.30 (2.48–4.40) NA Comoros 37.7 1.44 (1.29–1.63) 0.65 (0.58–0.73) 1.31 (1.17–1.47) Congo (Brazzaville) 2011–12 26.2 2.55 (2.24–2.92) 1.91 (1.66–2.19) 3.21 (2.78–3.69) Congo Republic, 2007 11.4 2.56 (2.14–3.06) 1.92 (1.62–2.28) 2.74 (2.30–3.28) Cote d’Ivoire, 2011–12 25.7 2.56 (2.14–3.06) 1.14 (0.96–1.35) 2.65 (2.31–3.04) Ethiopia, 2011 5.9 7.94 (6.95–9.07) 3.71 (3.27–4.21) 7.86 (6.66–9.28) Gabon, 2012 44.9 1.24 (1.10–1.40) 1.06 (0.95–1.18) 1.73 (1.53–1.96) Ghana, 2008 30.3 3.01 (2.64–3.43) 1.75 (1.54–1.99) 4.12 (3.61–4.70) Guinean, 2005 19.5 3.02 (2.60–3.50) 1.53 (1.29–1.83) 3.30 (2.82–3.85) Kenya, 2008 25.2 2.49 (2.25–2.76) 2.07 (1.87–2.29) 3.13 (2.82–3.48) Lesotho, 2009 42.6 1.65 (1.43–1.91) 1.32 (1.16–1.50) 2.07 (1.82–2.36) Liberia, 2007 20.2 1.95 (1.73–2.19) 1.61 (1.41–1.83) 2.22 (1.98–2.50) Madagascar, 2008–09 6.3 3.28 (2.76–3.90) 2.91 (2.44–3.46) 4.66 (3.80–5.71) Malawi, 2010 17.7 2.14 (1.84–2.50) 1.61 (1.40–1.86) 2.19 (1.94–2.48) Mali, 2006 17.5 3.34 (2.90–3.84) 1.44 (1.21–1.72) 3.10 (2.68–3.59) Mozambique, 2011 16.7 3.02 (2.77–3.30) 1.84 (1.67–2.01) 4.53 (4.10–5.01) Namibia, 2006–07 28.5 2.14 (1.95–2.34) 1.27 (1.15–1.40) 2.76 (2.52–3.03) Niger, 2006 17.1 3.97 (3.44–4.57) 1.77 (1.46–2.15) 3.18 (2.74–3.69) Nigeria, 2013 25.1 2.11 (2.02–2.21) 1.67 (1.59–1.75) 2.90 (2.76–3.04) Rwanda, 2010 17.5 2.10 (1.82–2.43) 1.68 (1.45–1.95) 2.33 (2.05–2.64) Sao Tome, 2008–09 34.8 1.29 (1.09–1.53) 0.79 (0.66–0.96) 1.43 (1.20–1.70) Senegal, 2010–11 21.3 2.18 (1.90–2.50) 0.77 (0.65–0.93) 1.90 (1.66–2.19) Sierra Leon, 2008 29.9 1.59 (1.38–1.84) 1.29 (1.10–1.53) 1.38 (1.19–1.59) Swaziland, 2006–07 51.0 1.32 (1.16–1.49) 1.13 (1.01–1.27) 1.63 (1.46–1.83) Tanzania, 2010 21.3 2.64 (2.38–2.94) 1.56 (1.41–1.74) 3.62 (3.27–4.01) Uganda, 2011 19.2 3.20 (2.63–3.89) 2.18 (1.79–2.65) 4.68 (3.77–5.82) Zambia, 2007 20.0 2.72 (2.41–3.07) 1.78 (1.58–2.01) 3.15 (2.78–3.57) Zimbabwe, 2010–11 31.9 2.06 (1.88–2.26) 1.16 (1.05–1.28) 2.18 (1.98–2.39) Pooled value for the region 22.5 2.35 (2.31–2.40) 1.81 (1.78–1.85) 2.45 (2.40–2.50) NA data not available aPercentages of overweight and obesity cases obesity in Africa has been largely attributed to the ris- ing level of urbanisation in the region and its attendant global nutrition transition [19]. Urbanization in Africa is increasing rapidly, for example, for the first time, the Ghana Population and Housing Census shows that more Ghanaians are living in urban areas than rural areas [20]. African countries are projected to have 50 % urbanisation by 2020 [21]. Authors’ contributions Authors’ contributions SN and DTD conceptualized the study, developed the analytical strategy. SN conducted the statistical analysis and drafted the methods and result sections. DTD drafted the introduction and discussion sections and contributed to the methods sections. PK contributed in cleaning the data and statistical analysis. SN and DTD together wrote the first draft of the report. All authors did the critical revision of the manuscript and approved the final version. j g [ ] We also found socioeconomic differences in over- weight and obesity by level of education and wealth to the detriment of those with lower socioeconomic status in most countries across Africa similar to those found in other previous studies [12, 30]. Studies have argued that cultural norms that favour fatter body size contribute significantly to the SES differences in overweight and obesity in developing countries, particularly in Africa [31]. Women of higher SES may have the resources and knowledge of the importance of physical activity and healthy diet but they also face several socio- cultural barriers that may prevent them from putting those into use [10, 30]. Received: 24 July 2015 Accepted: 7 January 2016 Received: 24 July 2015 Accepted: 7 January 2016 Received: 24 July 2015 Accepted: 7 January 2016 Received: 24 July 2015 Accepted: 7 January 2016 Discussion The growing urbanization comes along with sedentary lifestyle, "motorised culture", availability of refined foods and physical in- activity related recreations such as cinema houses and video games are all implicated in the rising overweight and obesity in Sub-Saharan Africa. These risk factors for obesity are similar to those found in several studies across the world as accounting for the increasing over- weight and obesity epidemic in developing countries and the stall in the phenomenon in developed countries [22–25]. obesity in Africa has been largely attributed to the ris- ing level of urbanisation in the region and its attendant global nutrition transition [19]. Urbanization in Africa is increasing rapidly, for example, for the first time, the Ghana Population and Housing Census shows that more Ghanaians are living in urban areas than rural areas [20]. African countries are projected to have 50 % urbanisation by 2020 [21]. The growing urbanization comes along with sedentary lifestyle, "motorised Neupane et al. BMC Public Health (2016) 16:30 Page 8 of 9 Page 8 of 9 Our study found differences in overweight and obesity to the disadvantage of those in urban settings similar to a recent study [26]. These rural-urban disparities could be explained by the differences in lifestyle and dietary pattern between urban and rural dwellers in Africa. In rural areas, residents mainly eat fresh food from the farm, and mostly green and fresh fruits and vegetables are available from backyards. Therefore, the dietary pat- tern of rural folks, although unintentional, tends to be healthy compared to that of urban folks. Apart from diet, the lifestyle of urban residents is tilted towards westernization and the blind adoption of the so-called western lifestyle. There are growing numbers of western food outlets and urban residents perceive eating at such joints as a sign of affluence. Transnational fast-food companies in the region are aggressively exploiting this perception [21]. study personnel with similar measurement equipment which is another strengths of our study. There are also some limitations in this study which are worth discuss- ing. Firstly, we did not have data on waist circumference which would have allowed examination of trends in ab- dominal obesity. Additionally, no data were available on behavioural or other factors that could have explained the prevalence of overweight across the countries. Acknowledgements The authors are thankful to all the participants who participated in this study and all the members who took part in the data collection process. We thank Measure DHS for granting us the permission to use the data for the study. There was no funding source for this study. SN and DTD had full access to the DHS data and final responsibility to submit the report. Conclusions Women of higher SES may have the resources and knowledge of the importance of physical activity and healthy diet but they also face several socio- cultural barriers that may prevent them from putting those into use [10, 30]. The nature of occupation prevailing in rural and urban setting in Africa also contributes to the differences in obesity and overweight between the two settings. In most rural settings the main form of occupation is still non-mechanized agriculture and physical activity based vocations such as fishing, small scale mining and lum- bering. An occupation related physical activity is a known protective factor against obesity [27]. In the urban area, however, there is increasing shift from high physical activity based occupations such as construction and working in factories to sedentary and service based occupation. On the other hand, the growing urbanisa- tion coupled with climate change and other socioeco- nomic transition have raised food prices in most developing countries and it is possible that rural women are just unable to afford enough food to feed on [28, 29]. 1. Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2014;384:766–81. Competing interest Competing interest The authors declare that they have no competing interests. Conclusions The prevalence of overweight and obesity varied highly between the countries and wealth index (rich vs. poor) was found to be the strongest predictor in most of the countries. Owing to the well-known risk of overweight and obesity, the emerging epidemic in Africa needs to be addressed early enough to prevent its related morbidity and mortality. A number of interventions can be imple- mented. The fast growing cities should include in their development plans the creation of safe pedestrian walk- ways and bicycle lanes to enable pedestrians and cyclist feel safe. This may encourage walk and cycling in cities. There should be national and regional campaigns to en- courage consumptions of healthy foods and physical ac- tivity. This should include the provision of healthy foods at school and nutritional education in schools and com- munities. Interventions that will aim at addressing the socio-cultural barriers to maintaining healthy body size can also contribute to fighting the overweight and obesity epidemic in Africa. p p [ ] The nature of occupation prevailing in rural and urban setting in Africa also contributes to the differences in obesity and overweight between the two settings. In most rural settings the main form of occupation is still non-mechanized agriculture and physical activity based vocations such as fishing, small scale mining and lum- bering. An occupation related physical activity is a known protective factor against obesity [27]. In the urban area, however, there is increasing shift from high physical activity based occupations such as construction and working in factories to sedentary and service based occupation. On the other hand, the growing urbanisa- tion coupled with climate change and other socioeco- nomic transition have raised food prices in most developing countries and it is possible that rural women are just unable to afford enough food to feed on [28, 29]. We also found socioeconomic differences in over- weight and obesity by level of education and wealth to the detriment of those with lower socioeconomic status in most countries across Africa similar to those found in other previous studies [12, 30]. Studies have argued that cultural norms that favour fatter body size contribute significantly to the SES differences in overweight and obesity in developing countries, particularly in Africa [31]. Author details 1 1School of Health Sciences, FI – 33014 University of Tampere, Tampere, Finland. 2Department of Population and Health, University of Cape Coast, Private Mail Bag, University Post Office, Cape Coast, Ghana. Reporting national-level rates of overweight and obes- ity using nationally representative data is a major strength of our study, therefore the findings are generalizable to the respective countries. In DHS sur- veys, each country followed standardised procedure to collect the data using validated questionnaire, therefore the data are comparable between the countries. The data on the body weight and height was measured by trained Neupane et al. BMC Public Health (2016) 16:30 Neupane et al. BMC Public Health (2016) 16:30 Page 9 of 9 2. Stevens GA, Singh GM, Lu Y, Danaei G, Lin JK, Finucane MM, et al. National, regional, and global trends in adult overweight and obesity prevalences. Popul Health Metr. 2012;10:22. 3. Lim SS, Vos T, Flaxman AD, Danaei G, Shibuya K, Adair-Rohani H, et al. 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https://openalex.org/W2963847546	https://ora.ox.ac.uk/objects/uuid:f07586d4-47c0-40d1-80c6-e6d352a54f2a/download_file?safe_filename=BurnetalVoR2019.pdf&file_format=application%2Fpdf&type_of_work=Journal+article	English	null	Cr2Te3 Thin Films for Integration in Magnetic Topological Insulator Heterostructures	Scientific reports	2,019	cc-by	7,476	Cr2Te3 Thin Films for Integration in Magnetic Topological Insulator Heterostructures D. M. Burn 1, L. B. Duffy2,3, R. Fujita1,2, S. L. Zhang2, A. I. Figueroa1,4, J. Herrero-Martin5 G. van der Laan 1 &T. Hesjedal 2 Received: 28 March 2019 Accepted: 12 July 2019 Published: xx xx xxxx Chromium telluride compounds are promising ferromagnets for proximity coupling to magnetic topological insulators (MTIs) of the Cr-doped (Bi,Sb)2(Se,Te)3 class of materials as they share the same elements, thus simplifying thin film growth, as well as due to their compatible crystal structure. Recently, it has been demonstrated that high quality (001)-oriented Cr2Te3 thin films with perpendicular magnetic anisotropy can be grown on c-plane sapphire substrate. Here, we present a magnetic and soft x-ray absorption spectroscopy study of the chemical and magnetic properties of Cr2Te3 thin films. X-ray magnetic circular dichroism (XMCD) measured at the Cr L2,3 edges gives information about the local electronic and magnetic structure of the Cr ions. We further demonstrate the overgrowth of Cr2Te3 (001) thin films by high-quality Cr-doped Sb2Te3 films. The magnetic properties of the layers have been characterized and our results provide a starting point for refining the physical models of the complex magnetic ordering in Cr2Te3 thin films, and their integration into advanced MTI heterostructures for quantum device applications. Ferromagnetic materials, which are compatible with semiconductors in terms of their crystal structure and dep- osition conditions, have been intensely studied for applications in spintronics1. Apart from traditional semicon- ductor spintronics, the combination of magnetic materials and topological insulators (TIs) has been a promising route for observing new quantum effects at more easily accessible temperatures2,3. Transition metal doped mag- netic TIs (MTIs) grown by molecular beam epitaxy (MBE), such as Cr-doped (Bi,Sb)2Te3, have been pivotal for observing the quantum anomalous Hall effect4.h gf The lattice-matched antiferromagnet CrSb and ferromagnetic Cr2Ge2Te6 have been shown to be ideal for the combination with Cr-doped (Bi,Sb)2Te3 MTI layers in heterostructures and superlattices, allowing for the engi- neering of their electronic and magnetic properties3,5. A key advantage of CrSb is the fact that no additional elements are needed for their MBE growth. On the other hand, chromium telluride compounds are promis- ing ferromagnets for the integration with Cr-doped (Bi,Sb)2Te3 as they also share the same elements and due to their compatible crystal structure. Recently, the epitaxial growth of high quality (001)-oriented Cr2Te3 thin films with perpendicular magnetic anisotropy has been demonstrated on c-plane sapphire and Si(111) substrates using MBE6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Cr2Te3 Thin Films for Integration in Magnetic Topological Insulator Heterostructures D. M. Burn 1, L. B. Duffy2,3, R. Fujita1,2, S. L. Zhang2, A. I. Figueroa1,4, J. Herrero-Martin5 G. van der Laan 1 &T. Hesjedal 2 1: CrI atoms have no direct neighbors in the ab plane, but are accompanied by CrIII atoms in the neighboring Cr layers above and below. CrII atoms, on the other hand, are the neighbor of CrIII in the filled Cr layers, however, not accompanied by any Cr atoms along the c-axis in the adjacent Cr layers. While the Cr sites at the layers con- taining CrII and CrIII are completely filled, the other layers are only partially occupied by CrI atoms with vacancies. From the crystal structure in Fig. 1, the exchange interaction between CrII and CrIII is expected to be large as they are close together. On the other hand, the interaction of CrI with the more distant CrII and CrIII ions is expected to be relatively weak because its nearest neighbor is Te.i Fig. 1: CrI atoms have no direct neighbors in the ab plane, but are accompanied by CrIII atoms in the neighboring Cr layers above and below. CrII atoms, on the other hand, are the neighbor of CrIII in the filled Cr layers, however, not accompanied by any Cr atoms along the c-axis in the adjacent Cr layers. While the Cr sites at the layers con- taining CrII and CrIII are completely filled, the other layers are only partially occupied by CrI atoms with vacancies. From the crystal structure in Fig. 1, the exchange interaction between CrII and CrIII is expected to be large as they are close together. On the other hand, the interaction of CrI with the more distant CrII and CrIII ions is expected to be relatively weak because its nearest neighbor is Te.i y g In the form of thin films, the most relevant compounds are the half-metal CrTe (δ = 0, zincblende), Cr3Te4 (δ = 0.25, monoclinic), and Cr2Te3 (δ = 0.33, trigonal), which are all metallic ferromagnets with ordering temper- atures ranging from 100–340K9–12. Cr2Te3 is of particular interest in the context of MBE-grown thin films with a reported TC ≈ 183K. Neutron diffraction showed that the magnetic moments of Cr in the fully occupied layers are ferromagnetically aligned and have an average value of 2.6 μB/atom, while the Cr atoms in the partially filled lay- ers are assumed to have only a small moment10. The interlayer coupling along the c-axis is weak. Cr2Te3 Thin Films for Integration in Magnetic Topological Insulator Heterostructures D. M. Burn 1, L. B. Duffy2,3, R. Fujita1,2, S. L. Zhang2, A. I. Figueroa1,4, J. Herrero-Martin5 G. van der Laan 1 &T. Hesjedal 2 For Cr2Te3, the electronic band-structure calculations show that Cr 3d–Te 5p covalency and Cr d 3 z 2–Cr d 3 z 2 overlap along the c-axis are the most important interactions. The magnetic polarization of Te is antiparallel to the Cr moment with calculated average values of μCr = 3.30 μB/Cr and μTe = −0.18 μB/Te, respectively9. g μCr μB μTe μB p y However, the relatively complicated magnetic structure of Cr2Te3 is not fully understood yet, in parts owing to the various ways the spins of the three distinct Cr sites can couple13. In fact, from electron spin resonance meas- urements of bulk crystals, it was claimed that the true Curie temperature is 335K, and that the generally reported TC of 198K is only another type of magnetic transition point14.t C y yp g p Here, we present a magnetic and soft x-ray absorption spectroscopy study of the chemical and magnetic prop- erties of Cr2Te3 thin films. We further demonstrate the successful growth of Cr2Te3/Cr:Sb2Te3 heterostructures, opening the door for advanced heterostructures combining proximity coupling to ferromagnetic layers. Cr2Te3 Thin Films for Integration in Magnetic Topological Insulator Heterostructures D. M. Burn 1, L. B. Duffy2,3, R. Fujita1,2, S. L. Zhang2, A. I. Figueroa1,4, J. Herrero-Martin5 G. van der Laan 1 &T. Hesjedal 2 A systematic study of the structural and magnetic properties of Cr2Te3 films on CdTe(001) sub- strates showed that the epitaxial relationship of the film with the substrate depends on the Cr:Te flux ratio during growth7. Cr2Te3 has also been grown in the form of nanorods using a high-temperature organic-solution-phase method8.hi The chromium tellurides with metal-deficient NiAs-type crystal structures, Cr1−δTe, have in common a (dis- torted) hexagonal close packing of Te atoms, with Cr atoms in octahedral interstices. The crystal structure of Cr2Te3 (see Fig. 1) can be described in the space group P c 31 or D d 3 2 (#163 in the International Tables of Crystallography), with the atoms on the special positions9. The lattice parameters are a = 6.814 Å and c = 12.073 Å10. The unit cell appears layered and is characterized by an alternating sequence of Cr and Te layers, while Cr vacancies occur in every second metal layer. Consequently, there are distinct Cr positions as shown in 1Magnetic Spectroscopy Group, Diamond Light Source, Didcot, OX11 0DE, United Kingdom. 2Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford, OX1 3PU, United Kingdom. 3ISIS, Rutherford Appleton Laboratory, Science and Technology Facilities Council, Oxon, OX11 0QX, United Kingdom. 4Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, Campus UAB, Barcelona, 08193, Spain. 5CELLS-Divisió Experiments, ALBA Synchrotron Light Source, E-08290 Cerdanyola del Vallès, Barcelona, Catalonia, Spain. Correspondence and requests for materials should be addressed to T.H. (email: Thorsten.Hesjedal@physics.ox.ac.uk) Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 1 www.nature.com/scientificreports/ Figure 1. Crystal structure of Cr2Te3. The unit cell is characterized by an alternating sequence of Cr and Te layers, with Cr vacancies occurring in every other Cr layer. The distinct Cr atoms, Cr I in the vacancy layer, as well as Cr II and Cr III in the fully occupied layer, are labeled. Note that the roman numbers in the subscripts refer to the different sites and not to the valencies of Cr. Figure 1. Crystal structure of Cr2Te3. The unit cell is characterized by an alternating sequence of Cr and Te layers, with Cr vacancies occurring in every other Cr layer. The distinct Cr atoms, Cr I in the vacancy layer, as well as Cr II and Cr III in the fully occupied layer, are labeled. Note that the roman numbers in the subscripts refer to the different sites and not to the valencies of Cr. Fig. Results and Discussionh Structural properties. The structural quality of the samples was characterized using XRD. The results for both single-layer Cr2Te3 and Cr2Te3/Cr:Sb2Te3 are shown in Fig. 2. The spectrum for single-layer Cr2Te3 (Fig. 2a) is characterized by film and sapphire substrate peaks, whereas the Cr2Te3/Cr:Sb2Te3 spectrum (Fig. 2b) addition- ally shows Sb2Te3 related peaks. These are shifted to higher angles with respect to undoped Sb2Te3 as Cr doping reduces the lattice spacing15–17. Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 2 www.nature.com/scientificreports/ Figure 2. X-ray diffraction characterization. (a), Cr2Te3, and (b), Cr:Sb2Te3/Cr2Te3 thin film samples on c- plane sapphire. The peaks from the Al2O3 substrate, and the Cr2Te3 and Cr:Sb2Te3 thin films, have been indexed (shown in red, green, and cyan, respectively). The small peak at 2θ = 38° in (b) is originating from the Al sample holder. On the right-hand side, 1 × 1 μm2 AFM scans show the morphology of the Cr2Te3 and Cr:Sb2Te3 surfaces, respectively, with the latter showing the characteristic triangular strictures. Figure 2. X-ray diffraction characterization. (a), Cr2Te3, and (b), Cr:Sb2Te3/Cr2Te3 thin film samples on c- plane sapphire. The peaks from the Al2O3 substrate, and the Cr2Te3 and Cr:Sb2Te3 thin films, have been indexed (shown in red, green, and cyan, respectively). The small peak at 2θ = 38° in (b) is originating from the Al sample holder. On the right-hand side, 1 × 1 μm2 AFM scans show the morphology of the Cr2Te3 and Cr:Sb2Te3 surfaces, respectively, with the latter showing the characteristic triangular strictures. Figure 2. X-ray diffraction characterization. (a), Cr2Te3, and (b), Cr:Sb2Te3/Cr2Te3 thin film samples on c- plane sapphire. The peaks from the Al2O3 substrate, and the Cr2Te3 and Cr:Sb2Te3 thin films, have been indexed (shown in red, green, and cyan, respectively). The small peak at 2θ = 38° in (b) is originating from the Al sample holder. On the right-hand side, 1 × 1 μm2 AFM scans show the morphology of the Cr2Te3 and Cr:Sb2Te3 surfaces, respectively, with the latter showing the characteristic triangular strictures. Figure 3. Magnetization as a function of applied out-of-plane field. (a), Cr2Te3, and (b), Cr2Te3/Cr:Sb2Te3. The measurements were performed at various temperatures as indicated. In (a) the average moment per Cr is f Figure 3. Magnetization as a function of applied out-of-plane field. (a), Cr2Te3, and (b), Cr2Te3/Cr:Sb2Te3. The measurements were performed at various temperatures as indicated. Results and Discussionh In (a) the average moment per Cr is shown, whilst in (b) the units are arbitrary as both layers contain Cr in different environments. Figure 3. Magnetization as a function of applied out-of-plane field. (a), Cr2Te3, and (b), Cr2Te3/Cr:Sb2Te3. The measurements were performed at various temperatures as indicated. In (a) the average moment per Cr is shown, whilst in (b) the units are arbitrary as both layers contain Cr in different environments. Magnetic behavior. The magnetic characterization of the films was performed through SQUID measure- ments as a function of both applied magnetic field and temperature. Figure 3a,b show hysteresis loops of the magnetization as a function of applied magnetic field for Cr2Te3 and Cr2Te3/Cr:Sb2Te3, respectively. The Cr2Te3 films show square hysteresis loops with the magnetization reversing to its saturated state dur- ing a sharp transition over a narrow field range (Fig. 3a). At 3K, the magnetization is saturated at 3.6 μB/Cr and a coercive field of 600 mT is required to reverse the magnetization At the higher temperature of 140K, the Magnetic behavior. The magnetic characterization of the films was performed through SQUID measure- ments as a function of both applied magnetic field and temperature. Figure 3a,b show hysteresis loops of the magnetization as a function of applied magnetic field for Cr2Te3 and Cr2Te3/Cr:Sb2Te3, respectively.hi Magnetic behavior. The magnetic characterization of the films was performed through SQUID measure- ments as a function of both applied magnetic field and temperature. Figure 3a,b show hysteresis loops of the magnetization as a function of applied magnetic field for Cr2Te3 and Cr2Te3/Cr:Sb2Te3, respectively. The Cr2Te3 films show square hysteresis loops with the magnetization reversing to its saturated state dur- i h t iti fi ld (Fi 3 ) At 3K th ti ti i t t d t 3 6 /C ments as a function of both applied magnetic field and temperature. Figure 3a,b show hysteresis loops of the magnetization as a function of applied magnetic field for Cr2Te3 and Cr2Te3/Cr:Sb2Te3, respectively. The Cr2Te3 films show square hysteresis loops with the magnetization reversing to its saturated state dur- ing a sharp transition over a narrow field range (Fig. 3a). Results and Discussionh This is attributed to a magnetic component in the Cr:Sb2Te3 film with a lower Curie temperature. Further SQUID magnetometry measurements were carried out to investigate the change in magnetic behavior at different temperatures. Figure 4a,b show both the magnetization and its derivative with respect to temperature as a function of increasing temperature after field-cooling in a field of 6T, respectively. At low temperatures after field cooling, Cr2Te3 has a large moment which decreases with increasing temperature. The Curie temperature, as defined as the peak in the derivative, is ~16K. The Cr2Te3/Cr:Sb2Te3 sample shows a similar variation of the magnetization with temperature, with a similar TC of ~15K. However, there are additional subtle features, such as kinks at 70K and 160K (see Fig. 4).hi g The variation in magnetization as a function of temperature from the field cooled measurements is consistent with the saturation magnetization found in Fig. 3. Furthermore, the feature at 70K indicates the point at which the two-step magnetization reversal occurs in Fig. 3b. X-ray magnetic circular dichroism. Before showing the results from a clean sample, we first present the results from a surface-oxidized sample, in order to show the effect of oxidation on the spectra. Figure 5a shows the Cr L2,3 edge of a ~100-nm-thick Cr2Te3 film measured with surface-sensitive TEY detection. Comparison with a Cr2O3 reference sample (Fig. 5b) clearly indicates that the Cr2Te3 film surface is oxidized (due to aging). Oxidation leads to an additional Cr3+ peak. The photon energy of this peak is 1.5 eV higher than the photon energy of the proper Cr peak.h Figure 5c shows the spectra across the Cr L2,3 edges in LY mode. These spectra represent an average over the entire thin film heterostructure. The background variations in the LY arise from the x-ray absorption near edge structure (XANES) O K edge above ~543 eV from the substrate sapphire (Al2O3)18. Furthermore, the Cr L2,3 edges coincide with Te M4,5 edges, resulting in a sloping background of the XAS. The integrated intensity ratio of Te M4,5/Cr L2,3 XAS is estimated to be ~7.5%19, meaning that the Te contribution is small.h Figure 5d shows the XMCD simultaneously detected with TEY and LY. The spectra have been normalized to the maximum of the Cr L3 XMCD signal. Results and Discussionh At 3K, the magnetization is saturated at 3.6 μB/Cr and a coercive field of 600 mT is required to reverse the magnetization At the higher temperature of 140K, the g pp gi 2 3 2 3 2 3 p y The Cr2Te3 films show square hysteresis loops with the magnetization reversing to its saturated state dur- ng a sharp transition over a narrow field range (Fig. 3a). At 3K, the magnetization is saturated at 3.6 μB/Cr nd a coercive field of 600 mT is required to reverse the magnetization At the higher temperature of 140K, the Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 3 www.nature.com/scientificreports/ Figure 4. Temperature dependence of the magnetization. (a), M(T), and (b), its derivative, measured with SQUID for Cr2Te3 and Cr2Te3/Cr:Sb2Te3 samples. The measurements were performed in a field of 20 mT with increasing temperature after preparation in a 6T field-cooled state. The moment for single-layer Cr2Te3 is shown in μB/Cr, while for the bilayer sample, the units are arbitrary due to the mixed Cr environments. Kinks at ~70K and 160K in the Cr2Te3/Cr:Sb2Te3 derivative plot are indicated by arrows. Figure 4. Temperature dependence of the magnetization. (a), M(T), and (b), its derivative, measured with SQUID for Cr2Te3 and Cr2Te3/Cr:Sb2Te3 samples. The measurements were performed in a field of 20 mT with increasing temperature after preparation in a 6T field-cooled state. The moment for single-layer Cr2Te3 is shown in μB/Cr, while for the bilayer sample, the units are arbitrary due to the mixed Cr environments. Kinks at ~70K and 160K in the Cr2Te3/Cr:Sb2Te3 derivative plot are indicated by arrows. oercivity reduces to 180 mT and is accompanied by a decrease in the saturation magnetization as the temperature pproaches TC. Th b b l h l l h dd l f d The Cr2Te3/Cr:Sb2Te3 bilayers show a more complex magnetization reversal with additional features associated with the MTI layer). At 140K, the magnetization reversals in Fig. 3a,b look similar with a sharp reversal at a low coercive field of 180 mT. This suggests the magnetic response is dominated by the Cr2Te3 layer at this temperature. ih gg g y y When the temperature is reduced, the magnetization reversal associated with the Cr2Te3 layer occurs at an increased coercive field, consistent with the measurements on single-layer Cr2Te3. However, a second magnetiza- tion reversal process arises with a low coercivity. Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 Results and Discussionh As expected, the overall XMCD intensity of the L3 peak is negative and that of the L2 peak positive, resulting from the spin-orbit interaction of the 2p core levels20. Although anti- ferromagnetic Cr2O3 has zero XMCD signal, the surface-sensitive TEY of Cr2Te3 XMCD shows a more detailed structure at the high end of the Cr L3 edge (577–580 eV). These differences in the XMCD are small in relation to those in the XAS. This means that the oxidized surface hardly contributes to the magnetic signal, and it is likely to be mainly antiferromagnetic as in the case of Cr2O3.i y g 2 3 Figure 6 shows the XAS and XMCD of a non-oxidized Cr2Te3 thin film. In the XMCD a small negative Te M5 signal is expected at ~572.7 eV as we earlier reported for Cr:Sb2Te3 21. In Fig. 6, this is however untraceable in 4 www.nature.com/scientificreports/ Figure 5. Experimental Cr L2,3 XAS and XMCD of a surface-oxidized, ~100-nm-thick film of Cr2Te3. Measurement carried out in a 6T field at 3K with 30° grazing incidence angle of the x-rays. (a), The total- electron yield (TEY) spectra for μ+ and μ− (field parallel and antiparallel to incident photon helicity, respectively). (b), For comparison, the Cr2O3 XAS, which quite well matches the second peak in the TEY of Cr2Te3, which demonstrates that the film is oxidized at the surface. (c), The luminescence yield (LY) spectra for μ+ and μ−, which show no oxidation. (d), The XMCD measured in TEY and LY. Spectra are normalized to the size of the maximum XMCD signal. Figure 5. Experimental Cr L2,3 XAS and XMCD of a surface-oxidized, ~100-nm-thick film of Cr2Te3. Measurement carried out in a 6T field at 3K with 30° grazing incidence angle of the x-rays. (a), The total- electron yield (TEY) spectra for μ+ and μ− (field parallel and antiparallel to incident photon helicity, respectively). (b), For comparison, the Cr2O3 XAS, which quite well matches the second peak in the TEY of Cr2Te3, which demonstrates that the film is oxidized at the surface. (c), The luminescence yield (LY) spectra for μ+ and μ−, which show no oxidation. (d), The XMCD measured in TEY and LY. Spectra are normalized to the size of the maximum XMCD signal. the coinciding Cr multiplet structure. Results and Discussionh The azimuthal quantum numbers of the orbitals in these electric-dipole transitions are 3d → 5p for Te and 2p → 3d for Cr, i.e., opposite. Consequently the Te and Cr moments are aligned antiparallel17,20 as expected from band structure calculations9. For comparison, theoretical calculations for the Cr L2,3 XAS and XMCD are also shown in Fig. 6, and are discussed below. Multiplet calculations. We employed atomic multiplet theory to calculate the electric-dipole transitions from 3dn to 2p53dn+1 22,23. For this purpose, the wave functions of the configurations of the initial and the final state are obtained in intermediate coupling using Cowan’s atomic Hartree-Fock (HF) code with relativistic cor- rections24,25. The 2p-3d and 3d-3d Coulomb and exchange interactions are included in the atomic electrostatic interactions. To account for the intra-atomic screening, their atomic HF value is reduced by 30%22. By mixing the 3dn with + d L 3 n 1 configurations with a transfer integral, V, hybridization effects are included. L represents a hole in the overlapping Te 5p orbitals.h pp g p The local ground state of Cr is taken as a coherent mixture of ψ(3d3) and ψ d L (3 ) 4 states. Similar to the calcu- lation by Yaji et al.19, we obtain a mixed ground state of 54% Cr3+ d3 and 46% Cr2+ d L 4 using the parameters Δ ≡ − E d L E d (3 ) (3 ) i 4 3 = 0 eV. As the presence of a core hole reduces the energy of the 3d states, Δf defined as − E p d L E p d (2 3 ) (2 3 ) 5 4 5 3 is −1 eV, the octahedral crystal field of 10Dq is 1.5 eV, and the mixing transfer integral V is 1.5 eV. To account for intrinsic lifetime broadening and instrumental broadening, the calculated Cr L3 (L2) line spectra are broadened by a Lorentzian and a Gaussian function, respectively. The Lorentzian has a half-width at half-maximum of Γ = 0.3 eV (0.4 eV) and the Gaussian a standard deviation of σ = 0.15 eV. The calculated XAS and XMCD are shown in Fig. 6.h g The obtained covalent character of Cr2Te3 can be ascribed to the hybridization between the Cr d(eg) and Te 5p bands, which are located just above and below the Fermi level, respectively. Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 5 www.nature.com/scientificreports/ Figure 6. Results and Discussionh Cr L2,3 spectra of a clean, ~100-nm-thick film of Cr2Te3. Measurement carried out in a 6T field at 3K with 30° grazing incidence angle of the x-rays. (a), The XAS (average of μ+ and μ−) obtained from multiplet calculations and as measured in LY. (b), The XMCD (μ+ − μ−) as calculated and as measured in LY. The red lines at 572.7 eV mark the location of the small Te M5 peak expected in the LY data, which is not clearly evident here. Spectra are normalized to the size of the maximum XMCD signal. Figure 6. Cr L2,3 spectra of a clean, ~100-nm-thick film of Cr2Te3. Measurement carried out in a 6T field at 3K with 30° grazing incidence angle of the x-rays. (a), The XAS (average of μ+ and μ−) obtained from multiplet calculations and as measured in LY. (b), The XMCD (μ+ − μ−) as calculated and as measured in LY. The red lines at 572.7 eV mark the location of the small Te M5 peak expected in the LY data, which is not clearly evident here. Spectra are normalized to the size of the maximum XMCD signal. The Cr state for the covalent compound Cr2Te3 (54% d3 and 46% d4 character in the wave function) resembles that of (V,Cr)xSb2−xTe3 (ref.26), but strongly differs from that of CrxSb2−xTe3 (ref.17) and CrxBi2−xSe3 (refs15,27). In the latter, Sb and Bi are substitutionally replaced by Cr. In this case, Cr is nominally divalent and has 30% d3 and 70% d4 character. The Cr state for the covalent compound Cr2Te3 (54% d3 and 46% d4 character in the wave function) resembles that of (V,Cr)xSb2−xTe3 (ref.26), but strongly differs from that of CrxSb2−xTe3 (ref.17) and CrxBi2−xSe3 (refs15,27). In the latter, Sb and Bi are substitutionally replaced by Cr. In this case, Cr is nominally divalent and has 30% d3 and 70% d4 character. Using the sum rules, the orbital and spin magnetic moments are obtained from the integrated L2,3 XAS and XMCD intensities28,29. For obtaining the spin moment from the sum rules, a correction factor has to be taken into account to include the jj mixing between the 2p3/2 and 2p1/2 manifolds15. This correction can result in a substantial error bar for Cr. Results and Discussionh Therefore, we determined instead the spin and orbital moments from the calculated ground state, which gives μspin ≈ 3.5 μB/Cr (i.e., high spin moment) and μorb ≈ −0.1 μB/Cr (i.e., opposite sign). XMCD magnetization loops. The XMCD furthermore provides a means to explore the element-specific hysteresis loops within our multilayer samples20. Figure 7 shows the Cr magnetization as a function of applied field at 3K with the photon energy tuned to the maximum of the XMCD at the Cr L3 edge. Both the loop shape and coercivity of the Cr XMCD hysteresis loop are consistent with the SQUID measurements of the bulk sample shown in Fig. 3a. This shows the magnetization of the Cr dominates the magnetization in the bulk sample.h h We note also that the XMCD hysteresis loop is present on a curved background. This is likely to result from the XMCD measurements being performed at an angle of 30°. Therefore, there is a component of the in-plane loop which also includes a continual rotation of the moments into the field direction at higher fields.i i gi Measurements of the Cr magnetization as a function of temperature were also performed on the Cr2Te3 film and are shown, along with the derivative dM/dT, in Fig. 8a,b. Here, the LY and TEY signals are collected simul- taneously. The solid lines present a polynomial fit to the data. The behavior of the magnetization in Cr shows the same trend as observed in the SQUID measurements (Fig. 4) except for the TC measured by x-rays appears slightly higher. This is likely to be due to temperature lag in the instrumentation. The TC, as obtained from dM/dT, for the TEY signal is ~8K lower than for the LY signal, possibly pointing towards an enhanced magnetic ordering temperature at the surface compared to the bulk. Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 6 www.nature.com/scientificreports/ Figure 7. Asymmetry in the luminescence yield of Cr2Te3 measured at the Cr L3 edge. The data represent the element-specific magnetization for Cr as a function of the applied magnetic field component out-of-plane of the sample. Measurements are performed at 3K with an x-ray incidence angle of 30°. Figure 7. Asymmetry in the luminescence yield of Cr2Te3 measured at the Cr L3 edge. The data represent the element-specific magnetization for Cr as a function of the applied magnetic field component out-of-plane of the sample. Results and Discussionh Measurements are performed at 3K with an x-ray incidence angle of 30°. Figure 8. Element-specific magnetization of Cr2Te3 measured at the Cr L3 edge. (a), Luminescence yield (LY) and total-electron yield (TEY) as a function of temperature, and (b), their derivatives. The sample was initially field-cooled in 6T before measuring from 10 to 250K with a constant applied field of 20 mT with an x-ray incidence angle of 30°. Figure 8. Element-specific magnetization of Cr2Te3 measured at the Cr L3 edge. (a), Luminescence yield (LY) and total-electron yield (TEY) as a function of temperature, and (b), their derivatives. The sample was initially field-cooled in 6T before measuring from 10 to 250K with a constant applied field of 20 mT with an x-ray incidence angle of 30°. Methods l The XMCD was obtained by taking the difference between XAS spectra with the photon helicity vector par- allel (μ+) and antiparallel (μ−) to the applied magnetic field, respectively. The degree of circular polarization is 100% and for the sign convention, see ref.20. XMCD measurements were performed after first field-cooling to base temperature (~3K) in a 6T field. The sample was mounted at a grazing incidence angle of 30° with the applied magnetic field of ±6T along the x-ray beam direction. The XMCD results are obtained from an average over four μ+ and four μ− scans of the photon energy across the absorption edges.ii X-ray absorption spectroscopy and x-ray magnetic circular dichroism. X-ray absorption spec- troscopy (XAS) and x-ray magnetic circular dichroism (XMCD) measurements were performed on beamline 29 (BOREAS) at the ALBA synchrotron in Barcelona, Spain31. XAS was simultaneously measured at the Cr L2,3 edge in total-electron-yield (TEY) mode and luminescence-yield (LY) mode (see Fig. 9 for a schematic of the measurement modes). The Cr L2,3 photon energy region coincides with that of the Te M4,5 edges. TEY provides surface-sensitive measurements with a probing depth of 3–5 nm20. On the other hand, luminescence-yield (LY) mode probes the entire thin film sample. In the latter mode, the transmitted x-rays that are not absorbed in the sample stack give rise to x-ray excited optical luminescence in the sapphire substrate. The emitted optical photons exit through a hole in the back of the sample holder and are detected by a photodiode21. The XAS spectra were calculated by taking the negative logarithm of the LY intensity after normalizing it by the incident beam intensity.hf y g g g yt g y y The XMCD was obtained by taking the difference between XAS spectra with the photon helicity vector par- allel (μ+) and antiparallel (μ−) to the applied magnetic field, respectively. The degree of circular polarization is 100% and for the sign convention, see ref.20. XMCD measurements were performed after first field-cooling to base temperature (~3K) in a 6T field. The sample was mounted at a grazing incidence angle of 30° with the applied magnetic field of ±6T along the x-ray beam direction. The XMCD results are obtained from an average over four μ+ and four μ− scans of the photon energy across the absorption edges.ii Element-specific measurements of the magnetization vs. field (M-H) and magnetization vs. Conclusions We have studied the structural and magnetic properties of MBE-grown Cr2Te3 films on c-plane sapphire, in com- parison with Cr2Te3/Cr:Sb2Te3 bilayer samples. The field and temperature dependence of the magnetization has been explored through SQUID magnetometry where a ferromagnetic response in Cr2Te3 arises below TC = 150K with a coercivity increasing with decreasing temperature. In bilayer samples with Cr:Sb2Te3, a second transition temperature at ~7K shows an onset of a two-step magnetization reversal process with the reversal of the MTI layer at a lower field. Using soft x-ray absorption spectroscopy we determined the chemical and magnetic properties of Cr2Te3 thin films. The field and temperature dependent Cr XMCD matches that of the bulk magnetometry meas- urements and confirms the full moment originates on the Cr sites. Comparison of the Cr L2,3 spectral shapes with multiplet calculations gives a hybridized Cr state of 54% Cr3+ 3d3 and 46% Cr2+ 3d4 character in an octahedral crystal-field symmetry with spin and orbital moments of μspin ≈ 3.5 μB/Cr and μorb ≈ −0.1 μB/Cr. Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 7 www.nature.com/scientificreports/ Figure 9. X-ray absorption spectroscopy and x-ray magnetic circular dichroism measurements. Schematic setup for simultaneous detection of total-electron yield (TEY) and luminescence yield (LY) in the vacuum chamber of the magnet. In TEY, the drain current (compensation current) is a measure for the amount of emitted electron due to x-ray absorption. TEY is surface-sensitive. In LY, which probes the entire thin-film sample, the transmitted x-rays that are not absorbed in the sample stack give rise to x-ray excited optical luminescence in the sapphire substrate. The emitted optical photons exit through a hole in the back of the sample holder and are detected by a photodiode. Figure 9. X-ray absorption spectroscopy and x-ray magnetic circular dichroism measurements. Schematic setup for simultaneous detection of total-electron yield (TEY) and luminescence yield (LY) in the vacuum chamber of the magnet. In TEY, the drain current (compensation current) is a measure for the amount of emitted electron due to x-ray absorption. TEY is surface-sensitive. In LY, which probes the entire thin-film sample, the transmitted x-rays that are not absorbed in the sample stack give rise to x-ray excited optical luminescence in the sapphire substrate. The emitted optical photons exit through a hole in the back of the sample holder and are detected by a photodiode. Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 Methods l Structural characterization. The thin film samples were grown by MBE on 1/4-2” diameter, c-plane sap- phire wafers. This study focuses on Cr2Te3 thin films (typical thickness 100 nm) which were grown in (001) ori- entation at a substrate temperature of 400 °C and at a rate of ~1.7 nm/min on Al2O3(0001). Some of the films were further overgrown with ~3 nm thick Cr-doped Sb2Te3 at a substrate temperature of 250 °C, as described in detail in ref.30. CrxSb2−xTe3 is an MTI with out-of-plane magnetic anisotropy and a Cr concentration-dependent transition temperature of up to 125K. The nominal Cr concentration was x = 0.25. X-ray diffraction (XRD) was carried out on a Bruker D8 diffractometer using incident Cu-Kα1 radiation. The XRD measurements provide an indication of the Cr-Te phase and the crystalline quality of the films. Atomic force microscopy (AFM) was used to characterize the surface morphology of the samples. Magnetometry. Magnetic characterization of the samples was performed through SQUID magnetometry with a field applied out-of-plane, along the c-axis of the sample. After field-cooling in a field of 6T, the magnet- ization as a function of increasing temperature from 3 to 300K was measured in a magnetic field of 20 mT. The magnetization as a function of applied field was also measured at various temperatures above and below TC. X-ray absorption spectroscopy and x-ray magnetic circular dichroism. X-ray absorption spec- troscopy (XAS) and x-ray magnetic circular dichroism (XMCD) measurements were performed on beamline 29 (BOREAS) at the ALBA synchrotron in Barcelona, Spain31. XAS was simultaneously measured at the Cr L2,3 edge in total-electron-yield (TEY) mode and luminescence-yield (LY) mode (see Fig. 9 for a schematic of the measurement modes). The Cr L2,3 photon energy region coincides with that of the Te M4,5 edges. TEY provides surface-sensitive measurements with a probing depth of 3–5 nm20. On the other hand, luminescence-yield (LY) mode probes the entire thin film sample. In the latter mode, the transmitted x-rays that are not absorbed in the sample stack give rise to x-ray excited optical luminescence in the sapphire substrate. The emitted optical photons exit through a hole in the back of the sample holder and are detected by a photodiode21. The XAS spectra were calculated by taking the negative logarithm of the LY intensity after normalizing it by the incident beam intensity. References The theory of atomic structure and spectra (Univ of California Press, Berkeley, 1981).h h y f p y 5. van der Laan, G. & Thole, B. T. Strong magnetic x-ray dichroism in 2p absorption spectra of 3d transition-metal ions. Phys. Rev. B 43, 13401–13411 (1991).fi 6. Duffy, L. B., Figueroa, A. I., van der Laan, G. & Hesjedal, T. Codoping of Sb2Te3 thin films with V and Cr. Phys. Rev. Mater. 1, 064409 (2017). ( ) 7. Baker, A. A. et al. Magnetic proximity-enhanced Curie temperature of Cr-doped Bi2Se3 thin films. Phys. Rev. B 92, 094420 (2015). Th l d l d h b f b l h Baker, A. A. et al. Magnetic proximity-enhanced Curie temperature 8. Thole, B. T., Carra, P., Sette, F. & van der Laan, G. X-ray circular dichroism as a probe of orbital magnetization. Phys. Rev. Lett. 68 1943 (1992).hi 29. Carra, P., Thole, B. T., Altarelli, M. & Wang, X. X-ray circular dichroism and local magnetic fields. Phys. Rev. Lett. 70, 694 (199 30 C lli M I t L J t l St t l l t i d ti i ti ti f ti d i i MBE C Sb T hi 30. Collins-McIntyre, L. J. et al. Structural, electronic, and magnetic investigation of magnetic ordering in MBE-grown CrxSb2−xTe3 films. EPL (Europhys. Lett.) 115, 27006 (2016). i p y 31. Barla, A. et al. Design and performance of BOREAS, the beamline for resonant X-ray absorption and scattering experiments at the ALBA synchrotron light source. J. Synchrotron Rad. 23, 1507–1517 (2016). i 1. Barla, A. et al. Design and performance of BOREAS, the beamline for resonant X-ray absorption and scattering experiments at the ALBA synchrotron light source. J. Synchrotron Rad. 23, 1507–1517 (2016). Acknowledgements g We acknowledge the CELLS-ALBA Synchrotron at Barcelona, Spain, on beamline BL29 (BOREAS) under proposal 2017092446. This publication arises from research funded by the John Fell Oxford University Press (OUP) Research Fund. RCaH is acknowledged for their hospitality. L.B.D. was supported by the Science and Technology Facilities Council and the Engineering and Physical Sciences Research Council through a Doctoral Training Award. References References 1. Dietl, T. Semiconductor spintronics, In Modern Aspects of Spin Physics, edited by Pötz, W., Hohenester, U. & Fabian, J. pp. 1–46 (Springer Berlin Heidelberg, 2007). p g g 2. Lee, I. et al. Davis, Imaging Dirac-mass disorder from magnetic dopant atoms in the ferromagnetic topological insulato Cr1−x(Bi0.1Sb0.9)2−xTe3. Proc. Natl. Acad. Sci. USA 112, 1316–1321 (2015). 3. He, Q. L. et al. Tailoring exchange couplings in magnetic topological-insulator/antiferromagnet heterostructures. Nat. Mater. 16 94–100 (2016).f ( ) 4. Chang, C.-Z. et al. Experimental observation of the quantum anomalous hall effect in a magnetic topological insulator. Science 340, 167–170 (2013).i Mogi, M. et al. Ferromagnetic insulator Cr2Ge2Te6 thin films with p g g 6i p p 6. Roy, A. et al. Perpendicular magnetic anisotropy and spin glass-like behavior in molecular beam epitaxy grown chromium telluride thin films. ACS Nano 9, 3772–3779 (2015).i i 7. Kanazawa, K. et al. Structural and magnetic properties of hexagonal Cr1−δTe films grown on CdTe(001) by molecular beam ep J. Cryst. Growth 415, 31–35 (2015). y 8. Wang, F. et al. Ferromagnetic Cr2Te3 nanorods with ultrahigh coercivity. Nanoscale 10, 11028–11033 (2018). 9. Dijkstra, J., Weitering, H. H., van Bruggen, C. F., Haas, C. & de Groot, R. A. Bandstructure calculations, and magneti properties of ferromagnetic chromium tellurides (CrTe, Cr3Te4, Cr2Te3). J. Phys.: Condens. Matter 1, 9141–9161 (198 9. Dijkstra, J., Weitering, H. H., van Bruggen, C. F., Haas, C. & de Groot, R. A. Bandstructure calculations, and magnetic and transp properties of ferromagnetic chromium tellurides (CrTe, Cr3Te4, Cr2Te3). J. Phys.: Condens. Matter 1, 9141–9161 (1989). 0 Andresen A F The Magnetic Structure of Cr2Te3 Cr3Te4 and Cr Te6 Acta Chem Scand 24 3495–3509 (1970) properties of ferromagnetic chromium tellurides (CrTe, Cr3Te4, Cr2Te3). J. Phys.: Condens. Matter 1, 9141–9161 (1989). 10. Andresen, A. F. The Magnetic Structure of Cr2Te3, Cr3Te4, and Cr5Te6. Acta Chem. Scand. 24, 3495–3509 (1970). properties of ferromagnetic chromium tellurides (CrTe, Cr3Te4, Cr2Te3). J. Phys.: Condens. Matter 1, 9141 9161 (1989). 10. Andresen, A. F. The Magnetic Structure of Cr2Te3, Cr3Te4, and Cr5Te6. Acta Chem. Scand. 24, 3495–3509 (1970).h 10. Andresen, A. F. The Magnetic Structure of Cr2Te3, Cr3Te4, and Cr5Te6. Acta Chem. Scand. 24, 3495–3509 (1970).h 10. Andresen, A. F. The Magnetic Structure of Cr2Te3, Cr3Te4, and C h g 1. Ipser, H., Komarek, K. L. & Klepp, K. O. Transition metal-chalcogen systems VIII: The Cr1−xTe phase diagram. J. Less Common. Methods l temperature (M-T) were also carried out using synchrotron x-rays with photon energy tuned to the Cr L3 edge. For the M-H measurements, the field was swept at a constant velocity taking alternating on- and off-edge measurements on the fly at a constant temperature of 3K. Similarly, for the M-T measurements, on the fly on- and off-edge 8 Scientific Reports \| (2019) 9:10793 \| https://doi.org/10.1038/s41598-019-47265-7 www.nature.com/scientificreports/ measurements were performed whilst ramping the temperature from 10K to 250K with a constant applied field of 20 mT. In both cases, the sample was initially field-cooled to 3K in a field of 6T. Measurements of the on-edge sig- nal were normalized against the off-edge signal and the asymmetry was obtained between repeat measurements with both μ+ and μ−. measurements were performed whilst ramping the temperature from 10K to 250K with a constant applied field of 20 mT. In both cases, the sample was initially field-cooled to 3K in a field of 6T. Measurements of the on-edge sig- nal were normalized against the off-edge signal and the asymmetry was obtained between repeat measurements with both μ+ and μ−. Author Contributions L.D., G.v.d.L. and T.H. conceived the idea and L.D. grew the samples and performed the XRD and SQUID experiments, and R.J. the AFM measurements. The XAS and XMCD measurements were carried out on beamline BL29 (BOREAS) at the CELLS-ALBA synchrotron by D.B., S.L.Z., A.I.F., J.H.-M., G.v.d.L. and T.H. D.B. performed the data analysis and G.v.d.L. carried out the ligand-field multiplet calculations. D.B. G.v.d.L. and T.H. wrote the paper with comments and input from all authors. All authors contributed to the discussions. Additional Information Th h d l References Met 92, 265–282 (1983). 2. Liu, Y. & Petrovic, C. Critical behavior of the quasi-two-dimensional weak itinerant ferromagnet trigonal chromium telluride Cr0.62Te. Phys. Rev. B 96, 134410 (2017).f y 3. Youn, S. J., Kwon, S. K. & Min, B. I. Correlation effect and magnetic moments in Cr2Te3. J. Appl. Phys. 101, 09G522 (2007). d h l h ( ) f Y. & Yuzuri, M. Magnetic Resonance on Cr2Te3. Jpn. J. Appl. Phys. 3 f 14. Konno, H., Adachi, Y. & Yuzuri, M. Magnetic Resonance on Cr2Te3. Jpn. J. Appl. Phys. 32, 308–310 (1993). g 3 p pp y 15. Figueroa, A. I. et al. Magnetic Cr doping of Bi2Se3: Evidence for divalent Cr from x-ray spectroscopy. Phys. Rev. B 90, 13 15. Figueroa, A. I. et al. Magnetic Cr doping of Bi2Se3: Evidence for divalent Cr from x-ray spectroscopy. Phys. Rev. B 90, 134402 (2014). 16. Figueroa, A. I. et al. Local Structure and Bonding of Transition Metal Dopants in Bi2Se3 Topological Insulator Thin Films. J. Phys. Chem. C 119, 17344–17351 (2015).fi 6. Figueroa, A. I. et al. Local Structure and Bonding of Transition Metal Dopants in Bi2Se3 Topological Insulator Thin Films. J. Phy Chem. C 119, 17344–17351 (2015).fi 17. Duffy, L. B. et al. Magnetic proximity coupling to Cr-doped Sb2Te3 thin films. Phys. Rev. B 95, 224422 (2017). f y g p y p g p 3i y 18. Henderson, G. S., Neuville, D. R. & Cormier, L. An O K-edge XANES study of glasses and crystals in the CaO–Al2O3–SiO2 (CAS) system. Chem. Geo. 259, 54–62 (2009).t y 9. Yaji, K. et al. Electronic structure of Cr1−δX (X=S,Te) studied by Cr 2p soft x-ray magnetic circular dichroism. Phys. Rev. B 70 064402 (2004). 20. van der Laan, G. & Figueroa, A. I. X-ray magnetic circular dichroism\|A versatile tool to study magnetism. Coord. Chem. Rev. 277–278, 95–129 (2014).f 21. Duffy, L. B. et al. Imposing long-range ferromagnetic order in rare-earth-doped magnetic topological insulator heterostructures. Phys. Rev. Mater. 2, 054201 (2018).h y 22. Thole, B. T. et al. 3d x-ray-absorption lines and the 3d94fn+1 multiplets of the lanthanides. Phys. Rev. B 32, 5107–5118 (1985). h 23. van der Laan, G. Hitchhiker’s Guide to Multiplet Calculations, Lect. Notes. Phys. 697, 143–200 (2006).h R. D. The theory of atomic structure and spectra (Univ of California P 24. Cowan, R. D. The theory of atomic structure and spectra (Univ o 24. Cowan, R. D. 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https://openalex.org/W2124330529	https://eprints.qut.edu.au/64192/1/64192.pdf	English	null	Physical activity patterns among South-Asian adults: a systematic review	The international journal of behavioural nutrition and physical activity	2,013	cc-by	9,036	This may be the author’s version of a work that was submitted/accepted for publication in the following source: Ranasinghe, Chathuranga, Ranasinghe, Priyanga, Mallika Arachchige, Ranil, & Misra, Anoop (2013) Physical activity patterns among South-Asian adults: a systematic review. International Journal of Behavioral Nutrition and Physical Activity, 10, pp. 1-11. This ﬁle was downloaded from: https://eprints.qut.edu.au/220121/ This may be the author’s version of a work that was submitted/accepted for publication in the following source: Ranasinghe, Chathuranga, Ranasinghe, Priyanga, Mallika Arachchige, Ranil, & Misra, Anoop (2013) Physical activity patterns among South-Asian adults: a systematic review. International Journal of Behavioral Nutrition and Physical Activity, 10, pp. 1-11. This ﬁle was downloaded from: https://eprints.qut.edu.au/220121/ This may be the author’s version of a work that was submitted/acc for publication in the following source: Ranasinghe, Chathuranga, Ranasinghe, Priyanga, Mallika Arach Ranil, & Misra, Anoop (2013) Physical activity patterns among South-Asian adults: a systematic r International Journal of Behavioral Nutrition and Physical Activity, 1 1-11. This ﬁle was downloaded from: https://eprints.qut.edu.au/220121/ c⃝Consult author(s) regarding copyright matters This work is covered by copyright. Unless the document is being made available Creative Commons Licence, you must assume that re-use is limited to personal u that permission from the copyright owner must be obtained for all other uses. If th ment is available under a Creative Commons License (or other speciﬁed license) th to the Licence for details of permitted re-use. 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Unless the document is being made available under a Creative Commons Licence, you must assume that re-use is limited to personal use and that permission from the copyright owner must be obtained for all other uses. If the docu- ment is available under a Creative Commons License (or other speciﬁed license) then refer to the Licence for details of permitted re-use. It is a condition of access that users recog- nise and abide by the legal requirements associated with these rights. If you believe that this work infringes copyright please provide details by email to qut.copyright@qut.edu.au License: Creative Commons: Attribution 2.5 Notice: Please note that this document may not be the Version of Record (i.e. published version) of the work. Author manuscript versions (as Sub- mitted for peer review or as Accepted for publication after peer review) can be identiﬁed by an absence of publisher branding and/or typeset appear- ance. If there is any doubt, please refer to the published source. https://doi.org/10.1186/1479-5868-10-116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 6 Keywords: Physical activity, Inactivity, South Asia, Adults Studies in the United Kingdom have shown that the risk of diabetes is 3 to 5 times higher for immigrants from Bangladesh, India and Pakistan compared with the native white Caucasian population, with an associated increased risk of complications, morbidity and mortality [3]. South Asia has the highest number of patients with diabetes and the prevalence of diabetes among adults is over 10% in many parts of the region [4]. This increased metabolic risk among South Asians appears to be multi-factorial, where unhealthy dietary habits and physical inactivity are coupled with genetic predisposition [5]. Physical inactivity increases the risk of developing abdominal adiposity, diabetes and cardiovascular disease [6]. Furthermore, physical inactivity is considered the fourth leading risk factor for global Physical activity patterns among South-Asian adults: a systematic review Chathuranga D Ranasinghe1, Priyanga Ranasinghe2, Ranil Jayawardena3 and Anoop Misra4 Chathuranga D Ranasinghe1, Priyanga Ranasinghe2, Ranil Jayawardena3 and Anoop Misra4 © 2013 Ranasinghe 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: chath_r@yahoo.com.au 1Allied Health Sciences Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka Full list of author information is available at the end of the article Abstract Physical activity (PA) has many beneficial physical and mental health effects. Physical inactivity is considered the fourth leading risk factor for global mortality. At present there are no systematic reviews on PA patterns among South Asian adults residing in the region. The present study aims to systematically evaluate studies on PA patterns in South Asian countries. A five-staged comprehensive search of the literature was conducted in Medline, Web of Science and SciVerse Scopus using keywords ‘Exercise’, ‘Walking’, ‘Physical activity’, ‘Inactivity’, ‘Physical Activity Questionnaire’, ‘International Physical Activity Questionnaire’, ‘IPAQ’, ‘Global Physical Activity Questionnaire’ and ‘GPAQ’, combined with individual country names. The search was restricted to English language articles conducted in humans and published before 31st December 2012. To obtain additional data a manual search of the reference lists of articles was performed. Data were also retrieved from the search of relevant web sites and online resources. The total number of hits obtained from the initial search was 1,771. The total number of research articles included in the present review is eleven (India-8, Sri Lanka-2, Pakistan-1). In addition, eleven country reports (Nepal-3, Bangladesh-2, India-2, Sri Lanka-2, Bhutan-1, Maldives-1) of World Health Organization STEPS survey from the South-Asian countries were retrieved online. In the research articles the overall prevalence of inactivity was as follows; India (18.5%-88.4%), Pakistan (60.1%) and Sri Lanka (11.0%-31.8%). STEPS survey reports were available from all countries except Pakistan. Overall in majority of STEPS surveys females were more inactive compared to males. Furthermore, leisure related inactivity was >75% in studies reporting inactivity in this domain and people were more active in transport domain when compared with the other domains. In conclusion, our results show that there is a wide variation in the prevalence of physical inactivity among South-Asian adults within and between countries. Furthermore, physical inactivity in South Asian adults was associated with several socio-demographic characteristics. Majority of South Asian adults were inactive during their leisure time. These Factors need to be considered when planning future interventions and research aimed at improving PA in the region. Keywords: Physical activity, Inactivity, South Asia, Adults Search strategy A fi d A five staged comprehensive search of the literature was conducted in the following databases; PubMed® (U.S. National Library of Medicine, USA), Web of Science® [v.5.4] (Thomson Reuters, USA) and SciVerse Scopus® (Elsevier Properties S.A, USA) for studies published before 31st December 2012. During the first stage the above databases were searched using the following search criteria. The Medline database was searched using the MeSH (Medical Subject Headings) terms ‘Exercise’ and ‘Walking’, with keywords ‘Physical activity’, ‘Inactivity’, ‘Physical Activity Questionnaire’, ‘International Physical Activity Questionnaire’, ‘IPAQ’, ‘Global Physical Activity Questionnaire’ and ‘GPAQ’. The search terms were com- bined with the names of the individual South-Asian countries; ‘Sri Lanka’, ‘Lanka’, ‘Ceylon’, ‘India’, ‘Bangladesh’, ‘Pakistan’, ‘Nepal’, ‘Bhutan’, ‘Maldives’. The Web of Science database was searched using all of the above search terms in article topic. In the SciVerse Scopus database the above terms were searched in the article title, abstract or keywords. In the United Kingdom the levels of physical activity have long been known to be lower in the South Asian people than in the general white Caucasian population [14]. The desire to walk, cycle and participate in sports and recreational activity is low among South Asians in comparison with the general population in United Kingdom [14]. Data from the Health Survey for England found that South Asians were 60% less likely than native Caucasians to meet government recommendations for physical activity [15]. Likewise, a systematic review by Fischbacher et al. (2004) examined physical activity levels among South Asians and compared it to the general United Kingdom population and noted that rates were 50–75% less in South Asians [16]. Lack of understanding about benefits, communication gap with health care professionals, cultural beliefs and lack of culturally sensi- tive facilities are some of the potential barriers for physical activity in South Asians [17]. Swaminathan and Vaz, systematically reviewed the physical activity level of children in India [18]. Although there are several stud- ies from individual South Asian countries on the level of physical activity among adults in each country, at present there are no comprehensive systematic reviews on physical activity patterns among South Asian adults residing in the region. The aim of this exercise is to systematically evaluate published work on physical activ- ity from individual South Asian countries and summarize as for a common region enabling comparison with other regions. Introduction South Asia, commonly known as the Indian sub-continent, is home to almost one-fifth of the world’s population and is comprised of many diverse ethnic, linguistic and religious groups [1]. Altogether there are 7 countries in the region namely; Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan and Sri Lanka. Although there are signifi- cant cultural differences between regional countries, South Asians are an inherently high-risk group for developing abdominal adiposity, diabetes, cardiovascular diseases [2]. * Correspondence: chath_r@yahoo.com.au 1Allied Health Sciences Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka Full list of author information is available at the end of the article Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 2 of 11 Page 2 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Search strategy A fi d In the second stage the total hits obtained from searching these three databases were pooled together and duplicates were removed. This was followed by screening of the retrieved articles by reading the article title in the third stage and abstracts in stage four. In the fifth stage individual manuscripts were screened, and those not satisfying inclusion criteria (given below) were excluded. To obtain additional data a manual search of the reference lists of articles selected in stage five was performed. Wherever possible forward citations of the studies retrieved during the literature search was traced and screened for possible inclusion. Furthermore, data were also retrieved from the search of relevant web sites and online resources. This search process was conducted independently by two reviewers (PR and RJ) and the final group of articles to be included in the review was deter- mined after an iterative consensus process. The initial literature search using the above search criteria identified the following number of articles in the respective databases; Medline® (n = 121), Web of Science® (n = 424) and SciVerse Scopus® (n = 1226). The search strategy is summarized in Figure 1. Methods mortality causing an estimated 3.2 million annual deaths (6% of global deaths) [7]. A systematic review of published studies reporting physical activity among South Asian adults residing in South Asia was undertaken in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement (Additional file 1) [19]. Physical activity is defined as any bodily movement produced by skeletal muscles that substantially elevates energy expenditure [8]. It is an established fact that physical activity has many beneficial health effects. Regular (≥3 times per week) moderate intensity physical activity such as brisk walking, dancing and gardening decreases the risk of non-communicable disease [9]. Adequate phys- ical activity is also known to increase mental well being [10]. The Harvard alumni study has shown that the alumni mortality rates were significantly lower among those who were physically active, even after adjusting for other lifestyle risk factors [10]. Regular physical activity has been reported to lower blood pressure in adults with hypertension [11]. Physical activity assists in weight loss or a reduction in visceral fat, which could ultimately help in reducing blood pressure [12]. Participation in regular physical activity improves blood glucose control by increasing insulin sensitivity and can prevent or delay onset of type 2 diabetes [13]. Hence it is evident that regular physical activity has numerous cardio-metabolic beneficial effects and the ongoing worldwide epidemic of cardiovascular and metabolic disease is evidence to the fact that the general population is physically inactive. Results The total number of research studies included in the present review is eleven (India-8, Sri Lanka-2, Pakistan-1). In addition, country reports (n = 11, Nepal-3, Bangladesh-2, India-2, Sri Lanka-2, Bhutan-1, Maldives-1) of World Health Organization (WHO) STEPS survey for non-communicable disease risk factors for the South-Asian countries were re- trieved by searching the WHO website (http://www.who. int/chp/steps/reports/en/index.html). The sample size in research studies varied from 416–6,940 and the study setting for research studies were urban (n = 2), rural (n = 3) or both (n = 6). In the STEPS surveys sample sizes were between 2,026 and 44,491. Most research studies used self designed interviewer administered questionnaires Quality assessment adults (>15 years of age), b) Cross‐sectional study design or being the first phase of a longitudinal study, c) Geographic- ally and temporally defined population from any of the South Asian countries mentioned above, d) Being an original study presenting data on physical activity, e) Evaluating physical activity using questionnaires, f) Published in English, or with detailed summaries in English and g) Peer-reviewed fully published research papers. Studies limited to adults engaged in a particular profession, based in hospitals/institutions and confined to those with diagnosed illnesses were excluded. In addition conference proceedings, editorials, commentaries and book chapters/book reviews were excluded. All included research studies were assessed for quality and assigned a rating of either “good” (6–7 points), “fair” (4–5 points) or “poor” (1–3 points) by two reviewers (DCR and RJ). The following seven criteria were used and each one was given a point; 1) appropriateness of research design, 2) appropriate recruitment strategy, 3) adequate response rate (>50%) 4) representativeness of sample, 5) usage of objective and reliable measures to assess physical activity 6) power calculation/justification of numbers and 7) appropriateness of statistical analysis. Inclusion and exclusion criteria The following inclusion criteria were used; a) Population- based studies among healthy non-institutionalized human Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 3 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 3 of 11 Figure 1 Summarized search strategy. Figure 1 Summarized search strategy. Findings from research studies d h ll l In India the overall prevalence of inactivity was 18.5%- 88.4% (Table 1). In Indian males the prevalence of inactivity was 12.7%-66.2%. Majority of the studies (n = 5, 62.5%) reported a prevalence of inactivity < 23% in Indian males. In Indian females the inactivity prevalence was 17.0%-79.6%, while majority of the studies (n = 5, 62.5%) reported it to be > 39.5%. The prevalence of inactivity in urban areas of India was reported as 20.7%-88.7%, while in rural areas it was 6.6%-88.1%. One study conducted in rural India reported that physical inactivity was more at leisure time (males - 85.2%, females - 97.3%), and at work (male - 57.2%, female - 59.9%) and less during transport (male - 18.8%, female - 45.7%) [23]. A similar study in both urban and rural areas of India reported inactivity more at leisure time (74.0%) and less at work (31.0%) [24]. There were two research studies on physical activity from Sri Lanka [30,31]. One study was conducted on a nationally representative sample and the other in the most populous western province of Sri Lanka. Both studies looked at urban and rural settings and Inactivity was defined using a uniformed method (IPAQ). The prevalence of Inactivity was 11% in the national sample and 31.8% in In India the overall prevalence of inactivity was 18.5%- 88.4% (Table 1). In Indian males the prevalence of inactivity was 12.7%-66.2%. Majority of the studies (n = 5, 62.5%) reported a prevalence of inactivity < 23% in Indian males. In Indian females the inactivity prevalence was 17.0%-79.6%, while majority of the studies (n = 5, 62.5%) reported it to be > 39.5%. The prevalence of inactivity in urban areas of India was reported as 20.7%-88.7%, while in rural areas it was 6.6%-88.1%. One study conducted in rural India reported that physical inactivity was more at leisure time (males - 85.2%, females - 97.3%), and at work (male - 57.2%, female - 59.9%) and less during transport (male - 18.8%, female - 45.7%) [23]. A similar study in both urban and rural areas of India reported inactivity more at leisure time (74.0%) and less at work (31.0%) [24]. Only one STEPS survey has been carried out in Bhutan in the capital city Thimphu (2007, n = 2,484). The overall inactivity prevalence was 58.6% and females (69.6%) were more inactive than males (49.8%). Findings from STEPS surveys In the STEPS survey reports there was uniformity in the sampling and the definition of inactivity. Overall in majority of studies females were more inactive compared to males (Table 2). Furthermore, leisure related inactivity was >75% in studies reporting inactivity in this domain and people were more active in the transport domain when compared with other domains (Table 2). Two surveys were done in 2002 (n = 11,409) and 2009 (n = 9,275) in Bangladesh. In 2002, overall prevalence of physical inactivity was 52.3%. However, in this survey physical activity was categorised as light, moderate, heavy based on a likert scale; always, usually, sometimes and never. Inactivity was defined as people who have never done light/moderate activities, which was different to the criteria used in all other WHO STEPS surveys. In the national sample (2009) the overall inactivity prevalence was 27%, with more females (41.3%) being inactive than males (10.5%) (Table 2). Data extraction A separate assessment is not included for the WHO STEPS surveys conducted according to the uniform STEPS study protocol which satisfies all of the above seven criterion used for quality assessment. Definition of inactivity How sedentary lifestyle or inactivity was defined varied significantly in the different studies that used self designed questionnaires to evaluate physical activity (Table 1). Phys- ical Activity Level (PAL) (Estimated total energy expend- iture per 24 hrs / basal metabolic rate) was calculated in 3 studies and a PAL for inactivity was defined (<1.5/<1.69) [20-22]. However, there was a wide variation in the def- inition of physical inactivity in the other studies (Table 1). In the WHO STEPS surveys using the GPAQ, the total of the categorical score in the 3 domains (work, leisure time and transport) classifies an individual into three categor- ies; ‘Inactive’, ‘Moderately active’ and ‘Highly active’. The ‘Inactive’ category includes those who do not perform any physical activity or those reporting some activity, but not enough to meet other categories. The same definition was used in the IPAQ for the ‘Inactive’ category. Data extraction Data were extracted from the included studies by one reviewer using a standardised form and checked for accuracy by a second reviewer. The data extracted from each study were: a) study details (lead author, country/ city, year published/year of survey), b) methods (sample size, sampling method, age of subjects in years and type of questionnaire used, definitions), and c) physical activity data (all adults, males, females and domain specific data). Discrepancies in the extracted data were resolved by discussion, with involvement of a third reviewer when necessary. Page 4 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 the other study conducted in western province (Table 1). Males (14.6% and 38.5%) were more inactive than females (8.7% and 24.7%). Inactivity of urban adults was 35.2% and higher than rural adults (27.6%) in the study from Western province (Table 1). In the National sample; female gender (OR:2.1), age > 70 (OR:3.8), urban-living (OR:2.5), Muslim ethnicity (Sri Lankan Moor) (OR:2.7), tertiary education (OR:3.6), obesity (OR:1.8), diabetes (OR:1.6), hypertension (OR:1.2) and metabolic syndrome (OR:1.3) all significantly increased odds of being ‘inactive’ [31]. The only study on physical activity conducted in urban Pakistan using IPAQ showed that prevalence of inactivity was 60.1% (males-52.1%, females-69.8%) [29]. for evaluation of physical activity (n = 7). International Physical Activity Questionnaire (IPAQ) was used by 3 research studies and 1 study used WHO STEPS survey tool. All WHO STEPS surveys used Global Physical Activity Questionnaire (GPAQ) for the evaluation of physical activity. Majority of the research studies received a “good” (n = 5) or “fair” (n = 3) ranking on the quality assessment, while only 3 were ranked “poor” (Additional file 2). A separate assessment is not included for the WHO STEPS surveys conducted according to the uniform STEPS study protocol which satisfies all of the above seven criterion used for quality assessment. for evaluation of physical activity (n = 7). International Physical Activity Questionnaire (IPAQ) was used by 3 research studies and 1 study used WHO STEPS survey tool. All WHO STEPS surveys used Global Physical Activity Questionnaire (GPAQ) for the evaluation of physical activity. Majority of the research studies received a “good” (n = 5) or “fair” (n = 3) ranking on the quality assessment, while only 3 were ranked “poor” (Additional file 2). Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 , Rural, M 579, (Ma s, Urban uster sa 416 (M es racterist 782 (mal s Urban minister ys, carryi activities y calcula dex inclu y onnaire, zed, 1.40–1.69 onnaire, physical nactive: hour per week exercise Findings from research studies d h ll l Two large STEPS surveys were done in India in 2003–2005 (6 states, n = 44,491) and 2007–2008 (7 states, n = 39,064). In 2003 the overall prevalence of physical inactivity was 15.8% and in 2007 it was 72.3%. This variation could be attributed to different states used in the two surveys. However Kerala and Maharashtra states were evaluated in both surveys, and the overall prevalence of physical inactivity in Kerala/ Maharashtra has increased from 6.7%/6.8% in 2003 to 75.8%/81.2% in 2007. Male and Female physical inactivity in 2003 was 12.6% and 18.9% respectively. In 2007 preva- lence of Male and Female physical inactivity was 58.5% and 75.7% respectively. Overall females were more inactive than males and urban residents were more inactive than rural (Table 2). The only STEPS survey conducted in Maldives (2004) has been conducted in its’ capital city Malē (n = 2026). p g g at work, home or tran discretionary time es 3,768, Females rban, rural and ng Interviewer administe PAL was calculated an PAL <1.40 extremely in sedentary/lightly active s 260, Females 260), nd rural, Random Interviewer administe Inactive: Sedentary job exercise or cycling, M Sedentary job and som physical exercise and/o OR Standing job and n or cycling ural po at work, discretio Interview PAL was PAL <1.4 sedentary Interview Inactive: exercise Sedentar physical OR Stand or cycling Findings from research studies d h ll l More than 90% (Males 91.2%, Females 94.6%) at work, home or transport and discretionary time ●In males inactivity was 19.7%, while in females it was 17.0% 6,447, (Males 3,768, Females 7-76 yrs, Urban, rural and xed sampling Interviewer administered questionnaire, PAL was calculated and categorized, PAL <1.40 extremely inactive, PAL 1.40–1.69 sedentary/lightly active ●Extreme inactivity prevalence 9.7% (Males 7.4%, Females 12.9%), ●Sedentary/lightly active prevalence 62.1% (Males 58.8%, Females 66.7%) 520, (Males 260, Females 260), rs, Urban and rural, Random Interviewer administered questionnaire, Inactive: Sedentary job and no physical exercise or cycling, Moderately inactive: Sedentary job and some but <1 hour physical exercise and/or cycling per week OR Standing job and no physical exercise or cycling ●Prevalence of inactivity 29.4%* (Males 12.7%, Females 46.1%, Urban 29.6%, Rural 29.2%), ●Prevalence of moderate inactivity 21.5%,* (Males 25.7%, Females 17.3%, Urban 30.0%, Rural 13.1%), ●Inactivity was more in Urban and in females, ●Urban females waist circumference reduced (p < 0.05) with increased physical activity, ●BMI showed a steady decline from inactivity to activity ,825, (Males 650, Females 1175), Rural population Interviewer administered questionnaire, Sedentary lifestyle: those who had never ●40.0% had a sedentary lifestyle (Males 40.8%, Females 39.7%) males it was ales 7.4%, e prevalence .7%, Females valence of %, Females ctivity was females with ed a steady e in all s 85.2%, 8.8%, 57.2% ghest in roup 2.9%, ore than 0%), more 8.9%, n 1.64 in an n the r old not d and cised vity lev 30-1.56 > 58 yr w ove tionary ●Wo ercise les, ● n/day ross ag 6 yrs ag 5%) and w nactivity m %), and le %), ●Ina - 59.9%; ● 4 yrs) and prevalen 1.9% ), ● poration 2 ●Inactivit (31.0%), ● %, RR = 1 killed wo (32.0%, R 3%, RR = prevalenc nder diffe activity w nactivity p %), ●Sed 58.8%, F of inactiv n 29.6%, 5%) and w nactivity m %), and le %), ●Ina - 59.9%; ● 64 yrs) and prevalen 1.9% ), ● poration 2 ●Inactivit (31.0%), ● 7%, RR = 1 killed wo (32.0%, R 3%, RR = prevalenc ender diffe activity w nactivity p %), ●Sed 58.8%, F of inactiv n 29 6%, Females 39.7%) Res ●P and olde fem mal you disc cho exe 40.9 high (rec R a o f m y d c e 4 ( e, e + ure e y onnaire, zed, 1.40–1.69 onnaire, hysical nactive: hour per week exercise ose who had never minister ys, carry activities y calcula dex inclu g home or tran nary time er administer calculated an 0 extremely in /lightly active er administer Sedentary job or cycling, Mo y job and som exercise and/o ng job and n ools an ministe y level diture/E ), Seden ools an ministe y level diture/E ), Seden y y y essm rview sical a rgy ex tabolic L) <1.5 Interview Inactive: light/sede travel, Ina Composit Interview Inactive: light/sede travel, Ina Composit Sedentar at work, discretio Interview PAL was PAL <1.4 sedentar Interview Inactive: exercise Sedentar physical OR Stand or cycling 441), ent pling les 3689), atified 188) pling les 3689), atified 188) pulation s 3,768, Females rban, rural and ng 260, Females 260), d rural, Random ural po 447, (M -76 yrs ed sam 20, (Ma Urban http://www.ijbnpa.org/content/10/1/116 http://www.ijbnpa.org/content/10/1/116 (Males 52.1%, Females 69.8%) ●Females were significantly more inactive than males (OR: 2.1, 95% CI 1.5–3.1, p < 0.001) ●Prevalence of inactivity 31.8%* (Males 38.5%, Females 24.7%), ●Inactivity in urban adults 35.2% (Males 41.0%, Females 29.0), ●Inactivity in rural adults (Males 35.0%, Females 19.0%), ●Physical inactivity had a significant association with high BMI among women irrespective of their urban or rural living ●Prevalence of inactivity 11.0% (Males 14.6%, Females 8.7%) th 2% th 2% ral with % an %, 35.2% l rban or %, rban or %, an 88.7% on incre activity 6 e inactiv < 0.001) * (Males rban ad activity irrespective of thei ivity 11.0% (Males 1 n 88.7% on incre ctivity e inactiv 0.001) (Males ban ad activity ve of thei % (Males 1 4% (U perte ce of %) tly m –3.1, y 31.8 vity in 9.0), ● Exercise .1%), ●P ck of exer Males 52.1 Females ales (OR: Prevalen males 24 Males 41.0 ults (Ma BMI amon rural livin ●Prevalen Females 8 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 7 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 7 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Table 2 Summary of findings from STEPS surveys Country Year of study Sample characteristics Results Inactive (%) Inactivity in each domain (%) Male Female Urban Rural Total Work Transport Recreation Bangladesh 2002 Sample size 11,409 NR NR 50.1# 56.7# 52# (Male 5,625, Female 5,784) Age 25–64 yrs Urban and rural In capital city - Dhaka Bangladesh 2009-2010 Sample size 9,275 10.5 41.3 NR NR 27.0 45.7 44.5 81.9 (Male 4,312, Female 4,963) Age > 25 yrs Urban and rural Bhutan 2007 Sample size 2,484 49.8 69.6 58.6 NA 58.6 69.0 63.2 78.7 (Male 1,138, Female 1,346) Age 25–74 yrs Urban In capital city - Thimpu India 2003-2005 Sample size 44,491 (Male 21,871, Female 22,620) Age 15–64 yrs Urban and rural 6 States in India 1. Assam 8.3 10.7 26.1* 1.6* 9.5* 28.3* 24.6* NR 2. Delhi 25.5 15.5 26.3* NR 20.4* 62.9* 39.3* NR 3. Haryana 16.9 47.6 38.3* 24.6* 32.7* 78.4* 41.9* NR 4. Kerala 4.8 4.3 7.6* 5.9* 6.7* 18.4* 31.8* NR 5. Maharashtra 7.7 6.1 14.9* 1.3* 6.8* 24.5* 21.1* NR 6. http://www.ijbnpa.org/content/10/1/116 Tamil Nadu 16.9 27.2 29.1* 19.9* 22.0* 90.8* 25.1* NR India 2007-2008 Sample size 38,064 (Male 16,891, Female 21,173) Age 24–64 yrs Urban and rural 7 states in India 1. Andhra Pradesh 55.9 79.7 77.5 63.8 67.7 NR NR NR 2.Kerala 64.7 86.2 70.8 74.5 75.8 NR NR NR 3. Madhya Pradesh 33.5 52.0 68.3 31.8 42.3 NR NR NR 4. Maharashtra 75.4 87.7 86.1 77.2 81.2 NR NR NR 5. Mizoram 60.9 82.4 79.1 62.5 71.1 NR NR NR 6. Tamil Nadu 57.3 74.2 79.4 61.6 65.8 NR NR NR 7. Uttarakhand 64.6 69.7 91.6 57.6 67.1 NR NR NR Maldives 2004 Sample size 2,026 NR NR NR NA NR 93.2* NR NR (Male 934, Female 1,092) Age 25–64 yrs Urban (In Male) Table 2 Summary of findings from STEPS surveys Country Year of study Sample characteristics Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 8 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 8 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 8 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 8 of 11 Table 2 Summary of findings from STEPS surveys (Continued) Nepal 2003 Sample size 2,030 73.6 90.9 NR NA 82.3* 82.3* 27.6* 94.5* (Male 1,010, Female 1,020) Age 25–64 yrs Urban Nepal 2004-2005 Sample size 7,792 NR NR NR NR NR 51.5 19.1 86.0 (Male 3,674, Female 4,118) Age 15–64 yrs Urban and rural Nepal 2007 Sample size 4,328 5.2 5.9 NR NR 5.5 10.6 19.0 83.2 (Male 1,907, Female 2,421) Age 15–64 yrs Urban and rural 15 of 75 districts Sri Lanka 2003 Sample size 3,000 12.1 19.1 NR NR 15.6 58.3* NR 94.8* (Male 1,500, Female 1,500) Age 15–74 yrs Urban One of nine provinces - Western Sri Lanka 2006 Sample size 11,680 17.9 31.9 NR NR 25.0 NR NR NR (Male 5,765, Female 5,915) Age 24–64 yrs Urban and rural 5 random districts out of all 25 districts * - calculated from available data; NR – Not reported; NA – Not applicable. http://www.ijbnpa.org/content/10/1/116 Table 2 Summary of findings from STEPS surveys (Continued) - calculated from available data; NR – Not reported; NA – Not applic of the respondents reported that they are physically inactive at work. In contrast STEPS survey data on physical activity were available from all countries except Pakistan. All research studies from the region collectively evaluated physical activity of 26,360 adults, while STEPS surveys contained data on 136,579 adults. We observed a marked variation between research studies in the definition of physical inactivity and in the questionnaires used. Hence, comparisons between studies were undertaken with caution. This barrier was overcome in the STEPS surveys as they used the GPAQ to evaluate physical activity. It is important that future researchers try and adhere to this uniformity in order to derive intra- and inter-regional comparable data and observe secular trends in physical activity. The present study mainly focuses on the preva- lence of physical inactivity and its variations with gender and area of residence. We also tried to identify inactivity levels in different domains (work, transport, leisure) as occupations, transport methods, social and cultural values differ in this region compared to other regions and the developed world. Three STEPS surveys have been conducted in Nepal. In 2003 only an urban sample was taken from the capital city (n = 2,030). In later STEPS surveys both urban and rural samples were studied (2004 n = 7,792 and 2007 n = 4,328). The 2003 survey reported an overall inactivity of 82.9% (Males 74.6%, Females 91.2%) in the urban residents. However in contrast the 2007 national sample (conducted in 15 out of 75 districts) reported that overall inactivity was only 5.5% (Males 5.2%, Females 5.9%) (Table 2). In Sri Lanka two STEPS surveys were done in 2003 (single province, n = 3,000) and 2006 (five districts, n = 11,680). In 2003 overall prevalence of physical inactivity was 15.6% and in 2006 it has increased to 25%. Females (2003–19.1%, 2006–31.9%) were more inactive than males (2003–12.1%, 2006–17.9%) in both studies. Discussion To our knowledge this is the first systematic review evaluating physical inactivity and physical activity patterns among adults from South Asian countries. Published research articles evaluating physical activity prevalence were only available from India, Sri Lanka and Pakistan. We observed several socio-economical factors associated with physical inactivity. Skilled workers and professionals were more inactive than unskilled workers in the region [24]. Similarly, higher education was a significant factor Page 9 of 11 associated with physical inactivity [31]. Among South Indians, Vaz et al. reported that people engaging in rec- reational exercise were inactive in other domains [20,21]. Gender is also an important factor to determining physical activity levels of a population. Several studies reported higher physical inactivity among South Asian women [23,25,29,31,32]. Cultural expectations may restrict the participation of women in certain forms of physical activity in some religious and ethnic groups in the region. The traditional role of South Asian women in taking care of household work and supporting extended family members may limit the time available for them to engage in physical activity, in particularly leisure time physical activities. Low physical activity is one of the contributing risk factors for the higher obesity levels seen among Asian Indians [33]. A qualitative study conducted among South Asians living in United Kingdom found that understanding external motivators and social context of their lives is very crucial for developing successful physical activity interventions [34]. Hence, although exercise is promoted in public health campaigns to increase the overall physical activity level of a population, it is important to under- stand socio-economical factors associated with different populations in order to deliver effective physical activity interventions. which were studied in both surveys also showed marked variation. This could be due to differences in the sampling frames used. However such dramatic increases in inactiv- ity over a short period warrants further investigation. The only surveys conducted in urban capitals of Maldives and Bhutan had significantly higher prevalence of inactivity. In Bhutan inactivity was lower compared to Maldives due to it being mostly an agrarian society with nearly 50% of the labour force engaged in agriculture as compared to Maldives where most are involved in the tourism industry [37]. The most active regional country in the STEPS surveys was Nepal, possibly due to 80% of the Nepalese population being involved in agriculture [38]. Discussion In all regional countries work and transport related activity was higher compared to leisure time activity. Hence it is important to consider individual country profiles, proportions of urbanity and cultures when delivering public health mes- sages focused on leisure time activity. Comparable nationally representative country data are available from the WHO World Health Survey (IPAQ questionnaire) conducted in 2002–2003, where the male and female inactivity prevalence was as follows; India 9.3% & 15.2%, Nepal 6.7% & 9.7% and Sri Lanka 7.3% & 13.8% [39]. The data are fairly comparable to those from WHO STEPS surveys in India (2003) and Nepal (2007). In contrast in Sri Lanka inactivity has markedly increased from the 2002 World Health Survey to the 2006 STEPS survey. Several studies show that IPAQ overestimates inactivity prevalence and under estimates prevalence in rural areas of developing countries [32,40,41]. The STEPS surveys have also been conducted in 80 developing countries around the world allowing for a meaningful comparison across regions [35]. In the Western Pacific region (19 countries) inactivity prevalence ranged from 7.5% in Mongolia to 75.3% in Cook Islands with majority of countries falling between 40%-66%. Eastern Mediterranean (14 countries) prevalence of inactivity ranged from 30%-60%. In Africa (31 countries) lowest prevalence of inactivity was observed in Mozambique (6.4%) and highest in Zimbabwe (79.3%). In nearly half of the African countries inactivity was <29%. East Asian countries like Myanmar (12.7%) and Indonesia (22.6%) had prevalence’s of inactivity comparable with the South Asia and Africans. With the available data we observe that the persons residing in South Asian region are more active compared with the Western Pacific and Mediterranean countries, lying in parallel with East Asia and Africa. In developing countries with agrarian economies inactivity was low irrespective of the region (Nepal, Mongolia, Malawi, Mozambique and Cambodia). However the inactivity prevalence seems to be on the rise in most Western Pacific countries due to recent economic transitions. Hence, countries in the South Asian region in economic transition also need public health planning to enable the people to maintain their already existing active The WHO STEPwise approach was introduced for chronic disease risk factor surveillance in order to close the gap in the availability of data from developing countries and to strengthen their capacity to conduct such surveillances [35]. The GPAQ used in these surveys not only gave uniformity to the gathered data, it also allowed meaningful comparisons. Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Discussion The GPAQ is known to be reliable for surveys in developing countries, adaptable to incorporate cultural differences [36]. The initial STEPS surveys from the region were mostly in sub-populations and confined to logistically favourable areas like capitals. Sri Lanka (2006), Nepal (2007) and Bangladesh (2009) (Inactivity: 25%, 5.5% and 27% respectively) had nationally representative samples in subsequent surveys. Prevalence of overall inactivity reduced in these subsequent surveys as the sample dispersed from populations in urban capitals to the rural parts of the countries. This highlights the effect of urbanity on physical activity. However, in the Sri Lankan STEPS surveys inactivity prevalence has increased in the second survey (2006), which used a national sample. This is probably due to the initial survey (2003) being conducted in more rural areas of the Western province of Sri Lanka, in contrast to Nepal and Bangladesh where the initial surveys were from more urbanised capitals. The two large STPES surveys (2003, 2007) from India contrasted in the prevalence of inactivity, possibly attribut- able to subjective variations in data collection, inter-state variations and differences in time and season of data col- lection. However, data from Kerala and Maharashtra states Page 10 of 11 life styles. We also observe that females were more inactive in the South Asian region when compared to males, a finding which is seen in most other regions and even in developed countries. leisure time. These factors need to be considered when planning future interventions and research aimed at improving PA in the region. However, future researchers should try and adhere to uniformity in definitions and assessment tools in order to derive intra- and inter-regional comparable data and observe secular trends. The International Prevalence Study conducted in 2002– 2004 using IPAQ categorized inactivity to be least in China (6.9%), New Zealand (12.2%), Canada (13.7%), USA (15.9%) and Australia (17.2%) [42]. We observe that South Asian activity levels were in parallel or sometimes better than these developed countries which have longer physical activity promotion strategies, mainly targeted at improving leisure time physical activity. However with increased urbanisation and busy work schedules the leisure time is also reduced. In the South Asian region we observed that activity during work and transport is higher than leisure time activity. Promoting leisure time activity has also become a challenge in South Asia due to cultural and attitudinal barriers. Discussion Hence public health messages in the region should be directed at improving all domains of activity with work and transport policies of the countries supporting them. Competing interests The authors declare that they have no competing interests. Received: 7 April 2013 Accepted: 9 October 2013 Published: 12 October 2013 Received: 7 April 2013 Accepted: 9 October 2013 Published: 12 October 2013 References 1. Gupta M, Singh N, Verma S: South Asians and cardiovascular risk: What clinicians should know. Circulation 2006, 113(25):e924–e929. 1. Gupta M, Singh N, Verma S: South Asians and cardiovascular risk: What clinicians should know. Circulation 2006, 113(25):e924–e929. 2. Eapen D, Kalra GL, Merchant N, Arora A, Khan BV: Metabolic syndrome and cardiovascular disease in South Asians. Vasc Health Risk Manag 2009, 5:731–743. 2. Eapen D, Kalra GL, Merchant N, Arora A, Khan BV: Metabolic syndrome and cardiovascular disease in South Asians. Vasc Health Risk Manag 2009, 5:731–743. 3. Erens B, Primatesta P, Prior G: The health of minority ethnic groups ′99 methodology & documentation. London: The health survey for england; 2001. 4 Jayawardena R Ranasinghe P Byrne NM Soares MJ Katulanda P Hills AP: 3. Erens B, Primatesta P, Prior G: The health of minority ethnic groups ′99 methodology & documentation. London: The health survey for england; 2001. 4. Jayawardena R, Ranasinghe P, Byrne NM, Soares MJ, Katulanda P, Hills AP: Prevalence and trends of the diabetes epidemic in South Asia: a systematic review and meta-analysis. BMC Public Health 2012, 12:380. 5. Gupta M, Brister S: Is South Asian ethnicity an independent cardiovascular risk factor? Can J Cardiol 2006 22(3):193 197 4. Jayawardena R, Ranasinghe P, Byrne NM, Soares MJ, Katulanda P, Hills AP: Prevalence and trends of the diabetes epidemic in South Asia: a systematic review and meta-analysis. BMC Public Health 2012, 12:380. 5. Gupta M, Brister S: Is South Asian ethnicity an independent cardiovascular risk factor? Can J Cardiol 2006, 22(3):193–197. 6. Qin L, Corpeleijn E, Jiang C, Thomas GN, Schooling CM, Zhang W, Cheng KK, Leung GM, Stolk RP, Lam TH: Physical activity, adiposity, and diabetes risk in middle-aged and older Chinese population: The Guangzhou Biobank Cohort Study. Diab Care 2010, 33(11):2342–2348. Authors’ contributions One of the strengths of the current systematic review is the comprehensive and easily replicable search strategy applied to 3 major medical databases. In addition we systematically selected the studies through the application of well-defined inclusion/exclusion criteria. We would also like to highlight several limitations in the present review. Firstly, most of studies were limited to regional populations. As South Asian countries have considerably diverse ethnic groups, the regional findings may not be generalized to whole country or for the entire South Asian region. Secondly, there is no uniformity on physical activity assessment tools. Although several of studies have used IPAQ, differences in the formats used limit comparability. For instance, Arambepola et al. has used longer version of IPAQ whereas Katulanda et al. has used a short version [30,31]. Thirdly, there is clear heterogeneity on the defin- ition of physical inactivity/sedentary life style and physical activity levels. Furthermore, only limited number of articles reported data on the sub-domains of physical activity (work, transport and leisure). In addition, although many studies have shown that resistance exercise improves over- all health and that it is beneficial for South Asians with Diabetes at present there is only very limited data available for South Asians [43,44]. DC, PR and RJ made substantial contribution to conception and study design. DC, PR and RJ were involved in data collection. DC, PR, RJ and AM were involved in refining the study design, statistical analysis and drafting the manuscript. DC, PR, RJ and AM critically revised the manuscript. All authors read and approved the final manuscript. Additional files Additional file 1: PRISMA 2009 checklist. Additional file 2: Assessment of Quality of the included Research Studies. Additional file 1: PRISMA 2009 checklist. Additional file 2: Assessment of Quality of the included Research Studies. Abbreviations Abbreviations GPAQ: Global physical activity questionnaire; IPAQ: International physical activity questionnaire; MeSH: Medical subject heading; MET: Metabolic equivalents; OR: Odds ratio; PAL: Physical activity level; PRISMA: Preferred reporting in systematic reviews & Meta-Analysis; WHO: World Health Organization. Author details 1 1Allied Health Sciences Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka. 2Department of Pharmacology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka. 3Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia. 4Fortis-C-DOC Centre of Excellence for Diabetes, Metabolic Diseases and Endocrinology, Chirag Enclave, New Delhi, India. Received: 7 April 2013 Accepted: 9 October 2013 Published: 12 October 2013 Conclusions Jepson R, Harris FM, Bowes A, Robertson R, Avan G, Sheikh A: Physical activity in South Asians: An in-depth qualitative study to explore motivations and facilitators. PLoS One 2012, 7(10):e45333. 12. Reaven PD, Barrett-Connor E, Edelstein S: Relation between leisure-time physical activity and blood pressure in older women. Circulation 1991, 83(2):559–565. 35. World Health Organization: WHO Chronic diseases and health promotion, STEPS country reports. http://www.who.int/chp/steps/reports/en/index.html. 13. Hamman RF, Wing RR, Edelstein SL, Lachin JM, Bray GA, Delahanty L, Hoskin M, Kriska AM, Mayer-Davis EJ, Pi-Sunyer X, et al: Effect of weight loss with lifestyle intervention on risk of diabetes. Diabetes Care 2006, 29(9):2102–2107. 36. Armstrong TA, Bull FC: Development of the Global Physical Activity Questionnaire (GPAQ). J Public Health 2006, 14:66–70. Questionnaire (GPAQ). J Public Health 2006, 14:66–70. 37. Central Intelligence Agency: The world factbook; 2013. https://www.cia.gov/ library/publications/the-world-factbook/index.html. 14. Zaman MJ, Jemni M: South Asians, physical exercise and heart disease. Heart 2011, 97(8):607–609. 38. Ertur O: The need for a national urbanization policy in Nepal. Asia Pac Popul J 1994, 9(3):19–36. 15. Williams ED, Stamatakis E, Chandola T, Hamer M: Assessment of physical activity levels in South Asians in the UK: Findings from the Health Survey for England. J Epidemiol Community Health 2011, 65(6):517–521. 39. Guthold R, Ono T, Strong KL, Chatterji S, Morabia A: Worldwide variability in physical inactivity a 51-country survey. Am J Prev Med 2008, 34(6):486–494. 16. Fischbacher CM, Hunt S, Alexander L: How physically active are South Asians in the United Kingdom? A literature review. J Public Health (Oxf) 2004, 26(3):250–258. 40. Ainsworth BE, Macera CA, Jones DA: Comparison of the 2001 BRFSS and the IPAQ physical activity questionnaires. Med Sci Sports Exerc 2006, 38:1584–1592. 17. Horne M, Tierney S: What are the barriers and facilitators to exercise and physical activity uptake and adherence among South Asian older adults: A systematic review of qualitative studies. Prev Med 2012, 55(4):276–284. 41. Craig CL, Marshall AL, Sjöström M: International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc 2003, 35:1381–1395. 18. Swaminathan S, Vaz M: Childhood physical activity, sports and exercise and noncommunicable disease: A special focus on India. Indian J Pediatr 2013, 80(1):63–70. 42. Bauman A, Bull F, Chey T, Craig CL, Ainsworth BE, Sallis JF, Bowles HR, Hagstromer M, Sjostrom M, Pratt M, et al: The international prevalence study on physical activity: results from 20 countries. Int J Behav Nutr Phys Act 2009, 6:21. Conclusions y , ( ) 7. World Health Organization: Global health risks: Mortality and burden of disease attributable to selected major risks. Geneva: World Health Organization; 2009. There is a wide variation in the prevalence of physical inactivity among South-Asian adults within and between countries. Hence it is difficult to comment about the overall prevalence of physical inactivity in the region. In the South Asian regions females, skilled workers, professionals and those with higher education were more inactive. Majority of South Asian adults were inactive during their 8. Aust NZJ, MedCaspersen CJ, Powell KE, Christenson GM: Physical activity, exercise, and physical fitness: Definitions and distinctions for health-related research. Public Health Rep 1985, 100(2):126–131. 9. Willett WC, Koplan JP, Nugent R, Dusenbury C, Puska P, Gaziano TA: 8. Aust NZJ, MedCaspersen CJ, Powell KE, Christenson GM: Physical activity, exercise, and physical fitness: Definitions and distinctions for health-related research. Public Health Rep 1985, 100(2):126–131. 9. Willett WC, Koplan JP, Nugent R, Dusenbury C, Puska P, Gaziano TA: Prevention of chronic disease by means of diet and lifestyle changes. In Disease control priorities in developing countries. 2nd edition. Edited by 9. Willett WC, Koplan JP, Nugent R, Dusenbury C, Puska P, Gaziano TA: Prevention of chronic disease by means of diet and lifestyle changes. In Disease control priorities in developing countries. 2nd edition. Edited by Page 11 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha P, Mills A. Washington (DC): Musgrove P; 2006. Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha P, Mills A. Washington (DC): Musgrove P; 2006. 32. Ekelund U, Sepp H, Barge S: Criterion-related validity of the last 7-day, short form of the International Physical Activity questionnaire in Swedish adults. Public Health Nutr 2006, 9:258–265. 10. Fox KR: The influence of physical activity on mental well-being. Public Health Nutr 1999, 2(3A):411–418. 33. Chopra SM, Misra A, Gulati S, Gupta R: Overweight, obesity and related non-communicable diseases in Asian Indian girls and women. Eur J Clin Nutr 2013, 67(7):688–696. 11. Ishikawa K, Ohta T, Zhang J, Hashimoto S, Tanaka H: Influence of age and gender on exercise training-induced blood pressure reduction in systemic hypertension. Am J Cardiol 1999, 84(2):192–196. 34. Conclusions 19. Moher D, Liberati A, Tetzlaff J, Altman DG, Group P: Preferred reporting items for systematic reviews and meta-analyses: The PRISMA statement. BMJ 2009, 339:b2535. 43. Hills AP, Shultz SP, Soares MJ, Byrne NM, Hunter GR, King NA, Misra A: Resistance training for obese, type 2 diabetic adults: a review of the evidence. Obes Rev 2010, 11(10):740–749. 20. Vaz M, Bharathi AV, Kurpad AV: Exercising’ but not active: Implications for physical activity counselling. Natl Med J India 2006, 19(6):345. 44. Misra A, Alappan NK, Vikram NK, Goel K, Gupta N, Mittal K, Bhatt S, Luthra K: Effect of supervised progressive resistance-exercise training protocol on insulin sensitivity, glycemia, lipids, and body composition in Asian Indians with type 2 diabetes. Diabetes Care 2008, 31(7):1282–1287. 21. Vaz M, Bharathi AV: Perceptions of the intensity of specific physical activities in Bangalore, South India: Implications for exercise prescription. J Assoc Physicians India 2004, 52:541–544. 22. Sullivan R, Kinra S, Ekelund U, Bharathi AV, Vaz M, Kurpad A, Collier T, Reddy KS, Prabhakaran D, Ben-Shlomo Y, et al: Socio-demographic patterning of physical activity across migrant groups in India: results from the Indian Migration Study. PLoS One 2011, 6(10):e24898. doi:10.1186/1479-5868-10-116 Cite this article as: Ranasinghe et al.: Physical activity patterns among South-Asian adults: a systematic review. International Journal of Behavioral Nutrition and Physical Activity 2013 10:116. 23. Krishnan A, Shah B, Lal V, Shukla DK, Paul E, Kapoor SK: Prevalence of risk factors for non-communicable disease in a rural area of Faridabad district of Haryana. Indian J Public Health 2008, 52(3):117–124. y 24. Sugathan TN, Soman CR, Sankaranarayanan K: Behavioural risk factors for 24. Sugathan TN, Soman CR, Sankaranarayanan K: Behavioural risk factors for non communicable diseases among adults in Kerala, India. Indian J Med Res 2008, 127(6):555–563. non communicable diseases among adults in Kerala, India. Indian J Med Res 2008, 127(6):555–563. 25. Agrawal VK, Basannar DR, Sing RP, Dutt M, Abraham D, Mustafa MS: Coronary risk factors in a rural community. Indian J Public Health 2006, 50(1):19–23. g g Coronary risk factors in a rural community. Indian J Public Health 2006, 50(1):19–23. 26. Mittal M, Arora M, Bachhel R, Kaur N, Sidhu RS: Physical activity, indices of obesity and mean arterial blood pressure: Does place of living matters? rural vs urban. J Clin Diagn Res 2011, 5(5):1038–1042. 27. Haldiya KR, Mathur ML, Sachdev R: Lifestyle-related risk factors for cardiovascular disease in a desert population of India. Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Conclusions Curr Sci 2010, 99(2):190–195. 28. Agrawal R, Chaturvedi M, Singh S, Gupta SC: An epidemiological study of dietary and exercise habits as correlates of hypertension in persons aged 45 years and above in Agra District. Indian J Comm Health 2012, 24(2):91–96. 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: 29. Khuwaja AK, Kadir MM: Gender differences and clustering pattern of behavioural risk factors for chronic non-communicable diseases: Community-based study from a developing country. Chronic Illn 2010, 6(3):163–170. • Convenient online submission 30. Arambepola C, Allender S, Ekanayake R, Fernando D: Urban living and obesity: Is it independent of its population and lifestyle characteristics? Trop Med Int Health 2008, 13(4):448–457. 31. Katulanda P, Jayawardana R, Ranasinghe P, Rezvi Sheriff M, Matthews DR: Physical activity patterns and correlates among adults from a developing country: The Sri Lanka Diabetes and Cardiovascular Study. Public Health Nutr 2013, 16(9):1684–1692. 31. Katulanda P, Jayawardana R, Ranasinghe P, Rezvi Sheriff M, Matthews DR: Physical activity patterns and correlates among adults from a developing country: The Sri Lanka Diabetes and Cardiovascular Study. Public Health Nutr 2013, 16(9):1684–1692.
https://openalex.org/W4200244620	https://bulletin.knob.nl/index.php/knob/article/download/728/753	Dutch	null	Estate landscapes in the Netherlands	Bulletin KNOB/Bulletin	2,021	cc-by	10,745	PAGINA’S 4-23 BUITENPLAATSEN- LANDSCHAPPEN IN NEDERLAND ‘GANSCHE STREKEN DES LANDS WAREN BEDEKT MET BUITENHUIZEN’ PAGINA’S 4-23 BUITENPLAATSEN- LANDSCHAPPEN IN NEDERLAND ‘GANSCHE STREKEN DES LANDS WAREN BEDEKT MET BUITENHUIZEN’ PAGINA’S 4-23 4 BUITENPLAATSEN- LANDSCHAPPEN IN NEDERLAND ‘GANSCHE STREKEN DES LANDS WAREN BEDEKT MET BUITENHUIZEN’ Hans Renes voor de vaste of tijdelijke woonplaatsen van stedelin gen in het buitengebied een groot aantal verschillende termen in gebruik. Martin van den Broeke beschrijft een rondreis door Zuid-Beveland in 1774, waarbij de reizigers een lange rij van kennissen bezochten op zomerverblijven die werden aangeduid met termen als lusthof, lustplaats, landhoeve, lusthoeve, zomer verblijfplaats, hofstede, hoeve, huis en zelfs boeren hoeve.6 Veel van die termen laten mooi zien hoe de stedelingen werden aangetrokken door het schijnbaar ongecompliceerde landleven. Tegelijk maken ze het leven van historische onderzoekers lastiger: de ver schillende termen overlapten elkaar en betekenissen veranderden in de loop van de tijd. De objecten zelf toonden ook een grote variatie: in omvang alleen al liep de reeks van de enorme, met buitenlandse land huizen vergelijkbare, buitenplaatsen van de stadhou ders tot de tuintjes van de lagere middenklasse.7 Een aanduiding als ‘hofstede’ kon bijvoorbeeld zowel dui den op een boerderij met herenkamer als op een grote buitenplaats.8 DE BUITENPLAATS: EEN INLEIDING In de uitgebreide literatuur over buitenplaatsen gaat veruit de meeste aandacht uit naar individuele huizen en tuinen. In de laatste decennia is daarbij meer inte resse gekomen voor de relatie tussen buitenplaatsen en hun omgeving. In dit artikel willen we nog een stap verder gaan en het begrip buitenplaatsenlandschap centraal stellen.1 Dat is een relatief nieuw begrip, dat nog niet zo eenvoudig te definiëren is. Een buitenplaats wordt door de Nederlandse Kaste lenstichting gedefinieerd als: ‘ieder verblijf, veelal met tuin en park en bijgebouwen, dat door de eigenaar werd gesticht met het oogmerk om voor kortere of langere tijd op het platteland te vertoeven’. Daaraan wordt toe gevoegd: ‘De stichting had ten doel de gebruikers van de rust en de landelijke omgeving te laten genieten. Tegelijk diende het als statussymbool en bood het de mogelijkheid het beheer te voeren over de eventueel aan de buitenplaats verbonden industriële, landbouw- en bosbouwbedrijven.’2 In deze definitie ontbreekt het argument van geldbelegging. In Amsterdam, net als eerder in Venetië, maakten de risicovolle investerin gen in handel en scheepvaart gaandeweg plaats voor zekerder beleggingen in onroerend goed en (in Amster dam) in aandelen.3 Een buitenplaats kon deel uitmaken van een land goed dat daarnaast landbouwgrond en bossen omvat te. Een landgoed beschikte daarmee over inkomsten die het voortbestaan veilig stelden. Daarentegen was een buitenplaats vooral een ‘plek van consumptie’, die weliswaar meestal wel enige inkomsten genereerde, maar waar toch altijd geld bij moest. b 1. Fragment van de plattegrond van Amsterdam door J. Blaeu, 1649. Dit toont de tuintjes van de middenklasse net buiten de stadsversterkingen, die ontstonden doordat een van de kenmerkende strookvormige weilanden werd verkaveld waarbij een centrale laan werd aangelegd met aan beide zijden tuintjes (Universiteitsbibliotheek Utrecht) PAGINA’S 4-23 4 BUITENPLAATSEN- LANDSCHAPPEN IN NEDERLAND ‘GANSCHE STREKEN DES LANDS WAREN BEDEKT MET BUITENHUIZEN’ Hans Renes De eigenaar van een buitenplaats was afhankelijk van inkomsten uit andere bronnen, zoals handel, industrie, grondstof fenwinning, koloniale exploitatie of een groot geërfd kapitaal.9 In de praktijk is de overgang tussen buiten plaats en landgoed vloeiend, omdat veel buitenplaat sen, bijvoorbeeld langs de Vecht, voortkwamen uit een boerderij waaraan een herenkamer of een landhuis werd toegevoegd. Naast de daardoor ontstane buiten plaats bleef de boerderij functioneren en voor inkom sten zorgen. Zo gedefinieerd vormen buitenplaatsen een verbin ding tussen stad en platteland. Het initiatief lag bij een stedeling die een deel van het jaar buiten de stad wilde wonen. Dat maakt dat we de term buitenplaats formeel niet kunnen gebruiken voor huizen in het landelijk gebied die het hoofdverblijf van de bewoners vormden, zoals de landhuizen die de centra vormden van land goederen en die het hele jaar bewoond waren.4 De scheidslijn is echter niet altijd duidelijk, zeker niet na dat in de loop van de negentiende eeuw steeds meer huizen die als buitenplaats waren gebouwd perma nent werden bewoond. Het laatste werd mogelijk ge maakt door verbeterd comfort (verwarming) en snel lere verbindingen. Misschien schieten we ons doel voorbij als de term buitenplaats op die huizen niet meer van toepassing zou zijn. Een andere onduidelijk heid betreft de minimale omvang. De definitie kan ook de vele volkstuinachtige complexen buiten de stad omvatten, waar de eigenaren ook wel eens langer dan een dag verbleven. In de praktijk worden die niet als buitenplaats aangeduid, maar de grens is vaag (afb. 1). Buitenplaatsen kunnen worden bekeken als indivi duele objecten, bestaande uit een huis en een aanslui tende tuinaanleg. Ze kunnen ook in hun ruimere con text worden beschreven, als deel van een landgoed of in samenhang met het omringende landschap. In het laatste geval kunnen we bijvoorbeeld kijken naar lanen en zichtassen. In de internationale literatuur bestaat hiervoor de term ‘estate landscape’,10 wat we zouden kunnen vertalen met de term buitenplaatslandschap of, in de terminologie van de provincies Utrecht en Zuid-Holland, buitenplaatsbiotoop.11 BULLETIN KNOB 2021 • 4 5 b De term buitenplaats is tegenwoordig algemeen voor een landhuis met tuinaanleg, maar kwam pas in de achttiende eeuw in gebruik en werd pas in de negen tiende eeuw de standaardaanduiding.5 Voordien was BULLETIN KNOB 2021 • 4 Al deze termen doen echter geen recht aan de situatie, die we nergens mooier dan in Nederland kunnen aan treffen, van een landschap dat wordt gekenmerkt door een aaneengesloten reeks buitenplaatsen.12 Voor een dergelijk landschap – het thema van deze bijdrage – geef ik daarom de voorkeur aan de term buitenplaatsen landschap.13 Dit kan worden gedefinieerd als een reeks 5 2. Walcheren in kaart gebracht door D.W.C. en A. Hattinga, 1749-1750 (Zeeuws Archief) 2. Walcheren in kaart gebracht door D.W.C. en A. Hattinga, 1749-1750 (Zeeuws Archief) plaatsennetwerk, geïntroduceerd door de landschaps architecte Dominique Blom.16 van naast elkaar gelegen buitenplaatsen die samen een landschapsarchitectonisch ensemble vormen.14 Binnen zo’n reeks kunnen de afzonderlijke buiten plaatsen of landgoederen door wegen en zichtassen met elkaar verbonden zijn of een gedeelde oorsprong hebben. We zullen hieronder enkele voorbeelden be spreken. Dergelijke linten werden ook in het verleden wel her kend; ze werden aangeduid als lustwarande17 of, met een verwijzing naar het pastorale ideaallandschap van de oude Grieken, als Arcadië.18 De term lustwarande is interessant. Het begrip warande komt sinds de late middeleeuwen voor als aanduiding van wildparken, besloten jachtgebieden. Meer specifiek was een waran de een wildpark waarin kleine dieren zoals konijnen werden gehouden. Dergelijke jachtgebieden kenmerk ten zich door een afwisselend ‘parkachtig’ landschap, dat ook esthetisch werd gewaardeerd.19 Bij een vroege vermelding van de term lustwarande, in de achttiende- eeuwse boekenreeks Tegenwoordige Staat der Vereenig- de Nederlanden als aanduiding voor de omgeving van BULLETIN KNOB 2021 • 4 • Directe omgeving van Amsterdam 100 • Watergraafsmeer 130 • Haarlem en omstreken 105 • Amstel, Gein, Angstel, Vecht, Bijlmermeer 150 • ’s-Graveland 26 Totaal 511 • Directe omgeving van Amsterdam 100 • Watergraafsmeer 130 • Haarlem en omstreken 105 • Amstel, Gein, Angstel, Vecht, Bijlmermeer 150 • ’s-Graveland 26 Totaal 511 VAN BUITENPLAATS NAAR BUITENPLAATSEN LANDSCHAP Er is tamelijk veel geschreven over de (mogelijke) rede nen om voor aanleg van buitenplaatsen voor bepaalde vestigingsplaatsen te kiezen. In sommige gevallen gaan buitenplaatsen terug tot ouder familiebezit, dat kon be staan uit een middeleeuws kasteel of landhuis, maar ook uit een enkele boerderij. Andere buitenplaatsen werden gebouwd door de eigenaar van een aangren zend industrieel bedrijf. Bij Utrecht stond bijvoorbeeld Rotsoord naast een steenfabriek en Zijdebalen bij een zijdefabriek. Door de voorkeur voor bereikbaarheid over water, de eis van een niet te grote afstand tot de stad en de aan trekkelijkheid van grenssituaties zoals de binnen duinrand, waar het vlakke landbouwgebied grensde aan de jachtgebieden in de wilde duinen, werden die vele buitenplaatsen geconcentreerd in een aantal lin ten. De linten van buitenplaatsen in laag Nederland zijn, voor zover bekend, uniek in de wereld. Voor buitens van stedelingen was de afstand van be lang die de eigenaar wilde afleggen tussen het huis in de stad en de buitenplaats.22 Concentraties van buiten plaatsen konden ontstaan waar een aantrekkelijk landschap samenviel met een goede bereikbaarheid vanuit een nabije stad. In veel gevallen begon zo’n lint met een klein aantal huizen annex tuinen en kwamen er in de loop van de tijd steeds meer buitenplaatsen bij. We zouden kun nen stellen dat buitenplaatsbezitters lijken op heden daagse toeristen die als reden voor een bezoek aan een Spaanse costa aangeven dat ze worden aangetrokken door het strand, het weer of zelfs het mooie land schap, maar die in feite vooral aangetrokken worden door andere toeristen. Dagboeken van buitenplaats bezitters tonen het eindeloze getuttel van de intensie ve onderlinge contacten tussen de lokale stedelingen op het platteland. Daarnaast had de concentratie van buitenplaatsen en landgoederen zeker ook praktische voordelen. De aanwezigheid van andere buitenplaat sen versterkte de hoge esthetische kwaliteit van het landschap. Ook was deskundig personeel makkelijker te vinden.27 Dat niet iedere stad evenveel buitenplaatsen heeft voortgebracht, had ook te maken met de stedelijke be volking zelf: nodig was vooral een elite van redelijke omvang die beschikte over voldoende financiën om zich buitenplaatsen te veroorloven. Ook was een sfeer nodig waarin de leden van deze elite elkaar stimuleer den om een huis buiten de stad te kopen of te bouwen. Internationaal vinden we dergelijke concentraties zo wel rond belangrijke stedelijke centra van handel en nijverheid als rond de grote hoven van vroegmoderne centrale staten. BULLETIN KNOB 2021 • 4 7 BULLETIN KNOB 2021 • 4 BULLETIN KNOB 2021 • 4 6 BULLETIN KNOB 2021 • 4 De belangstelling voor dergelijke buitenplaatsen landschappen groeit en dat leidt al direct tot de intro ductie van een groot aantal termen. De provincie Utrecht spreekt van een buitenplaatszone, Zuid- Holland van een landgoedzone (ietwat verwarrend omdat het in veel gevallen om buitenplaatsen gaat). Omdat de buitenplaatsen in zo’n zone meestal aan een weg of water liggen, is de term buitenplaatslint wel goed getroffen.15 Nog een andere term is buiten 6 de buitenplaats Honselaarsdijk bij Naaldwijk, lijkt de associatie met jacht nog aanwezig.20 wel uitzonderlijk groot. Roel Mulder gaf een over zicht van buitenplaatsbezitters per inkomensklasse in 1742. Van de Amsterdammers met inkomens tussen 4000 en 7000 gulden beschikte al een kwart over een buitenplaats, van degenen met inkomens boven 7000 gulden was dat twee derde (afb. 3).23 De buitenplaatsen zelf waren relatief klein, zeker in vergelijking met de Franse en de latere Engelse. 24 Alleen al op Walcheren lagen rond 1680 ruim vijftig buitenverblijven, een aan tal dat groeide naar ruim 130 in het midden van de achttiende eeuw. De meeste behoorden toe aan inwo ners van de steden Middelburg, Vlissingen en, in min dere mate, Veere.25 Marc Glaudemans schatte op basis van kaartstudie dat in het begin van de achttiende eeuw rond Amsterdam ruim vijfhonderd buitenplaat sen lagen.26 Hij geeft de volgende aantallen: Wel moeten we bij al deze termen bedenken dat de betreffende gebieden een oudere geschiedenis hebben en dat buitenplaatsen slechts een nieuwe laag aan brengen in een landschap dat al was ingericht. Vaak is het zelfs de oudere agrarische laag die uiteindelijk wint, waar de boerderijen de buitenplaatsen overle ven. De Beemster is werelderfgoed geworden op basis van de oorspronkelijke agrarische inrichting die nog steeds goed herkenbaar is. Het buitenplaatsenland schap vertegenwoordigde hier een kortstondige fase in de geschiedenis, die in de nominatie als werelderf goed nauwelijks een rol heeft gespeeld.21 De nadruk in onderstaande tekst ligt op twee perio den: de zeventiende en achttiende eeuw, gekenmerkt door geometrische tuinen en door een voorkeur voor het vlakke land, en de negentiende en vroege twintig ste eeuw, gekenmerkt door landschappelijke tuinen en een voorkeur voor de reliëfrijkere zandgebieden. BULLETIN KNOB 2021 • 4 De Nederlandse provincies Holland, Zeeland en Utrecht nemen internationaal en nationaal een eigen positie in door het enorme aantal buitenplaatsen (afb. 2). In de noordelijke Nederlanden was de welvaart zeker niet eerlijk over de bevolking verdeeld, maar de groep die zich buitenplaatsen kon veroorloven, was Toch is ook dit niet het hele verhaal. In een aantal gevallen gaan concentraties van buitenplaatsen terug op familiebanden, op de doelbewuste ontwikkeling 3. Kaart van de Amsteldijk met buitenplaatsen tussen de Utrechtse Poort en het Groote Loopveld (nu Ouderkerkerlaan) door E. Florijn, 1779 (Stadsarchief Amsterdam) 3. Kaart van de Amsteldijk met buitenplaatsen tussen de Utrechtse Poort en het Groote Loopveld (nu Ouderkerkerlaan) door E. Florijn, 1779 (Stadsarchief Amsterdam) et buitenplaatsen tussen de Utrechtse Poort en het Groote Loopveld (nu Ouderkerkerlaan) door E. Florijn, 1779 van buitenplaatsenlandschappen of, in een enkel ge val, komt zelfs een zekere eenheid tot stand door de werkzaamheden van een tuinarchitect.28 In de volgen de paragrafen nemen we een aantal voorbeelden on der de loep. Remmet van Luttervelt over de buitenplaatsen aan de Vecht en over de Stichtse Lustwarande.32 In de jaren zeventig lijkt de belangstelling voor een dergelijke regionale benadering te zijn toegenomen.33 Een belangrijke pionier was Henri van der Wyck, die al in de jaren zeventig kaarten publiceerde waarop de lijn- en vlakelementen waren geaccentueerd die be hoorden bij de verschillende buitenplaatsen in het ge bied. In 1977 publiceerde hij kaarten voor de Stichtse Lustwarande en Kennemerland, twee jaar later ge volgd door een soortgelijke kaart van de oostelijke Veluwezoom.34 De kaarten laten zien hoe de buiten plaatsen hier een bijna aaneengesloten gebied vorm den en hoe verschillende afzonderlijke buitens door lanen met elkaar waren verbonden. Van der Wyck ging daarmee verder dan de eerdere auteurs, door niet uit te gaan van gebieden met een groot aantal buitenplaat sen maar van een samenhangend geheel. Dit thema nummer is daarmee schatplichtig aan Van der Wyck. Contemporaine waarnemers herkenden de buiten plaatsenlandschappen al. In de achttiende eeuw be stond een markt voor platenboeken met titels als Verscheyde gesigten van de vermaarde rievier de buyten Amstel (een van de eerste, verschenen kort voor 1716), De zegepralende Vecht (1719), Het verheerlykt Water- graefs- of Diemer-Meer (1725)29 en Amstel’s Lustwaran- de, Rhynlands fraaiste gezichten (1732). Een mooi voor beeld is Het zegenpralent Kennemerlant (ca. BULLETIN KNOB 2021 • 4 BULLETIN KNOB 2021 • 4 8 BULLETIN KNOB 2021 • 4 BULLETIN KNOB 2021 • 4 1730), met een overzichtskaart en gravures van de afzonderlijke plaatsen, ‘alle naer ’t leven op het naeukeurighste en met de uiterste oplettenheit door H. de Leth in den jaere 1728 getekent, en dat wel zonder het minste stipje aen eenigh gebouw of tuinsieradie over te slaen’.30 Christian Bertram deed voor de huidige provincie Noord-Holland de interessante observatie dat derge lijke platenboeken pas verschenen nadat de buiten plaatsen en de bijbehorende tuinen in de periode 1700- 1730 op grotere schaal waren herbouwd.31 In de volgende paragrafen worden enkele Neder landse buitenplaatsenlandschappen uit de zeventien de, achttiende en negentiende eeuw beschreven. BUITENPLAATSENLANDSCHAPPEN VAN DE ZEVENTIENDE EN ACHTTIENDE EEUW In de twintigste eeuw werden buitenplaatsen het on derwerp van historisch onderzoek. Sindsdien is een enorm aantal historische publicaties verschenen, waarvan de meeste over een enkele buitenplaats gaan. Toch waren er al vroeg auteurs die zich op regionale schaal bewogen. Te denken valt aan de publicaties van Hoewel er zeker voorgangers aan te wijzen zijn, begon de glorietijd van de buitenplaatsen in het huidige Nederland in de zeventiende eeuw.35 Een aantal bui tenplaatsen gaat terug op middeleeuwse kastelen of ridderhofsteden, soms omdat de adellijke eigenaren 8 naar Weesp. Een andere route naar de Vecht liep via de Amstel naar Ouderkerk en vandaar via de Holendrecht naar Abcoude, en vervolgens over de Angstel en via de Nieuwe Wetering naar Nieuwersluis aan de Vecht.39 De spreiding van buitenplaatsen wijst erop dat de laatste verbinding het belangrijkst was. De Vecht zelf werd in het netwerk van trekvaarten opgenomen door de aan leg van een jaagpad in de jaren 1626-1628 (afb. 5 en 6).40 Wel dient opgemerkt dat de nadruk in de literatuur te sterk op watertransport ligt. Zeker over korte afstan den liet men zich ook wel per koets vervoeren. Een kaart van de buitenplaatsen rond Leiden maakt bij voorbeeld duidelijk dat de buitenplaatsen zowel langs wegen als langs de Oude Rijn (waar Leiderdorp in 1660 door een buitenlandse reiziger werd omschreven als ‘meer paleizen dan boerenhuizen’) en de trekvaarten lagen.41 Van de buitenplaatsen rond Den Haag werd een deel gebouwd aan de Vliet, maar ook hier lagen er veel aan landwegen, zeker nadat de belangrijkste uit valswegen in de loop van de zeventiende eeuw werden bestraat.42 In Zeeland lijkt het reizen van stad naar bui tenplaats vooral over land te hebben plaatsgevonden. De voorkeur ging daarbij uiteraard uit naar wegen die, door hun ligging op dijken en stroomruggen, een groot deel van het jaar begaanbaar waren.43 De grote zeventiende-eeuwse droogmakerijen wer met hun tijd meegingen, soms ook omdat stedelijke patriciërs niet alleen op zoek waren naar een plek op het platteland maar ook naar een adellijke status en uitstraling.36 Toch waren de buitenplaatsen van de ze ventiende eeuw ook iets nieuws. Het waren, zoals de term buitenplaats al laat zien, huizen die door de ste delijke elite werden gebouwd vanuit een behoefte om een deel van de tijd in het buitengebied door te bren gen. BUITENPLAATSENLANDSCHAPPEN VAN DE ZEVENTIENDE EN ACHTTIENDE EEUW Die buitenplaatsen waren over een groot deel van het land verspreid, maar een aantal gebieden was wel bijzonder in trek (afb. 4). naar Weesp. Een andere route naar de Vecht liep via de Amstel naar Ouderkerk en vandaar via de Holendrecht naar Abcoude, en vervolgens over de Angstel en via de Nieuwe Wetering naar Nieuwersluis aan de Vecht.39 De spreiding van buitenplaatsen wijst erop dat de laatste verbinding het belangrijkst was. De Vecht zelf werd in het netwerk van trekvaarten opgenomen door de aan leg van een jaagpad in de jaren 1626-1628 (afb. 5 en 6).40 Wel dient opgemerkt dat de nadruk in de literatuur te sterk op watertransport ligt. Zeker over korte afstan den liet men zich ook wel per koets vervoeren. Een kaart van de buitenplaatsen rond Leiden maakt bij voorbeeld duidelijk dat de buitenplaatsen zowel langs wegen als langs de Oude Rijn (waar Leiderdorp in 1660 door een buitenlandse reiziger werd omschreven als ‘meer paleizen dan boerenhuizen’) en de trekvaarten lagen.41 Van de buitenplaatsen rond Den Haag werd een deel gebouwd aan de Vliet, maar ook hier lagen er veel aan landwegen, zeker nadat de belangrijkste uit valswegen in de loop van de zeventiende eeuw werden bestraat.42 In Zeeland lijkt het reizen van stad naar bui tenplaats vooral over land te hebben plaatsgevonden. De voorkeur ging daarbij uiteraard uit naar wegen die, door hun ligging op dijken en stroomruggen, een groot deel van het jaar begaanbaar waren.43 b Het grootste deel van de buitenplaatsen lag aan be vaarbaar water, binnen een straal van dertig kilometer van een stad.37 Dat maakte het mogelijk om binnen een dag per boot van stadshuis naar buitenhuis te kun nen komen. De oevers van deze wateren waren meestal bezet door veel buitenplaatsen, wat ongetwijfeld het cultiveren van de nodige sociale contacten zal hebben vereenvoudigd. Belangrijke waterverbindingen waren rivieren zoals de Amstel en de Vecht. Voor Amsterdam was ook het IJ van betekenis, een zeearm die doorliep tot aan Velzen en Beverwijk. Door de drooglegging van het IJ (de IJpolders, rond 1872), de verstedelijking en de infrastructuur is de vroegere oriëntatie van de buiten plaatsen rond Velzen nu volkomen onherkenbaar.38 4. Kaart van een gedeelte van Noord-Kennemerland door H. de Leth, 1728. De buitenplaatsen rond Velsen lagen in een boog om het westelijke uiteinde van het IJ (de Wijkermeer) en waren alle via een kort kanaal met dat meer verbonden (Noord-Hollands Archief) BULLETIN KNOB 2021 • 4 De grote zeventiende-eeuwse droogmakerijen wer den gefinancierd door dezelfde kooplieden en patrici ërs die ook buitenplaatsen aanlegden. De investeer ders kregen land toegewezen in de nieuwe polders en beschikten daarmee over boerderijen waar ze een De natuurlijke wateren werden in het midden van de zeventiende eeuw aangevuld met een netwerk van trekvaarten. Vanuit Amsterdam liep er een langs de Watergraafsmeer naar Diemerbrug, om zich daar te vertakken in vaarten naar Muiden en via de Gaasp herenkamer aan konden bouwen en later een landhuis naast konden zetten. Ten slotte waren er de randen van de hogere zand gronden, zoals de duinen en de stuwwallen van het Gooi en de Veluwe. Die hogere gebieden zelf werden minder aantrekkelijk gevonden dan de rijke veen- en kleilandschappen, maar boden wel mogelijkheden voor jacht, een vorm van netwerkvermaak dat verge lijkbaar is met het huidige golf. Zowel aan de rand van het Gooi (’s-Graveland) als aan de duinrand (Elswout, Groenendaal) werd zand afgegraven.44 Dat leverde in komsten, een vlak stuk land en een waterverbinding (de vaart die nodig was voor de afvoer van het zand) en daarmee een goede basis voor een buitenplaats. g p De ontwikkeling van buitenplaatsen laat daarnaast de omvang en rijkdom van de stedelijke elites zien. In Holland was Amsterdam in de zeventiende en acht tiende eeuw veruit de grootste en rijkste stad en dat vertaalde zich in het enorme aantal buitenplaatsen binnen bereik van deze stad.45 Een tweede grote con centratie lag rond het bestuurscentrum Den Haag. Daarnaast lagen grote aantallen buitenplaatsen rond steden als Haarlem46, Leiden47, Delft, Rotterdam48 en Dordrecht49. De verschillende buitenplaatsenland schappen overlapten. Zo liep de Amsterdamse invloed tot aan Utrecht en lagen de buitenplaatsen rond Haar lem binnen de Amsterdamse invloedssfeer. Hier is interessant dat bijvoorbeeld de buitenplaatsen in Heemstede nabij Haarlem vaak door de lokale bevol king gesticht zijn, maar vanaf de jaren dertig van de zeventiende eeuw door Amsterdammers werden ge kocht en later samengevoegd tot grotere buitenplaat sen.50 In Zuid-Holland vloeiden de invloeden van Den Haag en Delft samen in het Westland.51 t Ook aan de Vecht verdrong de kapitaalkrachtige Am sterdamse elite de Utrechtse, zelfs tot vlak voor de poorten van Utrecht.52 De Utrechtse bovenlaag week uit naar gebieden aan de oostzijde van de stad, bijvoor beeld rond De Bilt, waar een hoeveelheid voormalig kloosterbezit ter beschikking kwam. Het ging om de Sint-Laurensabdij in Oostbroek, gesticht in 1121, die beschikte over een aanzienlijk en grotendeels aaneen gesloten grondbezit. Geëxploiteerd werd het grondbe zit vanuit een reeks kloosterboerderijen of ‘uithoven’. Toen het Onze Lieve Vrouweklooster (beter bekend als het Vrouwenklooster) van de abdij werd afgesplitst, ging een deel van de uithoven mee. Na de Reformatie werden de kloosters opgeheven en de bezittingen over gedaan aan de Staten van Utrecht. Die verkochten ge bouwen en landerijen grotendeels tussen 1640 en 1680 5. Gezicht vanaf de Ringdijk van de Watergraafsmeer in oostelijke richting naar de Diemerbrug door D. Stopendaal, 1725 (Stadsarchief Amsterdam) 5. Gezicht vanaf de Ringdijk van de Watergraafsmeer in oostelijke richting naar de Diemerbrug door D. Stopendaal, 1725 (Stadsarchief Amsterdam) 5. Gezicht vanaf de Ringdijk van de Watergraafsmeer in oostelijke richting naar de Diemerbrug door D. Stopendaal, 1725 (Stadsarchief Amsterdam) Amsterdam Rotterdam Den Haag Utrecht Groningen Noordbroek Nieuwe Schans Appingedam Delfzijl Uithuizen Warffum Ulrum Dokkum Leeuwarden Franeker Harlingen Sneek Assen Bolsward Workum Staveren Lemmer Hasselt Zwolle Kampen Enkhuizen Schagen Alkmaar Edam Monnikendam Haarlem Weesp Naarden Harderwijk Amersfoort Leiden Delft Maassluis Gouda Vreeswijk/Vianen Gorinchem Arnhem Nijmegen Dordrecht Muiden Hoorn Purmerend Zuidbroek Winschoten Veendam trekvaarten tot 1650 trekvaarten aangelegd 1650-1665 6. De aanleg van trekvaarten begon in laag Nederland rond 1630. In 1665 was een aaneengesloten netwerk tot stand gekomen (Ton Markus, Faculteit Geo wetenschappen Utrecht) BULLETIN KNOB 2021 • 4 11 Noordbroek Groningen Leeuwarden Zuidbroek Assen Sneek Workum Staveren Lemmer Enkhuizen Hoorn Alkmaar Hasselt Kampen Edam Purmerend trekvaarten tot 1650 trekvaarten aangelegd 1650-1665 Harderwijk Amsterdam Naarden BULLETIN KNOB 2021 • 4 Amersfoort Utrecht Den Haag Gouda Vreeswijk/Vianen Maassluis 6. De aanleg van trekvaarten begon in laag Nederland rond 1630. In 1665 was een aaneengesloten netwerk tot stand gekomen (Ton Markus, Faculteit Geo wetenschappen Utrecht) Nijmegen Gorinchem Dordrecht 11 7. Buitenplaatsen aan de noordoostzijde van Utrecht die teruggaan op voormalige kloosters en de bijbehorende uithoven, aangegeven op de manuscript-topografische kaart van ca. 1840. Het ingekleurde gebied in het midden is het huidige Utrecht Science Park (Ton Markus, Faculteit Geowetenschappen Utrecht) 7. Buitenplaatsen aan de noordoostzijde van Utrecht die teruggaan op voormalige kloosters en de bijbehorende uithoven, aangegeven op de manuscript-topografische kaart van ca. 1840. Het ingekleurde gebied in het midden is het huidige Utrecht Science Park (Ton Markus, Faculteit Geowetenschappen Utrecht) keld. Een van de mooiste, zij het onvoltooide, voorbeel den van zo’n planmatig buitenplaatsenlandschap is dat langs de weg van Utrecht naar Amersfoort, de ‘wegh der weegen’. Ontwerper hiervan was de archi tect van het Amsterdamse stadhuis, Jacob van Cam pen, die zich blijkbaar liet leiden door Italiaanse trak taten. Everard Meyster vergeleek de weg zelfs met de Via Appia bij Rome. De aanleg, waarschijnlijk op initi atief van de stad Amersfoort, begon in 1647 maar kwam pas in 1652 goed op gang. Aan het aanvankelijke plan voor de weg waren toen intussen vakken voor de ontwikkeling van buitenplaatsen toegevoegd. De weg en de vakken voor de buitenplaatsen, zeventien aan elke zijde, werden in het terrein uitgezet. De weg kreeg een breedte van maar liefst zestig meter en moest door de aangrenzende landeigenaren worden beplant (afb. 8). Het project is geen groot succes geworden: uit eindelijk zijn er slechts enkele huizen gebouwd.56 aan de regionale elite. Deze vormde de forse boerde rijen en stukken land in de loop van de tijd om tot bui tenplaatsen. Uit de Laurensabdij kwam het landgoed Oostbroek voort, uit het Vrouwenklooster de buiten plaats ’t Klooster, ook wel Koelenberg genoemd. De uithoven vormden de kern van landgoederen als Houdringe, Beerschoten en Vollenhoven in De Bilt en Nienoord bij Bunnik (afb. 7).53 8. De Amersfoortse- weg op de tweede editie van de Nieuwe kaart van den Lande van Utrecht, door B. du Roy, 1743 (Universiteitsbiblio- theek Utrecht) BULLETIN KNOB 2021 • 4 BULLETIN KNOB 2021 • 4 12 b Vergelijkbare ontwikkelingen vonden plaats rond Arnhem, waar de lokale elite de bezittingen van het klooster Mariënborn aan zich wist te trekken en daar vanaf 1640 een reeks buitenplaatsen stichtte.54 In Zee land werd het geestelijk grondbezit nog sneller ver kocht, al tussen 1576 en 1578, en ook daar vinden we een aantal buitenplaatsen op voormalig geestelijk grondbezit.55 BULLETIN KNOB 2021 • 4 Er zijn enkele gevallen bekend waarin een groep bui tenplaatsen aaneensluitend parallel werden ontwik 12 8. De Amersfoortse- weg op de tweede editie van de Nieuwe kaart van den Lande van Utrecht, door B. du Roy, 1743 (Universiteitsbiblio- theek Utrecht) BULLETIN KNOB 2021 • 4 13 13 Interessant als planmatige ontwikkeling is ook ’s-Graveland, aan de westzijde van het Gooi. De Staten gaven in 1625 aan een aantal Amsterdamse patriciërs octrooi om hier zand te winnen. Het gebied werd in 1634 in 27 kavels verdeeld, die werden verloot onder de deelnemers. Enkele jaren later kwam de zandwinning op gang, na de aanleg van de ’s-Gravelandsevaart (1638), die de afvoer van zand naar Amsterdam moge lijk maakte. De vaart had twee takken: een zuidelijke door de Horstermeer, die vooral werd gebruikt voor zandafvoer, en een noordelijke naar de Vecht bij Uiter meer. Op de laatste werd al in 1644 een trekschuit dienst op Amsterdam geopend.57 Het meeste zand zal in de eerste jaren zijn gewonnen, maar het gebied is nooit volledig afgegraven.58 begin al de bedoeling was. Als we de ontwikkeling van dit gebied in tijdbalken vatten, wordt duidelijk dat al snel na de afzanding de eerste ‘hofsteden’, vaak boer derijen met een herenkamer, werden gebouwd (afb. 9).59 Het lijkt erop dat de afzanders al vroeg, mis schien zelfs al direct, plannen hadden om het gebied na afzanding in te richten voor landbouw, met boerde rijen annex herenkamers (afb. 10). De bouw van echte herenhuizen begon pas veel later en toont een zeer sterke spreiding in de tijd. Het bekendste buitenplaatsenlandschap in Neder land is wel dat langs de Vecht (afb. 11). Interessant is dat de bouw van deze reeks buitenplaatsen, hoewel een Amsterdamse aangelegenheid, begon aan de noordzijde van de stad Utrecht. Al in 1608 kocht hier de handelaar in huiden en leer Jan Jacobsz. Bal (1541-1624) de boerderij De Gouden Hoeff in Maarssen. Later zou Bal zich in toespeling op zijn beroep Huyde coper noemen. BULLETIN KNOB 2021 • 4 In de vier decennia nadien kochten hij en zijn zoon Joan (1625-1704) meer land in deze Na de afgraving resteerde een vlak landschap, dat geschikt was voor landbouw maar ook voor de aanleg van geometrische tuinen. De meeste kavels vinden we later terug als buitenplaatsen. Net als in de droogma kerijen moeten we ons hier afvragen of dat vanaf het 9. De ontwikkeling van de buitenplaatsen in 's-Graveland (het gebied van de concessie van 1625) in tijdslijnen (Ton Markus, Faculteit Geowetenschappen Utrecht) 9. De ontwikkeling van de buitenplaatsen in 's-Graveland (het gebied van de concessie van 1625) in tijdslijnen (Ton Markus, Faculteit Geowetenschappen Utrecht) BULLETIN KNOB 2021 • 4 14 BULLETIN KNOB 2021 • 4 14 14 10. Brambergen in ’s-Graveland biedt nog een mooi voorbeeld van een boerderij met herenkamer, 1963 (foto G.J. Dukker, Rijksdienst voor het Cultureel Erfgoed) 10. Brambergen in ’s-Graveland biedt nog een mooi voorbeeld van een boerderij met herenkamer, 1963 (foto G.J. Dukker, Rijksdienst voor het Cultureel Erfgoed) 11. Ontwikkeling van de buitenplaatsen langs de Vecht (Ton Markus, Faculteit Geowetenschappen Utrecht) BULLETIN KNOB 2021 • 4 15 11. Ontwikkeling van de buitenplaatsen langs de Vecht (Ton Markus, Faculteit Geowetenschappen Utrecht) BULLETIN KNOB 2021 • 4 15 15 b 12. Kaart van Loenen met een aaneengesloten reeks buiten- plaatsen, kopergravure door C.C. van Bloemswaerdt, 1727 (Universiteitsbibliotheek Utrecht) b 12. Kaart van Loenen met een aaneengesloten reeks buiten- plaatsen, kopergravure door C.C. van Bloemswaerdt, 1727 (Universiteitsbibliotheek Utrecht) veel langzamer op gang. In de Watergraafsmeer, die droogviel in 1629, werden in de loop van de zeventien de eeuw hofsteden en buitenplaatsen gebouwd, maar volgde de grote doorbraak pas na 1700. Glaudemans vermeldt dat er op ‘een zeker moment’ 50 hofsteden, 31 buitenplaatsen en 53 (grotendeels openbare) ple ziertuinen lagen, waarvan er slechts 14 vóór 1700 en slechts drie vóór 1651 waren aangelegd.63 omgeving. De laatste, die zich later Joan Huydecoper van Maarsseveen liet noemen, bouwde de Gouden Hoeff in 1629 uit tot de buitenplaats Goudestein. Veel van het aangekochte land werd verkaveld en in delen verkocht voor de aanleg van buitenplaatsen. Het was een succesvolle projectontwikkeling. De reeks buiten plaatsen die hier tot stand kwam, droeg bij aan het prestige van de al aanwezige buitens.60 Onlangs konden we ook voor de Purmer vaststellen dat de ontwikkeling geleidelijk verliep (afb. 13. De buitenplaatsen in de Purmer in tijdslijnen (Ton Markus, Faculteit Geo wetenschappen Utrecht) BULLETIN KNOB 2021 • 4 13).64 In de eerste jaren nadat de Purmer in 1622 droogviel, werden twee buitenplaatsen en een ‘ridderhofstad’ gebouwd, al waren er mogelijk wel al snel enkele boerderijen met herenkamers. In 1700 lagen er acht buitenplaatsen, daarna nam het aantal langzaam toe, tot in het mid den van de achttiende eeuw het hoogste aantal van veertien werd bereikt. Daarna begon een geleidelijke afname, tot er in het midden van de negentiende eeuw nog maar één over was. Vanuit de oudste kern bij Maarssen werd het buiten plaatsenlint langs de Vecht uitgebreid en verdicht. De grootste dichtheid, met een aaneengesloten reeks buitenplaatsen, ontstond in Maarssen, Breukelen en Loenen. Verder naar het noorden begon de bouw van buitenplaatsen later, bleef de dichtheid geringer en werden de buitenplaatsen eerder weer afgebroken. Dat wijst erop dat de concentratie zichzelf versterkte en in stand hield (afb. 12). In Zeeland genoten de buitenplaatsen in Walcheren de grootste bekendheid. Hier ontstond een aaneenge sloten reeks buitenplaatsen aan de binnenduinrand. Deze zette zich voort in een boog via Middelburg naar Vlissingen.65 Op Schouwen-Duiveland zijn buiten plaatsen gebouwd door de stedelijke elite van Zierik zee, met een opvallende concentratie rond de plaats Noordgouwe, een dorp met op het hoogtepunt ruim dertig buitenplaatsen dat dan ook wel eens is aange duid als Noord-Gouws Arkadia (afb. 14). Ook hier zien we dat de concentratie in de loop van de tijd zelfs ster ker werd: de zeven resterende buitenplaatsen liggen in elkaars directe omgeving.66 Voor droogmakerijen als de Beemster, de Water graafsmeer en de Purmer wordt vaak verondersteld dat de aanleg van buitenplaatsen al snel na droogma king begon of zelfs al bij de droogmaking gepland was.61 Dat beeld klopt zeker niet in alle gevallen. In de Beemster kwam de aanleg redelijk snel op gang en stonden er in 1640, een generatie na de droogleg ging (1618-1612) al 52 ‘heerenhuizen’, waarvan er twin tig als hoofdverblijf dienden en de rest alleen in de zomermaanden bewoond was. Dat aantal bleef stabiel tot het midden van de achttiende eeuw. In de tachtig jaar nadien verdwenen alle buitenplaatsen.62 In de verschillende voorbeeldgebieden die we hier boven bespraken, zien we steeds opnieuw een groei in In andere droogmakerijen kwam de bouw echter 13. De buitenplaatsen in de Purmer in tijdslijnen (Ton Markus, Faculteit Geo wetenschappen Utrecht) BULLETIN KNOB 2021 • 4 18 13. De buitenplaatsen in de Purmer in tijdslijnen (Ton Markus, Faculteit Geo wetenschappen Utrecht) BULLETIN KNOB 2021 • 4 BULLETIN KNOB 2021 • 4 BULLETIN KNOB 2021 • 4 18 18 14. De buitenplaatsen rond Noordgouwe (Ton Markus, Faculteit Geowetenschappen Utrecht) de spoorlijn van Amsterdam via Utrecht en Drieber gen-Zeist naar Arnhem schiep de mogelijkheid om da gelijks heen een weer te reizen naar Amsterdam. Op de zuidrand van de Utrechtse Heuvelrug ontstond nu in korte tijd een aaneengesloten reeks buitenplaatsen en landgoederen, die al snel werd aangeduid als de Sticht se Lustwarande. Een vergelijkbare reeks lag op de zui delijke Veluwezoom (afb. 15). Ook hier bestonden al middeleeuwse kastelen, deels verbonden met de jacht op de Veluwe, deels met lokale adel. In de zeventiende en achttiende eeuw werd een deel van de kastelen ver bouwd tot buitenplaatsen en werden nieuwe buiten plaatsen gebouwd. In de negentiende eeuw ontstond ook hier een dichte en op sommige plaatsen aaneenge sloten reeks buitenplaatsen en landgoederen. Daarbij boden de zandlandschappen nieuwe moge lijkheden. De grote heidevelden waren lange tijd essen tieel geweest voor de landbouw op de zandgronden, als weidegrond voor koeien en schapen die mest lever den die werd vermengd met heideplaggen en waarmee de vruchtbaarheid van de bouwlanden langzaam werd verbeterd. De komst van alternatieve meststoffen, zo als chilisalpeter, maakte dat de vraag vanuit de land bouw naar heidevelden in de negentiende eeuw begon af te nemen. Gemeenten begonnen stukken heide te verkopen en boerenorganisaties (‘marken’) werkten toe naar verdeling van de heidevelden. De boeren had den echter geen mest en kapitaal om hun nieuwe bezit te ontginnen of te bebossen. In die omstandigheden konden rijke stedelingen voor weinig geld grote opper vlakten land kopen en de status van grootgrondbezit ter verwerven. 14. De buitenplaatsen rond Noordgouwe (Ton Markus, Faculteit Geowetenschappen Utrecht) de zeventiende en de eerste helft van de achttiende eeuw. Na een hoogtepunt in het midden van de acht tiende eeuw volgde een lange periode van verval en af braak. Vooral in het begin van de negentiende eeuw werden grote aantallen buitenplaatsen afgebroken en nam de landbouw de polders en rivieroevers weer over.67 DE NEERGANG VAN BUITENPLAATSENLANDSCHAPPEN De meeste literatuur over buitenplaatsen gaat over de perioden van ontstaan, groei en bloei. Het proces van verdwijnen van buitenplaatsenlandschappen is veel minder onderzocht. Dat geldt zeker voor de vraag waarom sommige buitenplaatsenlandschappen beter bewaard zijn gebleven dan andere. Toch heeft ook de ze fase haar eigen geografie. Van de buitenplaatsen landschappen uit de zeventiende en achttiende eeuw BUITENPLAATSLANDSCHAPPEN VAN DE NEGENTIENDE EN VROEGE TWINTIGSTE EEUW Toen de bouw van buitenplaatsen in het tweede kwart van de negentiende eeuw weer op gang kwam, was de landschappelijke voorkeur veranderd. De reliëfrijke zandgronden, die lange tijd vooral negatief waren ge waardeerd, werden nu juist aantrekkelijk gevonden. Ze waren ook eenvoudiger in te richten in de land schapsstijl, die in de negentiende eeuw de overheer sende mode in de tuinaanleg was. Een familie als de Huydecopers, die we al eerder tegenkwamen in de Vechtstreek, investeerde in het begin van de negen tiende eeuw in buitenplaatsen bij Zeist.68 De nieuwe eigenaren van buitenplaatsen kwamen ook in deze periode nog uit de grote steden. Op de zuidelijke Utrechtse Heuvelrug ging het om bankiers, industrië len en oud-kolonialen.69 15. Landgoederen en buitenplaatsen van de Zuidelijke Veluwezoom, op basis van Storms-Smeets 2011 15. Landgoederen en buitenplaatsen van de Zuidelijke Veluwezoom, op basis van Storms-Smeets 2011 BULLETIN KNOB 2021 • 4 19 BULLETIN KNOB 2021 • 4 Dit gebied en de zuidelijke Veluwezoom werden in het midden van de negentiende eeuw ontsloten door spoorwegen, later aangevuld met een dicht net van tramwegen en met straatwegen. Vooral de aanleg van 19 is bijvoorbeeld in delen van de Vechtstreek en ’s-Grave land nog relatief veel over, terwijl die in de droogmake rijen en in Zeeland vrijwel verdwenen zijn. Haag, die hun welvaart wat langer vasthielden, juist in de eerste decennia van de negentiende eeuw nog veel afbraak plaatsvond. Het hoogtepunt van de afbraak van buitenplaatsen in de Beemster en langs de Vecht lag in de eerste decennia van de negentiende eeuw. Voor de Purmer geldt hetzelfde, maar daar was de sloop al in de achttiende eeuw op gang gekomen.72 In Kennemerland verdween de kleinschalige buiten plaatscultuur om ten dele plaats te maken voor veel grotere buitenplaatsen. Mogelijk speelde ook een rol dat de concurrentie met de landbouw op de vruchtbare kleigronden van de droogmakerijen en Zeeland wat sterker was dan in de veengebieden. De oorzaken van deze verschillen zijn nog onduide lijk. Voor iedere afzonderlijke buitenplaats en voor elk buitenplaatsenlandschap zijn wel redenen aange voerd, zoals rond Den Haag het verdwijnen van het stadhouderlijke hof in 1795.70 Veel buitenplaatsen die direct buiten de steden lagen zijn, nadat de steden in Nederland rond 1860 weer gingen groeien, opge slokt door stedelijke uitbreidingen. Dat hoeft niet al tijd ongunstig te zijn; veel buitenplaatsen werden in de stedelijke bebouwing geïntegreerd als stadspark. Een voorbeeld zijn de overgebleven buitenplaatsen aan de Amstel. Die vormen geen aaneengesloten bui tenplaatsenlandschap meer, maar de grootste gaten waren al vóór het midden van de negentiende eeuw ge vallen. Van de negentiende-eeuwse buitenplaatsen lijkt meer over te zijn. De crisis van de jaren dertig heeft wel iswaar een einde gemaakt aan de aanleg van buiten plaatsen, maar de bestaande buitenplaatsen, en vooral de bijbehorende parken in landschapsstijl, werden zo wel door cultuur- als door natuurliefhebbers gewaar deerd. De Vereniging Natuurmonumenten, Staatsbos beheer en de provinciale landschappen, met name Het Geldersch en Het Utrechts Landschap, fungeerden als vangnet wanneer particuliere eigenaren het niet meer konden bolwerken. De bos- en parkrijke omgeving maakte het ook aantrekkelijk om buitenplaatsen te ‘verkavelen’ voor villabouw. De Stichtse Lustwarande en de zuidelijke Veluwezoom werden daarmee bereik baar voor de hogere middenklasse, terwijl het groene karakter redelijk bewaard bleef. Later werden de bui tenplaatsen ook gewilde locaties voor bedrijven die een elitaire uitstraling wensten. BULLETIN KNOB 2021 • 4 Dat heeft niet altijd gun stig uitgepakt: als het bedrijf geen succes had werd de buitenplaats slecht onderhouden, had het wel succes dan verdween een steeds groter deel van het park onder nieuwbouw en parkeerplaatsen. Datzelfde gebeurde bij veel buitenplaatsen die werden ingericht als zorgin stellingen.73 Ook het argument van de veranderde mode in tuin aanleg wordt wel genoemd: de omslag van de geome trische naar de landschappelijke tuinstijl maakte dat de voorkeur verschoof van de vlakke en welvarende landbouwgebieden naar de wildere landschappen van de stuwwallen.71 Dat is voor nieuwe buitenplaatsen en landgoederen zeker een belangrijke factor geweest, maar het verklaart de verschillende ontwikkelingen in de oude buitenplaatsenlandschappen onvoldoende. We moeten bedenken dat de landschapsstijl al rond 1770 in Nederland werd geïntroduceerd en de eerste vijftig jaar vooral werd toegepast binnen bestaande, voordien vaak geometrisch vormgegeven buitenplaat sen. Daarbij werden vijvers gegraven en werd het mate riaal gebruikt om heuvels aan te leggen. In ’s-Grave land kunnen we hier een zekere ironie in zien: het buitenplaatsenlandschap dat ooit gevormd was door de randen van het Gooi af te graven en te veranderen in een vlak gebied, werd nu opnieuw vergraven om juist weer reliëf te creëren. De buitenplaatsen sloten daar mee weer logisch aan op het Gooi, dat zich in de negen tiende eeuw juist ontwikkelde tot een geliefd woon oord. CONCLUSIE Nederland kende in het verleden verschillende gebie den met een grote dichtheid aan buitenplaatsen. Door voor een aantal buitenplaatsenlandschappen gedetail leerde tijdslijnen op te stellen, konden we aantonen dat veel concentraties van buitenplaatsen eerder geleide lijk dan planmatig zijn ontstaan. In veel gevallen vorm den deze buitenplaatsenlandschappen zich namelijk cumulatief; nieuwe buitenplaatsen werden aangetrok ken door oudere. Er zijn echter ook voorbeelden van groepen buitenplaatsen die vanaf het begin een sa menhang vertonen, met als mooiste voorbeeld de aan leg van de Amersfoortseweg met de aansluitende vor ming van kavels voor ontwikkeling van buitenplaatsen. Belangrijke buitenplaatsenlandschappen ontstonden in de zeventiende eeuw en bereikten hun hoogtepunt in het midden van de achttiende eeuw. Het meest uitge strekte buitenplaatsenlandschap lag in een ruime cir De algemene tendens tot afbraak wijst er echter op dat we ons niet moeten blindstaren op de motieven van afzonderlijke groepen eigenaren, maar dat er meer structurele ontwikkelingen waren. De verslechteren de economische situatie in de Republiek in de laatste decennia van de achttiende eeuw is zeker een van de hoofdoorzaken geweest. Tegelijk was de tweede helft van de achttiende eeuw juist een periode van welvaart in de landbouw, wat de concurrentie om de grond ver sterkte. BULLETIN KNOB 2021 • 4 20 BULLETIN KNOB 2021 • 4 Het zou interessant zijn om de chronologie van de afbraak nauwkeuriger in kaart te brengen. De indruk bestaat dat de afbraak van buitenplaatsen in Zeeland in de tweede helft van de achttiende eeuw al ver voort schreed, terwijl rond steden als Amsterdam en Den 20 kel rond Amsterdam, andere bevonden zich rond de andere grote steden en op Walcheren. De meeste waren goed bereikbaar over water, maar landtransport was belangrijker dan vaak wordt aangenomen. Na het mid den van de achttiende eeuw volgde een lange periode van neergang, met vooral in de decennia rond 1800 grootschalige afbraak. Nadien ontstonden weer nieu we buitenplaatsenlandschappen, maar nu vooral aan de randen van de Utrechtse Heuvelrug en de zuidelijke Veluwe en ontsloten door spoorlijnen en straatwegen. plaatsen heeft een ontwikkeling doorgemaakt waarbij steeds meer naar de grote samenhangen werd ge keken. Was de aandacht aanvankelijk gericht op de huizen, vanaf de jaren zeventig is er groeiende belang stelling voor de bijbehorende tuinen en parken. In die tijd beschreef Van der Wyck al de noodzaak om nog verder te kijken, naar buitenplaatsen in hun omgeving en naar groepen buitenplaatsen. 2 www.kastelen.nl/kastelen-nieuws-nks. php onder 15-11-2019 (geraadpleegd 22 juli 2021). een huis met tuin buiten de bebouwde kom. De term buitenplaats is voor hem specifieker: een hofstede die als zomer verblijf in gebruik is bij een stedeling en die is ingericht voor recreatie. 1 Het citaat in de titel is afkomstig van J. Huizinga, Nederland’s beschaving in de zeventiende eeuw. Een schets, Haarlem 1941, 174. noten Loosjes Pz., Hollands Arkadia, of Wandelingen in de Omstreeken van Haarlem, Haarlem 1805; R.J. Ligt helm, De Kralingse buitenplaatsen van de 16e tot de 21e eeuw. Een vergeten Arcadië, Woudrichem 2020; C. Bertram, Noord- Hollands Arcadia. Ruim 400 Noord- Hollandse buitenplaatsen in tekeningen, 17 Olde Meierink 2017 (noot 13); F. Vogel zang, ‘De zoektocht naar de Stichtse Lustwarande’, Oud-Utrecht 94 (2021) 4, 9-13. De wandelende dominee Jacobus Craandijk paste de term lustwarande toe op de omgeving van Wassenaar, op Walcheren en op de Zuidelijke Utrechtse Heuvelrug: J. Craandijk, Wandelingen door Nederland met pen en potlood, delen 5 Haarlem 1880 en 6 Haarlem 1882 11 11 G.A. Verschuure-Stuip, ‘De buiten plaatsbiotoop of landgoedbiotoop. Nieuwe allianties in de bescherming van buitenplaatsen en landgoederen’ (Zuid-Holland en Utrecht), Vitruvius nr. 33 (2015), 18-23. Voor beide provincies is de biotoop een instrument voor de bescherming van samenhangende bui tenplaatsen en landgoederen. 4 Vogelzang 2014 (noot 3). 5 M. van den Broeke (red.), Buitenplaatsen in het Westland. Met smaak en tot voor- deel aangelegd, Heemstede 2018, 14-15. 5 M. van den Broeke (red.), Buitenplaatsen in het Westland. Met smaak en tot voor- deel aangelegd, Heemstede 2018, 14-15. C. Bertram, ‘Groenendaal als cultuur historische schatkamer. Buitenplaatsen tussen Heemstede en De Glip, 1600- 1913’, in: Groenendaal, van buitenplaats tot wandelbos, Heemstede 2013, 69-119, 70. CONCLUSIE Pas in de eenentwin tigste eeuw werkte dat door in beleid voor respectieve lijk buitenplaatsbiotopen en buitenplaatszones. De term buitenplaatsenlandschappen, die in de afgelopen jaren steeds meer ingang vindt, maakt duidelijk dat onderzoek en bescherming nog een stap verder moe ten gaan door zich te richten op samenhangende groe pen buitenplaatsen. Die zouden als één landschap pelijk ensemble moeten worden gezien.74 Meer dan afzonderlijke buitenplaatsen zijn deze samenhangen de buitenplaatsenlandschappen dragers van de land schappelijke kwaliteit. Vooral de buitenplaatsenlandschappen van de zeven tiende en achttiende eeuw zijn van internationaal be lang. Enkele daarvan zijn nog goed herkenbaar. De mooiste voorbeelden zijn wel die langs de Vecht en die van ’s-Graveland. Buitenplaatsenlandschappen vragen om een regio nale aanpak, die de laatste jaren langzaam vorm krijgt. De aandacht voor – en de bescherming van – buiten S. Nijhuis, ‘GIS-toepassingen in onder zoek naar buitenplaatsenlandschappen’, Bulletin KNOB 115 (2016) 3, 147-164; G. Verschuure-Stuip m.m.v. H. Renes, ‘Hollandse buitenplaatsenlandschap pen. Buitenplaatsen en hun relatie met het landschap (1609-1672)’, in: Kuiper en Olde Meierink 2015 (noot 3), 42-65. Verschuure-Stuip en Renes 2015 (noot 13), 50. Een buitenplaatsenlandschap verschilt van een landgoederenland schap omdat het laatste, waarin huis en tuin omringd worden door de bijbe horende boerderijen, velden en bossen, alleen al daardoor verspreid in het land schap liggen. Is het bij een buitenplaat senlandschap vooral de aaneenschake ling huizen en tuinen die een gevoel van samenhang oproepen, bij een landgoe derenlandschap zijn dat de parken, bossen en landgoedboerderijen een huis met tuin buiten de bebouwde kom. De term buitenplaats is voor hem specifieker: een hofstede die als zomer verblijf in gebruik is bij een stedeling en die is ingericht voor recreatie. C. Bertram, ‘Groenendaal als cultuur historische schatkamer. Buitenplaatsen tussen Heemstede en De Glip, 1600- 1913’, in: Groenendaal, van buitenplaats tot wandelbos, Heemstede 2013, 69-119, 70. noten 14 3 Y. Kuiper, ‘Onderzoek naar de buiten plaats in de Gouden Eeuw. Een vogel vluchtperspectief’, in: Y.B. Kuiper en B. Olde Meierink (red.), Buitenplaatsen in de Gouden Eeuw. De rijkdom van het buitenleven in de Republiek, Hilversum 2015, 12-41, 17; F. Vogelzang, ‘Buiten plaatsen als exportproduct? Een relatie tussen Italië, de Zuidelijke Nederlanden en de Republiek’, Kasteel & Buitenplaats 16 (2014) 47, 3-8; F. Vogelzang, ‘Het nut tige en het aangename. Buitenplaatsen als investering?’, Kasteel & Buitenplaats 17 (2015), 48, 3-8. Toch zou naar de rol van belegging in landbouwgrond nog eens goed moeten worden gekeken: de meeste buitenplaatsen in de Noordelijke Nederlanden werden gebouwd in de periode 1650-1750, toen dergelijke beleg gingen juist minder lucratief waren door de lage prijzen voor landbouwproduc ten. 9 9 Bij grondstoffenwinning gaat het voor beeld om afzandingen, die elders in dit artikel aan bod komen. Langs de Oude Rijn was er ook een verband tussen buitenplaatsen en kleiwinning: G. van Oosterom, ‘Deftig wonen aan de Oude Rijn. De rol van kleiwinning op de ont wikkeling van een vergeten buiten plaatslandschap’, Het Nederlands Landschap. Tijdschrift voor Landschaps geschiedenis 34 (2016) 1, 12-21. bossen en landgoedboerderijen. 15 Olde Meierink 2017 (noot 13). 10 10 J. Finch en K. Giles, Estate landscapes. Design, improvement and power in the post-medieval landscape, Woodbridge 2007; J. Finch, K. Dyrmann en M. Frau sing (red.), Estate landscapes in northern Europe, Aarhus 2019, 13-14. 16 D. Blom, ‘Buiten in een stadslandschap. “Welk een moed om op zoo groote schaal bosch en park aan te leggen”’, in: Buitenplaatsen. Jaarboek Monumen- tenzorg 1998, Zwolle/Zeist 1998, 84-95. 17 Olde Meierink 2017 (noot 13); F. Vogel zang, ‘De zoektocht naar de Stichtse Lustwarande’, Oud-Utrecht 94 (2021) 4, 9-13. De wandelende dominee Jacobus Craandijk paste de term lustwarande toe op de omgeving van Wassenaar, op Walcheren en op de Zuidelijke Utrechtse Heuvelrug: J. Craandijk, Wandelingen door Nederland met pen en potlood, delen 5 Haarlem 1880 en 6 Haarlem 1882 17 Olde Meierink 2017 (noot 13); F. Vogel zang, ‘De zoektocht naar de Stichtse Lustwarande’, Oud-Utrecht 94 (2021) 4, 9-13. De wandelende dominee Jacobus Craandijk paste de term lustwarande toe op de omgeving van Wassenaar, op Walcheren en op de Zuidelijke Utrechtse Heuvelrug: J. Craandijk, Wandelingen door Nederland met pen en potlood, delen 5, Haarlem 1880, en 6, Haarlem 1882. 18 E. Storms-Smeets (red.), Gelders Arcadië. Atlas van een buitenplaatsenlandschap, Utrecht 2011; A. BULLETIN KNOB 2021 • 4 6 M. van den Broeke m.m.v. S. den Haan, Buitenplaatsen in Noordgouwe. Hofste- den, lusthoven en landhuizen, Delft 2014, 12. 6 M. van den Broeke m.m.v. S. den Haan, Buitenplaatsen in Noordgouwe. Hofste- den, lusthoven en landhuizen, Delft 2014, 12. BULLETIN KNOB 2021 • 4 5, Haarlem 1880, en 6, Haarlem 1882. 8 E. Storms-Smeets (red.), Gelders Arcadië. Atlas van een buitenplaatsenlandschap, Utrecht 2011; A. Loosjes Pz., Hollands Arkadia, of Wandelingen in de Omstreeken van Haarlem, Haarlem 1805; R.J. Ligt helm, De Kralingse buitenplaatsen van de 16e tot de 21e eeuw. Een vergeten Arcadië, Woudrichem 2020; C. Bertram, Noord- Hollands Arcadia. Ruim 400 Noord- Hollandse buitenplaatsen in tekeningen, 18 12 H. Ronnes, Bij nader inzien, de Neder- landse buitenplaats. Tussen herinnering, vergetelheid en ongemak, Groningen 2019, 4-5. 7 Deze ‘laantjes’ zijn beschreven door J.D.H. Harten, ‘Stedelijke invloeden op het Hollandse landschap in de 16de, 17de en 18de eeuw’, Holland 10 (1978), 114-134, 118-119. 7 Deze ‘laantjes’ zijn beschreven door J.D.H. Harten, ‘Stedelijke invloeden op het Hollandse landschap in de 16de, 17de en 18de eeuw’, Holland 10 (1978), 114-134, 118-119. 13 13 Van der Wyck gebruikte hiervoor de term buitenplaatslandschap. De meer voudsvorm wordt recentelijk ook vaker gebruikt. Zie bijvoorbeeld: B. Olde Meierink, ‘Buitenplaatslandschappen’, Kasteel & Buitenplaats 19 (2017) 58, 22-29; 8 M. van den Broeke, ‘Het pryeel van Zeeland’. Buitenplaatsen op Walcheren 1600-1820, Hilversum 2016, 27-28. Bertram gebruikt de term hofstede voor 21 prenten en kaarten uit de Provinciale Atlas Noord-Holland, Alphen aan den Rijn 2005; M. Glaudemans, Amsterdams Arcadia. De ontdekking van het achter- land, Nijmegen 2000. Beneden-Leeuwen 2017. Beneden-Leeuwen 2017. 32 R. van Luttervelt, De buitenplaatsen aan de Vecht, Lochem 1948; R. van Luttervelt, De Stichtse Lustwarande, Amsterdam 1949. 50 Bertram 2013 (noot 8), 75-77, 79, 82. 50 Bertram 2013 (noot 8), 75-77, 79, 82. 51 Van den Broeke 2018 (noot 5), 22. De kaart van Kruikius (1712) toont in het Westland zo’n honderd buitenplaatsen, waarvan er vijftig met naam worden vermeld: Van den Broeke 2018 (noot 5), 35. 33 H. Tromp en J. Six, De buitenplaatsen van ’s-Graveland, Zeist 1975. 19 H. Renes, ‘Wildparken. Landschappen van jacht en wildbeheer in internatio naal perspectief’, in: Y. Kuiper e.a. (red.), De jacht. Een cultuurgeschiedenis van jager, dier en landschap, Hilversum 2021, 216-246. 34 H.W.M. van der Wyck, ‘Voorstellen tot inventarisatie en klassificatie ter bescherming van buitenplaatsen en historische landschappen’, Groen 33 (1977) 2, 41-50; H.W.M. BULLETIN KNOB 2021 • 4 42 Van den Broeke 2018 (noot 5), 21. 43 Van den Broeke en Den Haan 2014 (noot 6), 10-11. 28 Zie bijvoorbeeld R. van der Laarse, ‘Amsterdam en Oranje. De politieke cultuur van kasteel en buitenplaats in Hollands Gouden Eeuw’, in: Kuiper en Olde Meierink 2015 (noot 3), 68-95. Voor groepen buitenplaatsen die ge domineerd worden door een enkele architect, zie E. van der Laan-Meijer, Het handschrift van L.P. Roodbaard. Ontwerpprincipes van Noord-Neder landse landschapsparken tot 1850, in voorbereiding. 44 A.G. van der Steur, ‘De afzanding van de Hollandse binnenduinen in de 17e eeuw’, Contactblad Regionale en Locale Geschiedenis 1 (1968) 58-62; Bertram 2013 (noot 8), 69-73. 58 J.L. Kloosterhuis, ‘Zandafgraving in het Gooi’, Boor en Spade 8 (1955), 126-131. 45 Glaudemans 2000 (noot 18); Ronnes 2019 (noot 12), 20. BULLETIN KNOB 2021 • 4 22 Olde Meierink 2017 (noot 13).b 22 Olde Meierink 2017 (noot 13).b 36 R.E. de Bruin, ‘Leven als een edelman. Kasteelbezit van Utrechtse burgers en patriciërs, 1600-1850’, Jaarboek Oud- Utrecht, 2009, 67-108. 23 R. Mulder, Op afbraak. De sloop van buitenplaatsen in de periode 1780-1830, Utrecht 2006 (doctoraalscriptie Taal- en Cultuurstudies), 9. 55 Van den Broeke 2016 (noot 8), 60-67; H.M. van den Berg 1993 (noot 53). 37 Glaudemans 2000 (noot 18), 58. 56 J.E. Abrahamse, ‘“Wegh der weegen.” Ontwerp en aanleg van de Amersfoortse weg. Een zeventiende-eeuws landinrich tingsproject door Jacob van Campen’, Flehite, Historisch Jaarboek voor Amers- foort en Omstreken 7 (2006), 73-97; J.E. Abrahamse en R. Blijdenstijn, Wegh der weegen. De ontwikkeling van de Amersfoortseweg 1647-2010, Utrecht/ Amsterdam 2010; J.E. Abrahamse, ‘A Roman road in the Dutch Republic. Jacob van Campen’s “Via Appia” in the countryside of Utrecht’, Journal of the Society of Architectural Historians 70 (2011) 4, 442-465. 24 Glaudemans 2000 (noot 18), 74. 38 J. van Geest en R. Sierksma (red.), Verloren Uitzicht. Bloei en teloorgang van het Wijkermeer, Amsterdam 2002. 39 Glaudemans 2000 (noot 18), 91. 25 Van den Broeke 2016 (noot 8), 34, 400. 26 Glaudemans 2000 (noot 18), 184. Tegen woordig zijn er nog 552 officieel erkende buitenplaatsen over: R.W.C. Dessing, De Amsterdamse buitenplaatsen. Een ver- geten stadsgeschiedenis, Utrecht 2015, 7. 39 Glaudemans 2000 (noot 18), 91. 40 J. Schuyf, ‘De Huydecopers als project ontwikkelaars in Maarssen’, in: J.E. Abrahamse e. a. (red.), Het landschap beschreven, Hilversum 2021, 169-176, 171. 27 Bij een onderzoek in het oudste kadaster van Amby, een dorp bij Maastricht met een reeks van buitenplaatsen, bleek bijvoorbeeld een opvallend groot aantal hoveniers in het dorp te wonen: H. Renes, ‘De terrassen aan de oostzijde van Maastricht. Mens en landschap in Amby, Heer en Heugem’, in: G.D. Majoor e.a. (red.), Natuurlijk Maastricht. Compacte stad in een weids landschap, Maastricht 2020, 98-115. Amsterdam 2010; J.E. Abrahamse, 41 H. Rijken, De Leidse Lustwarande; de geschiedenis van de tuinkunst op kastelen en buitenplaatsen rond Leiden, 1600-1800, Leiden 2005; S. Schama, Overvloed en onbehagen. De Nederlandse cultuur in de Gouden Eeuw, Amsterdam 1988, 298. 57 Olde Meierink 2017 (noot 13); J.D.H. Harten, ‘De genese van het Gooise cultuurlandschap’, K.N.A.G. Geografisch Tijdschrift 10 (1976), 93-116; J.D.H. Har ten, ‘De Nederlandse buitenplaats’, Historisch-Geografisch Tijdschrift 16 (1998), 178-187; U.M. Mehrtens, ’s-Grave- land en zijn buitenplaatsen, Zeist 1985. 42 Van den Broeke 2018 (noot 5), 21. BULLETIN KNOB 2021 • 4 van der Wyck, ‘Het historische landschap van de oostelijke Veluwezoom en Rosendael’, in G.G.[L.] Steur e.a. (red.), Acht zwerf stenen uit het Gelders landschap, Arnhem 1979, 71-116, hierin 72 (kaart) en 76 (term buitenplaatsenlandschap). De kaart maakte deel uit van een atlas, die echter onvoltooid is gebleven. 52 De Bruin 2009 (noot 37). ( ) 53 J.D.H. Harten, ‘Landhuizen en buiten plaatsen’, in: J.D.H. Harten e. a. (red.), De tuin van Utrecht. Geschiedenis en waarden van het landschap in het land inrichtingsgebied Groenraven-Oost, Utrecht 1992, 43-68; H.M. van den Berg, ‘De plaats waarop gij woont had gewijde bestemming. Buitenplaatsen op het terrein van middeleeuwse kloosters’, in: W. Denslagen e.a. (red.), Bouwkunst. Studies in vriendschap voor Kees Peeters, Amsterdam 1993, 63-75; H.M.J. Tromp, Buitenplaatsen bij De Bilt. Vollenhoven, Houdringe en Beerschoten, Zeist 1980; H. Renes, ‘De Uithof vóór De Uithof’, Oud-Utrecht 84 (2011), 35-39. Naast de genoemde buitenplaatsen is ook Sand wijck in De Bilt gesticht op land dat tot de abdij Oostbroek had behoord: H.M.J. Tromp, Sandwijck bij De Bilt, Zeist 1980. 20 Jan de Marre gebruikte in 1740 de term lustwaranden voor de omgeving van Batavia; A. Zuiderweg, ‘”Lustwaranden van aanminnelyken zwier”. Bataviase thuynen’, Cascade 19 (2010) 1, 23-34. Enkele jaren later besprak de Tegen woordige Staat de omgeving van Hon selaarsdijk als volgt: ‘De Lustwaranden rondsom dit Hof zyn zeer fraai. Behalve eene ruime wildbaan, vogelgaarde en oranjehuis, zijn hier schoone Dreeven en Lustbosschen …’; Tegenwoordige Staat der Verenigde Nederlanden 6 (1746), 595. In de laatste beschrijving worden nog de jachtlandschappen als eerste ge noemd, maar rond Batavia gaat het vooral over de tuinen rond de stad. 35 Voorgangers zijn bijvoorbeeld de con centraties van minilandgoederen rond het machtscentrum van de Utrechtse bisschop: J. van Doesburg e.a., ‘Kastelen in middeleeuwse veenontginningen’, Tijdschrift voor Historische Geografie 2 (2017) 4, 212-230. Zie ook Vogelzang 2014 (noot 3) en R. Meischke, ‘Buiten verblijven van Amsterdammers voor 1625’, Jaarboek van het Genootschap Amstelodamum 70 (1978), 82-106. Voorgangers zijn bijvoorbeeld de con centraties van minilandgoederen rond het machtscentrum van de Utrechtse bisschop: J. van Doesburg e.a., ‘Kastelen in middeleeuwse veenontginningen’, Tijdschrift voor Historische Geografie 2 (2017) 4, 212-230. Zie ook Vogelzang 2014 (noot 3) en R. Meischke, ‘Buiten verblijven van Amsterdammers voor 1625’, Jaarboek van het Genootschap Amstelodamum 70 (1978), 82-106. 21 whc.unesco.org/uploads/nominations/ 899.pdf. 54 E. Storms-Smeets, ‘Gelders Arcadië. Landgoederen en buitenplaatsen’, in: Storms-Smeets 2011 (noot 18), 32-39, 33. BULLETIN KNOB 2021 • 4 Mulder, ‘Herfsttij der buitenplaatsen’, Jaarboekje van het Oudheidkundig Genootschap 67 Mulder 2006 (noot 23); R. Mulder, ‘Herfsttij der buitenplaatsen’, Jaarboekje van het Oudheidkundig Genootschap De gegevens voor de Purmer zijn geba European Landscapes en voorzitter van het Netwerk Historisch Cultuurlandschap. Zijn publicaties gaan veelal over historische landschappen en erfgoed in Nederland en Europa. Prof. dr. J. Renes (1954) is historisch-geograaf en emeritus-hoogleraar Erfgoedstudies aan de Vrije Uni versiteit Amsterdam. Hij is redactielid van het Tijd- schrift voor Historische Geografie en het Journal of BULLETIN KNOB 2021 • 4 BULLETIN KNOB 2021 • 4 59 Over de problemen met de term ‘hof stede’ zie boven. In ’s-Graveland gaan we ervan uit dat de ‘hofsteden’ uit de eerste fase boerderijen met herenkamers wa ren. 46 A. Neuteboom, De Haarlemse buiten- plaats in het stadsbeeld, toen en nu (MA-thesis VU, Amsterdam 2019) 47 Rijken 2005 (noot 43). 47 Rijken 2005 (noot 43). 60 Schuyf 2021 (noot 40); J. Simonis, J. Kott man en H. van Bemmel (red.), Elsenburg, de verdwenen buitenplaats. Het ontstaan van het buitenleven aan de Vecht, Hilver sum 2020. 48 R.J. Ligthelm, De Kralingse buiten plaatsen van de 16e tot de 21e eeuw. Een vergeten Arcadië?, Woudrichem 2020. 30 H. de Leth (tekeningen) en M. Brouërius van Nidek (tekst), Het zegenpralent Kennemerlant, vertoont in veele heerelyke gezichten van deszelfs voornaemste lustplaetzen, adelyke huizen, dorp- en stede-gebouwen, Amsterdam 1629. 30 H. de Leth (tekeningen) en M. Brouërius van Nidek (tekst), Het zegenpralent Kennemerlant, vertoont in veele heerelyke gezichten van deszelfs voornaemste lustplaetzen, adelyke huizen, dorp- en stede-gebouwen, Amsterdam 1629. 49 C.J. van Rossum, ‘De voornaamste vermaaken der Dordtenaaren’. Een studie naar de opkomst en neergang van de buitenverblijven op het Eiland van Dordrecht (1600-1832), 61 Van der Laarse 2015 (noot 28), 80. 62 K. Bossaers, ‘Buitenplaatsen in de Beem ster’, De Nieuwe Schouwschuit. Tijdschrift 22 31 Bertram 2005 (noot 18), 7. seerd op het boek van C. Boschma- Aarnoudse, Buitenplaatsen in de Purmer. Investeren en buiten leven in een Noord- Hollandse polder, Wormerveer 2015. 65 Van den Broeke 2016 (noot 8), met een overzichtskaart met 139 buitenplaatsen op de binnenzijde van het omslag. 66 Van den Broeke en Den Haan 2014 (noot 6); M. van den Broeke, ‘Buitenplaatsen in Noordgouwe, 1820-1940. Notabele levensvormen van het Zierikzeese patriciaat’, in: C. Gietman e.a. (red.), Huis en habitus. Over kastelen, buiten- plaatsen en notabele levensvormen, Hilversum 2017, 269-283. 67 Mulder 2006 (noot 23); R. Mulder, ‘Herfsttij der buitenplaatsen’, Jaarboekje van het Oudheidkundig Genootschap ‘Niftarlake’ (2006), 39-56. ‘Niftarlake’ (2006), 39-56. seerd op het boek van C. Boschma- Aarnoudse, Buitenplaatsen in de Purmer. Investeren en buiten leven in een Noord- Hollandse polder, Wormerveer 2015. van het Historisch Genootschap Beemster 6 (november 2008), 17-20. Een aantal ‘plaijsirhuijsen’ komt ook al voor in de verponding van 1633: C.L. Nijst en L. den Boon, Geluk in de Beemster. Onderzoek naar buitenplaatsen, eendenkooien en molengangen, Alkmaar 2012. Katja W.J.M. hans renes BULLETIN KNOB 2021 • 4 In a few areas, the evolving density of country houses has been traced in a detailed chronological record. In most cases it reveals progressive growth towards a high point in the first half of the eighteenth century, after which a gradual decline sets in. However, in a number of areas growth was much more rapid, in par ticular along the River Vecht. BULLETIN KNOB 2021 • 4 Bossaers heeft een website onder houden met gedetailleerde gegevens over buitenplaatsen in de Beemster. Na haar overlijden in 2019 is die website echter verdwenen. van het Historisch Genootschap Beemster 6 (november 2008), 17-20. Een aantal ‘plaijsirhuijsen’ komt ook al voor in de verponding van 1633: C.L. Nijst en L. den Boon, Geluk in de Beemster. Onderzoek naar buitenplaatsen, eendenkooien en molengangen, Alkmaar 2012. Katja W.J.M. Bossaers heeft een website onder houden met gedetailleerde gegevens over buitenplaatsen in de Beemster. Na haar overlijden in 2019 is die website echter verdwenen. Niftarlake (2006), 39 56. 68 R. Blijdenstein [Blijdenstijn], ‘Negen tiende- en vroeg twintigste-eeuws tuin stijlen in zuid-oost Utrecht’, in: K.M. Veenland-Heineman (red.), Tuin & park. Historische buitenplaatsen in de provincie Utrecht, Utrecht 1992, 43-58. 69 Vogelzang 2021 (noot 17), 11. 70 Van den Broeke 2018 (noot 5), 41-44. 71 Zie bijvoorbeeld Olde Meierink 2017 (noot 13). 72 Mulder 2006 (noot 23). 73 Zie onder meer Bertram 2005 (noot 18), 8-9. 74 G. Verschuure-Stuip, Welgelegen. Analyse van Hollandse buitenplaatsen in hun landschappen (1630-1730), Delft 2019, 41-60. ft ( ), 39 5 68 R. Blijdenstein [Blijdenstijn], ‘Negen tiende- en vroeg twintigste-eeuws tuin stijlen in zuid-oost Utrecht’, in: K.M. Veenland-Heineman (red.), Tuin & park. Historische buitenplaatsen in de provincie Utrecht, Utrecht 1992, 43-58. 69 Vogelzang 2021 (noot 17), 11. 70 Van den Broeke 2018 (noot 5), 41-44. 71 Zie bijvoorbeeld Olde Meierink 2017 (noot 13). 72 Mulder 2006 (noot 23). 73 Zie onder meer Bertram 2005 (noot 18), 8-9. 74 G. Verschuure-Stuip, Welgelegen. Analyse van Hollandse buitenplaatsen in hun landschappen (1630-1730), Delft 2019, 41-60. 65 65 Van den Broeke 2016 (noot 8), met een overzichtskaart met 139 buitenplaatsen op de binnenzijde van het omslag. 66 66 Van den Broeke en Den Haan 2014 (noot 6); M. van den Broeke, ‘Buitenplaatsen in Noordgouwe, 1820-1940. Notabele levensvormen van het Zierikzeese patriciaat’, in: C. Gietman e.a. (red.), Huis en habitus. Over kastelen, buiten- plaatsen en notabele levensvormen, Hilversum 2017, 269-283. 70 Van den Broeke 2018 (noot 5), 41-44. 72 Mulder 2006 (noot 23). 63 Glaudemans 2000 (noot 18), 81-90. 63 Glaudemans 2000 (noot 18), 81-90. 67 64 H. Renes, ‘Water en buitenplaatsen’, in: C. Gietman e. a. (red.), Huis en habitus; over kastelen, buitenplaatsen en notabele levensvormen, Hilversum 2017, 98-113. 74 G. Verschuure-Stuip, Welgelegen. Analyse van Hollandse buitenplaatsen in hun landschappen (1630-1730), Delft 2019, 41-60. 67 Mulder 2006 (noot 23); R. hans renes In the past, country house research was mainly con cerned with individual houses and gardens. Yet, as ear ly as the seventeenth and eighteenth centuries, so ma ny country houses were being built around the major cities that they came to define the landscape. Genuine estate landscapes took shape along several rivers (Amstel, Vecht), along the inner edge of coastal dunes, and on newly reclaimed land. In the middle of the sev enteenth century, the rivers were augmented with a network of barge canals and soon they too were lined by a belt of country houses. The greatest density of country houses was to be found around Amsterdam, but other big cities in the provinces of Holland and Zee land had their fair share as well. Access was mostly by water, but in some areas, especially in Zeeland, country roads performed this role. The majority of country houses were built on or next to a farm, which generally continued to exist and, in many cases, survived the country house. decade of the nineteenth, large numbers of country houses were demolished and in many instances the land reverted to agriculture production. It appears that the decline set in earlier in Zeeland than in Holland, but regional differences in decline are not yet entirely clear. The second quarter of the nineteenth century saw the construction of a new generation of country houses, especially in the undulating sandy areas of the Utrecht se Heuvelrug and the southern part of the Veluwezoom, where railway lines provided access. The owners of this new crop of country houses laid out their gardens in the English landscape style. They also bought up vast, neighbouring heathlands from local councils or farm ers and planted them with trees. As a result, these country houses are quite different in character from those of the earlier period. In the past the concentrations of country houses dominated the landscape and even today, wherever they have survived to a substantial degree they contin ue to represent an important landscape quality. As such, protection and management should not be con fined to individual country houses but should extend to groups of country houses and their interrelation ships (in the form of visual axes, for example). In recent years, a number of provinces have already set a good example by formulating policies for country house bio topes and linear estate landscapes. BULLETIN KNOB 2021 • 4 Sustained growth was followed by decline. In the final decade of the eighteenth century and the first 23
https://openalex.org/W2965058899	https://europepmc.org/articles/pmc6745853?pdf=render	English	null	High aldehyde dehydrogenase activity at diagnosis predicts relapse in patients with t(8;21) acute myeloid leukemia	Cancer medicine	2,019	cc-by	5,851	O R I G I N A L R E S E A R C H O R I G I N A L R E S E A R C H Lu Yang \| Wen‐Min Chen \| Feng‐Ting Dao \| Yan‐Huan Zhang \| Ya‐Zhe Wang \| Yan Chang \| Yan‐Rong Liu \| Qian Jiang \| Xiao‐Hui Zhang \| Kai‐Yan Liu \| Xiao‐Jun Huang \| Ya‐Zhen Qin Lu Yang \| Wen‐Min Chen \| Feng‐Ting Dao \| Yan‐Huan Zhang \| Ya‐Zhe Wang \| Yan Chang \| Yan‐Rong Liu \| Qian Jiang \| Xiao‐Hui Zhang \| Kai‐Yan Liu \| Xiao‐Jun Huang \| Ya‐Zhen Qin Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing, China Abstract Abstract Acute myeloid leukemia (AML) with t(8;21) is a heterogeneous disease. Although the detection of minimal residual disease (MRD), which is indicated by RUNX1‐ RUNX1T1 transcript levels, plays a key role in directing treatment, risk stratification needs to be improved, and other markers need to be assessed. A total of 66 t(8;21) AML patients were tested for aldehyde dehydrogenase (ALDH) activity by flow cy- tometry at diagnosis, and 52 patients were followed up for a median of 20 (1‐34) months. The median percentage of CD34+ALDH+, CD34+CD38‐ALDH+, and CD34+CD38+ALDH+ cells among nucleated cells were 0.028%, 0.012%, and 0.0070%, respectively. The CD34+ALDH+‐H, CD34+CD38‐ALDH+‐H, and CD34+CD38+ALDH+‐H statuses (the percentage of cells that were higher than the individual cutoffs) were all significantly associated with a lower 2‐year re- lapse‐free survival (RFS) rate in both the whole cohort and adult patients (P = .015, .016, and .049; P = .014, .018, and .032). Patients with < 3‐log reduction in the RUNX1‐RUNX1T1 transcript level after the second consolidation therapy (defined as MRD‐H) had a significantly lower 2‐year RFS rate than patients with ≥ 3‐log reduction (MRD‐L) (P = .017). The CD34+ALDH+ status at diagnosis was then combined with the MRD status. CD34+ALDH+‐L/MRD‐H patients had similar 2‐ year RFS rates to both CD34+ALDH+‐L/MRD‐L and CD34+ALDH+‐H/MRD‐L patients (P = .50 and 1.0); and CD34+ALDH+‐H/MRD‐H patients had significantly lower 2‐year RFS rate compared with CD34+ALDH+‐L and/or MRD‐L patients (P < .0001). Multivariate analysis showed that CD34+ALDH+‐H/MRD‐H was an independent adverse prognostic factor for relapse. In conclusion, ALDH status at di- agnosis may improve MRD‐based risk stratification in t(8;21) AML, and concurrent high levels of CD34+ALDH+ at diagnosis and MRD predict relapse. Received: 26 February 2019 \| Revised: 19 June 2019 \| Accepted: 2 July 2019 DOI: 10.1002/cam4.2422 Received: 26 February 2019 \| Revised: 19 June 2019 \| Accepted: 2 July 2019 DOI: 10.1002/cam4.2422 Correspondence Ya‐Zhen Qin, Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, No. 11 Xizhimen South Street, Xicheng District, Beijing, China. Email: qin2000@aliyun.com Funding information Nature Science Foundation of China, Grant/ Award Numbers: 81570130 and 81870125 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. © 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 1 \| INTRODUCTION p p y Overall, 52 patients received treatment and were followed up at our hospital. As we previously reported, induction ther- apy consisted of 1‐2 cycles of the “3 + 7” regimen or the homoharringtonine, aclarubicin, and cytarabine regimen for the 37 adult patients, and cytarabine, idarubicin, and etopo- side were used for the 15 pediatric patients.6,7,28 Forty‐eight patients received consolidation therapy after achieving CR. Among them, 36 received an intermediate‐dose cytarabine‐ based chemotherapy only, and 12 received chemotherapy fol- lowed by allogeneic hematopoietic stem cell transplantation (allo‐HSCT) from a human leukocyte antigen (HLA)‐iden- tical sibling (n = 9) or an HLA haplotype‐matched relative (n = 3) in CR1. The allo‐HSCT conditioning regimen and graft‐versus‐host disease prophylaxis have been described previously.29 Dasatinib was used in four patients with c‐KIT mutations when the reduction of the RUNX1‐RUNX1T1 transcript levels was less than 3‐log after the second cycle of consolidation. The cutoff date for follow‐up was October 2018. This study was approved by the Ethics Committee of Peking University People's Hospital. All patients provided written informed consent in accordance with the Declaration of Helsinki to participate in the present study. Although t(8;21) acute myeloid leukemia (AML) is consid- ered to have a good prognosis, relapse occurs in up to 40% of patients treated with chemotherapy.1-6 Therefore, strat- ification is needed in order to guide appropriate treatment. Minimal residual disease (MRD) levels indicated by RUNX1‐ RUNX1T1 transcript levels as well as c‐KIT mutations have been demonstrated to be strong prognostic factors in t(8;21) AML.4-9 However, their risk predictions are not perfect, and other markers have yet to be evaluated. Leukemia stem cells (LSCs) may cause relapse from the complete remission (CR) state.10 CD34+CD38‐ is a putative immunophenotype of LSCs with the ability to generate leu- kemia in immunodeficient mice.11-13 However, this immuno- phenotype has been challenged because some studies have demonstrated that LSCs might exist in CD34+CD38+ and CD34‐ cells.14-16 Thus, this surface immunophenotype alone might be inadequate for identifying LSCs. 2.2 Bone marrow (BM) was aspirated from all 66 patients at diag- nosis. Red blood cells were lysed using ammonium chloride solution (STEMCELL Technologies, Vancouver, Canada). ALDH activity of nucleated cells was detected using an ALDEFLUOR Kit (STEMCELL Technologies), according to the manufacturer's instructions. Nucleated cells of each sample were adjusted to a concentration of 1 × 106/mL. The cells were incubated with the ALDH‐substrate BODIPY‐ aminoacetaldehyde with or without the ALDH inhibitor diethylaminobenzaldehyde (DEAB). Test and control cells were then incubated at 37°C for 30 minutes, centrifuged, and resuspended in ice‐cold ALDEFLUOR buffer. Cells were then incubated with fluorochrome‐labeled mouse anti‐human monoclonal antibodies for 30 minutes on ice and then resus- pended in ALDEFLUOR buffer. Testing and data acquisition were performed using a FACS Canto II (Becton Dickinson, San Jose CA, USA). Analysis was performed using Kaluza flow analysis software (Beckman Coulter, Brea, CA, USA). In the present study, we examined ALDH activity in 66 t(8;21) AML patients at diagnosis and evaluated its sole prognostic role and the impact of its combination with MRD on relapse. 1 \| INTRODUCTION Aldehyde dehydrogenases (ALDHs) are a family of cy- tosolic enzymes that are involved in various biological pro- cesses.17,18 Cells with high ALDH activity (ALDH+) have been identified as cancer stem cells in various solid tumors, including breast, lung, and ovarian cancer.19-21 Furthermore, it has been demonstrated that ALDH alone or in combina- tion with CD34 can be used to purify hematopoietic stem cells.22,23 In AML, some studies have shown that LSCs might be enriched among the ALDH+ subsets.22,24,25 A high per- centage of ALDH+ was demonstrated to be associated with adverse cytogenetic factors.24-27 Whether the percentage of ALDH+ is prognostic within the same cytogenetic risk group, such as t(8;21), is unknown. In addition, a clinical co- hort study in intermediate and high cytogenetic risk AML showed that patients with a high percentage of ALDH + had adverse outcomes.24,26,27 However, this effect has not been evaluated in t(8;21) AML patients to date. YANG et al 14 years at diagnosis were categorized as pediatric and adult patients, respectively. Funding information K E Y W O R D S aldehyde dehydrogenase, flow cytometry, minimal residual disease, relapse, RUNX1‐RUNX1T1, t(8;21) acute myeloid leukemia K E Y W O R D S aldehyde dehydrogenase, flow cytometry, minimal residual disease, relapse, RUNX1‐RUNX1T1, t(8;21) acute myeloid leukemia \| 5459 wileyonlinelibrary.com/journal/cam4 wileyonlinelibrary.com/journal/cam4 Cancer Medicine. 2019;8:5459–5467. 5460 2.1 \| Patients and treatment A total of 66 t(8;21) AML patients were enrolled in the pre- sent study. These patients were diagnosed at our hospital from September 2015 to July 2018. A t(8;21) AML diagno- sis was determined according to morphologic evaluation of bone marrow (BM) smears, immunophenotyping, cytoge- netics, and molecular analyses. In total, 44 (66.7%) patients were male. The median age of the patients at diagnosis was 38 (range: 2‐66) years. Patients younger than and older than The monoclonal antibodies that were used included CD45‐PerCP, CD34‐PE‐Cy7, CD38‐APC, and Lineage‐ APC‐H7/Cy7 (consisting of CD3, CD14, CD16, CD20, and CD36). CD16, CD20, and CD36 antibodies were purchased from BioLegend (San Diego, CA, USA), and the others were purchased from BD Biosciences (San Jose, CA, USA). The gating strategy is shown in Figure 1. An forward scattering/side scattering (FSC/SSC) plot was used to YANG et al 5461 \| 5461 YANG et al FIGURE 1 Gating strategy for flow cytometric testing of ALDH+, CD34+ALDH+, CD34+CD38‐ALDH +, and CD34+CD38+ALDH+ cells. Sequential gating is shown from A to E. (A) nucleated cells; (B) Lin- cells; (C) blast cells; (D) CD34+ cells; (E) CD34+CD38‐ and CD34+CD38+ cells. (F) ALDH+; (G) CD34+ALDH+; (H) CD34+CD38‐ALDH+; (I) CD34+CD38+ALDH+; (J) negative control FIGURE 1 Gating strategy for flow cytometric testing of ALDH+, CD34+ALDH+, CD34+CD38‐ALDH +, and CD34+CD38+ALDH+ cells. Sequential gating is shown from A to E. (A) nucleated cells; (B) Lin- cells; (C) blast cells; (D) CD34+ cells; (E) CD34+CD38‐ and CD34+CD38+ cells. (F) ALDH+; (G) CD34+ALDH+; (H) CD34+CD38‐ALDH+; (I) CD34+CD38+ALDH+; (J) negative control used to identify the optimal cutoff levels that best discrimi- nated patients with relapse. Survival functions were estimated using the Kaplan‐Meier method and were compared using the log‐rank test. Relapse‐free survival (RFS) was measured from the date when CR was achieved to relapse. Overall survival (OS) was measured from diagnosis to death (regardless of the cause) or patients were queried at the date of last follow‐up to determine whether they were still alive, or were censored on the date they were last known to be alive. Variables associ- ated with P < .2 in the univariate analysis were entered into a multivariate analysis performed by the Cox models. The level for a statistically significant difference was set at P < .05. The SPSS 19.0 software package (SPSS Inc), and GraphPad Prism 5 (GraphPad Software Inc) were used for data analysis. exclude cellular debris and nonviable cells (Figure 1A). 2.1 \| Patients and treatment Lymphocytes, monocytes, macrophages, natural killer cells, and erythrocytes were excluded by lineage‐negative (Lin‐) (Figure 1B), blast cells were defined as SSClow/CD45dim (Figure 1C), CD34+ was defined in CD34/SSC plot (Figure 1D), and CD34+CD38‐ and CD34+CD38+ were derived from CD34+ cells (Figure 1E). Cells with DEAB‐sensitive ALDH activity were defined as ALDH+ (Figure 1F‐I). 3.1 The characteristics of all patients at diagnosis are summa- rized in Table 1. A total of 27 (40.9%) patients had c‐KIT mutations. In the 39 patients who were followed up until the second consolidation therapy, 11 (28.2%) had < 3‐log reduc- tion in the RUNX1‐RUNX1T1 transcript level compared to baseline (>0.4%) and were defined as MRD‐H, and the other 28 patients had ≥ 3‐log reduction and were defined as MRD‐L.6 As we previously reported, cDNA was used to perform PCR to test for c‐KIT mutations in exons 17 and 8.7,8 A total of 52 patients were followed up for a median of 20 (1‐34) months. About 50 (96.2%) patients achieved CR after 1‐3 cycles of induction therapy, and seven (14.0%) of these patients relapsed, all of whom received chemo- therapy only. The 2‐year RFS rate of the 50 patients who 2.3 \| Detection of RUNX1‐RUNX1T1 transcript levels and c‐KIT mutation and MRD monitoring RNA extraction, complementary DNA (cDNA) synthesis, and TaqMan‐based real‐time quantitative PCR technology were used as described previously.6 The RUNX1‐RUNX1T1 transcript level was calculated as the percentage of RUNX1‐ RUNX1T1 transcript copies/ABL copies. The pretreatment baseline level of RUNX1‐RUNX1T1 transcripts was 388% in our laboratory. According to our previous reports, a high MRD level (MRD‐H) was defined as less than a 3‐log re- duction in RUNX1‐RUNX1T1 transcript level compared to baseline after the second cycle of consolidation chemother- apy (>0.4%).6 3.2 \| The percentage of ALDH+cells in t(8;21) AML patients at diagnosis Of all 66 patients, the median percentage of ALDH+cells among nucleated cells was 0.17% (range 0.0029%‐6.8%), and that were 0.028% (range 0.0013%‐3.0%), 0.012% (range 0.00028%‐0.62%), and 0.0070% (range 0%‐2.1%) for CD34+ALDH+, CD34+CD38‐ALDH+, and CD34+CD38+ALDH+ cells among nucleated cells, respectively. 3.4 \| Impact of the percentage of ALDH+cells on the achievement of CR In the entire cohort, the CR rate after the first course of induction was 76.9% (40/52). The percentages of ALDH+, CD34+ALDH+, CD34+CD38‐ALDH+, and CD34+CD38+ALDH+ cells all had no impact on the achievement of CR (H group vs L group: 92.3% vs 71.8%, P = .25; 73.3% vs 78.4%, P = .98; 76.5% vs 77.1%, P = 1.0; 70.8% vs 82.1%, P = .34). 3.5 \| Impact of the percentage of ALDH+ cells on relapse In the 48 follow‐up patients who achieved CR and received consolidation therapy, ALDH+‐H patients tended to have a lower 2‐year RFS rate than ALDH+‐L patients (65.6% [95% CI 32.0%‐85.6%] vs 84.7% [95% CI 57.0%‐95.2%], P = .062, Figure 2A). However, CD34+ALDH+‐H pa- tients had a significantly lower 2‐year RFS rate com- pared to CD34+ALDH+‐L patients (60.6% [95% CI 28.7%‐81.8%] vs 88.0% [95% CI 57.2%‐97.1%], P = .015, Figure 2B). Furthermore, both CD34+CD38‐ALDH+‐H and CD34+CD38+ALDH+‐H patients had a significantly lower 2‐year RFS rate compared to CD34+CD38‐ALDH+‐L and CD34+CD38+ALDH+‐L patients, respectively (61.9% [95% CI 30.7%‐82.3%] vs 88.0% [95% CI 57.2%‐97.1%], P = .016, Figure 2C; 70.1% [95% CI 44.8%‐85.4%] vs 88.9% [95% CI 43.3%‐98.4%], P = .049, Figure 2D). 2.4 Pairwise comparisons of the variables between groups were performed using the Fisher's exact test for categorical vari- ables. A receiver operating characteristic (ROC) curve was 5462 YANG et al TABLE 1 Characteristics of the patients at diagnosis TABLE 1 Characteristics of the patients at diagnosis Variables Value N 66 Age (y, median; range) 38 (2‐66) Sex (male/female) 44/22 White blood cell (WBC) count (×109/L; median; range) 8.8 (1.7‐58.2) Hemoglobin (g/L; median; range) 81 (42‐126) Platelet count (×109/L; median; range) 32.5 (2‐312) Blasts in BM (%, median; range) 51 (15‐89) Patients with cytogenetic abnormalities other than t(8;21) (n = 62) 41 (66.1%) RUNX1‐RUNX1T1 transcript level 555.0% (123.7%‐1880.5%) Patients with c‐KIT mutations 27 (40.9%) achieved CR was 78.3% (95% confidence interval (CI) 57.9%‐89.6%). Three (5.8%) of 52 patients died during fol- low‐up, and the 2‐year OS rate of the 52 patients was 92.6% (95%CI 78.3%‐97.6%). 3.4 \| Impact of the percentage of ALDH+cells on the achievement of CR 3.7 \| The impact of the combination of the percentage of CD34+ALDH+ cells with MRD on relapse By combining the percentage of CD34+ALDH+ cells with MRD levels, 39 patients were categorized into the follow- ing four groups: CD34+ALDH+‐L/MRD‐L (n = 21), CD34+ALDH+‐H/MRD‐L (n = 7), CD34+ALDH+‐L/ MRD‐H (n = 5), and CD34+ALDH+‐H/MRD‐H (n = 6). As shown in Figure 3B, the 2‐year RFS rate was sig- nificantly different among the four groups (P = .0002). In MRD‐H patients, CD34+ALDH+‐H/MRD‐H pa- tients had a significantly lower 2‐year RFS rate than CD34+ALDH+‐L/MRD‐H patients (33.3% [95% CI 4.6%‐67.6%] vs 100%, P = .036). Furthermore, CD34+ALDH+‐L/MRD‐H patients had a similar 2‐ year RFS rate to both CD34+ALDH+‐L/MRD‐L and CD34+ALDH+‐H/MRD‐L patients (100% vs 85.3%, P = .50; 100% vs 100%, P = 1.0). Therefore, these three groups were merged into the CD34+ALDH+‐L and/or MRD‐L group. As a result, CD34+ALDH+‐H/MRD‐H patients had a significantly lower 2‐year RFS rate com- pared to CD34+ALDH+‐L and/or MRD‐L patients (33.3% [95% CI 4.6%‐67.6%] vs 90.3% [95% CI 64.2%‐97.7%], P < .0001, Figure 3C). Similar results were observed if Because the percentages of both CD34+CD38‐ALDH+ and CD34+CD38+ALDH+ had an impact on relapse, the CD34+ALDH+ subset was used in the subsequent analysis. 3.3 \| Determination of the optimal cutoff values for patient grouping (A‐D) no censoring; (E‐H) censoring at the time of allo‐ HSCT; (A, E) ALDH+; (B, F) CD34+ALDH+; (C, G) CD34+CD38‐ALDH+; (D, H) CD34+CD38+ALDH+ \| 5463 5463 YANG et al FIGURE 2 RFS analysis of the 48 follow‐up patients based on ALDH+grouping. (A‐D) no censoring; (E‐H) censoring at the time of allo‐ HSCT; (A, E) ALDH+; (B, F) CD34+ALDH+; (C, G) CD34+CD38‐ALDH+; (D, H) CD34+CD38+ALDH+ [95% CI 37.9%‐95.6%], P = .022, Figure 2G). In addition, CD34+CD38+ALDH+‐H patients tended to have a lower 2‐ year RFS rate than CD34+CD38+ALDH+‐L patients (66.1% [95% CI 38.2%‐83.7%] vs 80.0% [95% CI 20.4%‐96.9%], P = .083, Figure 2H). [95% CI 37.9%‐95.6%], P = .022, Figure 2G). In addition, CD34+CD38+ALDH+‐H patients tended to have a lower 2‐ year RFS rate than CD34+CD38+ALDH+‐L patients (66.1% [95% CI 38.2%‐83.7%] vs 80.0% [95% CI 20.4%‐96.9%], P = .083, Figure 2H). a lower 2‐year RFS rate compared with that of treatment with allo‐HSCT (P = .070). In contrast, white blood cell (WBC) count, hemoglobin, platelet count, blast percentage in BM, cytogenetic abnormalities, other than t(8;21), CR achievement after the first induction therapy, and c‐KIT mutation status at diagnosis had no impact on the 2‐year RFS rate (all P > .05). Thirty‐three adult patients who achieved CR and re- ceived consolidation therapy were further analyzed. As shown in Figure S1, ALDH+‐H and CD34+ALDH+‐H patients individually had significantly lower 2‐year RFS rate compared with ALDH+‐L and CD34+ALDH+‐L patients (P = .035, Figure S1A; P = .014, Figure S1B). Furthermore, both CD34+CD38‐ALDH+‐H and CD34+CD38+ALDH+‐H patients had significantly lower 2‐year RFS rates compared with CD34+CD38‐ALDH+‐L and CD34+CD38+ALDH+‐L patients, respectively (P = .018, Figure S1C; P = .032, Figure S1D). Similar re- sults existed if the 11 patients who underwent allo‐HSCT were censored at the time of transplantation (Figure S1E‐H). 3.3 \| Determination of the optimal cutoff values for patient grouping The ROC curve analysis was performed in 48 follow‐up pa- tients who achieved CR and received consolidation therapy. Although the percentages of ALDH+, CD34+ALDH+, CD34+CD38‐ALDH+, and CD34+CD38+ALDH+ cells could not significantly differentiate patients who relapsed (P = .23, .065, .072, and .088), trends existed, and 0.34%, 0.065%, 0.024%, and 0.0094% were determined as the indi- vidual optimal cutoff values according to the Youden index. We referred to patients with percentages of ALDH+, CD34+ALDH+, CD34+CD38‐ALDH+, and CD34+CD38+ALDH+ cells that were higher than the cut- off values as ALDH+‐H, CD34+ALDH+‐H, CD34+CD38‐ ALDH+‐H, and CD34+CD38+ALDH+‐H, and the patients with percentages of cells that were lower than the cutoff values as ALDH+‐L, CD34+ALDH+‐L, CD34+CD38‐ALDH+‐L, and CD34+CD38+ALDH+‐L. Thus, among the 48 follow‐up patients, 13 (27.1%), 15 (31.3%), 16 (33.3%), and 23 (47.9%) patients were categorized as ALDH+‐H, CD34+ALDH+‐H, CD34+CD38‐ALDH+‐H, and CD34+CD38+ALDH+‐H, respectively. The ROC curve analysis was performed in 48 follow‐up pa- tients who achieved CR and received consolidation therapy. Although the percentages of ALDH+, CD34+ALDH+, CD34+CD38‐ALDH+, and CD34+CD38+ALDH+ cells could not significantly differentiate patients who relapsed (P = .23, .065, .072, and .088), trends existed, and 0.34%, 0.065%, 0.024%, and 0.0094% were determined as the indi- vidual optimal cutoff values according to the Youden index. We referred to patients with percentages of ALDH+, CD34+ALDH+, CD34+CD38‐ALDH+, and CD34+CD38+ALDH+ cells that were higher than the cut- off values as ALDH+‐H, CD34+ALDH+‐H, CD34+CD38‐ ALDH+‐H, and CD34+CD38+ALDH+‐H, and the patients with percentages of cells that were lower than the cutoff values as ALDH+‐L, CD34+ALDH+‐L, CD34+CD38‐ALDH+‐L, and CD34+CD38+ALDH+‐L. Thus, among the 48 follow‐up patients, 13 (27.1%), 15 (31.3%), 16 (33.3%), and 23 (47.9%) patients were categorized as ALDH+‐H, CD34+ALDH+‐H, CD34+CD38‐ALDH+‐H, and CD34+CD38+ALDH+‐H, respectively. Next, the 12 patients who underwent allo‐HSCT were cen- sored at the time of transplantation. ALDH+‐H patients had a similar 2‐year RFS rate to ALDH+‐L patients (64.3% [95% CI 29.8%‐85.1%] vs 76.4% [95% CI 38.3%‐92.7%], P = .14, Figure 2E). However, CD34+ALDH+‐H and CD34+CD38‐ ALDH+‐H patients were both individually significantly related to a lower 2‐year RFS rate compared with that of CD34+ALDH+‐L and CD34+CD38‐ALDH+‐L patients (55.4% [95% CI 21.2%‐79.9%] vs 81.0% [95% CI 37.8%‐95.5%], P = .027, Figure 2F; 54.5% [95% CI 20.4%‐79.4%] vs 81.2% \| 5463 YANG et al FIGURE 2 RFS analysis of the 48 follow‐up patients based on ALDH+grouping. 3.6 \| Univariate analysis of variables other than the percentage of ALDH+ cells Therefore, ALDH may improve MRD‐based risk stratification in t(8;21) AML. 3.6 \| Univariate analysis of variables other than the percentage of ALDH+ cells Among 39 patients who were followed up at least until the second course of consolidation therapy, MRD‐H patients had a significantly lower 2‐year RFS rate than MRD‐L patients (62.3% [95% CI 27.7%‐84.0%] vs 88.8% [95% CI 59.7%‐97.3%], P = .017, Table 2, Figure 3A), and pedi- atric patients had a significantly lower 2‐year RFS rate than adult patients (P = .032). In addition, treatment with chemotherapy only tended to be significantly related to 5464 YANG et al YANG et al 5464 \| YANG et al TABLE 2 Univariate analysis of relapse in the follow‐up patients (n = 48) Variables No. of patients 2‐year RFS rate (95% CI) HR (95% CI) P value Age Pediatric patients 15 100% 1.0 .032 Adult patients 33 65.3% (37.4%‐83.2%) 5.3 (1.2‐24.3) Sex Male 31 88.7% (69.0%‐96.2%) 1.0 .17 Female 17 57.3% (20.1%‐82.4%) 3.0 (0.62‐14.6) WBC count at diagnosis ≤ 10 × 109/L 28 85.9% (61.9%‐95.3%) 1.0 .33 > 10 × 109/L 20 67.9% (31.1%‐87.9%) 2.1 (0.47‐9.7) Hemoglobin ≤ 80 g/L 24 80.2% (48.5%‐93.5%) 1.0 .64 > 80 g/L 24 78.6% (51.5%‐91.6%) 1.4 (0.32‐6.4) Platelet count ≤ 35 × 109/L 23 81.9% (53.8%‐93.8%) 1.0 .94 > 35 × 109/L 25 74.2% (41.2%‐90.4%) 0.95 (0.21‐4.3) Blasts in BM ≤ 50% 24 71.0% (36.3%‐89.0%) 1.0 .79 > 50% 24 85.0% (60.4%‐94.9%) 0.82 (0.18‐3.6) Patients with cytogenetic abnormalities other than t(8;21) No 16 75.2% (40.7%‐91.4%) 1.0 .45 Yes 30 78.4% (49.3‐92.0) 0.53 (0.10‐2.7) c‐KIT gene Wild‐type 29 71.2% (44.6%‐86.7%) 1.0 .25 Mutation 19 93.3% (61.2%‐99.0%) 0.40 (0.085‐1.9) Treatment modality Allo‐HSCT 12 100% 1.0 .070 Chemotherapy only 36 68.9% (42.4%‐85.1%) 4.4 (0.89‐21.5) CR after the first induction therapy Yes 40 76.2% (52.3%‐89.3%) 1.0 .78 No 8 87.5% (38.7%‐98.1%) 0.76 (0.11‐5.2) MRD status MRD‐L 28 88.8% (59.7%‐97.3%) 1.0 .017 MRD‐H 11 62.3% (27.7%‐84.0%) 9.7 (1.5‐63.0) the patients who underwent allo‐HSCT were censored at the time of transplantation (20.8% [95% CI 0.87%‐59.5%] vs 85.3% [95% CI 47.7%‐96.7%], P < .0001, Figure 3D). the patients who underwent allo‐HSCT were censored at the time of transplantation (20.8% [95% CI 0.87%‐59.5%] vs 85.3% [95% CI 47.7%‐96.7%], P < .0001, Figure 3D). Of 11 MRD‐H patients, four patients relapsed. After con- sidering the percentage of CD34+ALDH+ cells along with MRD, five patients were placed into the CD34+ALDH‐L/ MRD‐H group and none of these patients relapsed; all four of the relapsed patients were placed into the CD34+ALDH‐H/ MRD‐H group. 4 \| DISCUSSION to successfully reconstitute leukemia by engrafting cells into immunodeficient mice.11-13 However, mouse engraftment could not be used in clinical practice due to its operational complexity. Thus, it is necessary to find feasible methods to identify LSCs and evaluate their clinical significance. AML with t(8;21) is a heterogeneous disease, and other prognostic factors need to be assessed in addition to MRD.2 In the present study, ALDH activity was evaluated in 66 t(8;21) AML patients at diagnosis. We found that high per- centages of CD34+ALDH+, CD34+CD38‐ALDH +, and CD34 + CD38+ALDH + cells as well as high MRD lev- els were significantly related to relapse. After combining the percentage of CD34+ALDH+ at diagnosis with MRD, patients were regrouped, and CD34+ALDH+‐H/MRD‐H status was an independent adverse prognostic factor for re- lapse in t(8;21) AML. ALDHs are an NAD(P)+‐dependent enzyme superfamily that are involved in various biological processes.17,18 Many reports have demonstrated that ALDH is expressed at high levels in cancer stem cells.19-21,25-27 In AML, ALDH was found to be highly expressed in LSCs and to be related to chemotherapy resistance.22,24-27 Several studies have evaluated the prognostic role of ALDH in AML. Cheung et al reported that in patients, a high percentage of ALDH+ cells was associated with adverse cytogenetic abnormalities.25 Ran et al demon- strated that AML patients with a higher percentage of ALDH+ cells had a high number of genetic and molec- ular risk factors and poor clinical outcomes.24,26 Hoang et al analyzed intermediate‐risk AML and found that patients with a high percentage of ALDH+ cells had shorter disease‐free survival and OS than patients with a low percentage of ALDH+ cells.27 In a prospective clin- ical study, Gerber et al reported that patients with a high percentage of CD34+CD38‐ALDH + cells manifested a significantly lower CR rate and lower event‐free and OS rates.33 A total of five t(8;21) AML and 13 AML pa- tients with favorable cytogenetic risk were included in the above five studies, and all of them were categorized as ALDH+‐L.24-27,33 It implied that among AML, patients with t(8;21) usually had low percentage of ALDH+, which is in consistent with their low cytogenetic risk. In the present study, similar prognostic significance of the percentage of ALDH+ was seen within t(8;21) AML; that is, a high percentage of CD34 + ALDH+ cells was cor- related with a low RFS rate. 3.8 \| CD34+ALDH+‐H/MRD‐H status independently predicts relapse CD34+ALDH+‐H/MRD‐H status, age, sex, and treat- ment modality were all used in a multivariate analysis of 39 patients who were followed up at least until the second course of consolidation therapy. The results showed that CD34+ALDH+‐H/MRD‐H status was the only independ- ent adverse prognostic factor for relapse (HR 27.5 [95% CI 3.1‐246.4], P = .0030). Of 11 MRD‐H patients, four patients relapsed. After con- sidering the percentage of CD34+ALDH+ cells along with MRD, five patients were placed into the CD34+ALDH‐L/ MRD‐H group and none of these patients relapsed; all four of the relapsed patients were placed into the CD34+ALDH‐H/ MRD‐H group. Therefore, ALDH may improve MRD‐based risk stratification in t(8;21) AML. \| 5465 YANG et al FIGURE 3 RFS of patients grouped by MRD (A); combination of CD34+ALDH+ and MRD (B); CD34+ALDH+‐L and/or MRD‐L and CD34+ALDH+‐H/MRD‐H (C); CD34+ALDH+‐L and/or MRD‐L and CD34+ALDH+‐H/MRD‐H with censoring at the time of allo‐HSCT (D) \| 5465 g 5465 YANG et al Since MRD is the most important prognostic molecular marker in t(8;21) AML, which was similarly demonstrated in the current study, we combined the ALDH with MRD parameters. We found that in patients, both CD34+CD38‐ ALDH+‐H and CD34+CD38+ALDH+‐H status had an adverse impact on the 2‐year RFS rate. CD34+CD38‐ is widely accepted as an immunophenotype of LSCs, but CD34+CD38+ cells were also found to have LSC char- acteristics.14 Pearce et al reported that the CD34+ and ALDH+ subpopulations in AML largely overlapped, and CD34+ALDH+ cells possess the CD34+CD38‐ and CD34+CD38+ phenotypes.22 Therefore, we did not consider CD38 expression, and we combined the CD34+ALDH+ and MRD parameters to categorize patients into four groups. The combined results showed that only patients with concurrent high levels of CD34+ALDH+ at diagnosis and MRD had a high relapse risk. Furthermore, CD34+ALDH+‐H/MRD‐H was found to be the only in- dependent adverse prognostic factor. Our results demon- strated the usefulness of ALDH for improving MRD‐based risk stratification in t(8;21) AML. REFERENCES 1. Schlenk RF, Benner A, Krauter J, et al. 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Nature. 1994;367(6464):645‐648. 4 \| DISCUSSION Over the past decade, molecular markers have been incorporated into the risk stratification system in t(8;21) AML. Several studies, including ours, have demonstrated that high MRD levels, which are indicated by less than a 3‐log reduction of RUNX1‐RUNX1T1 transcript levels during treatment, were poor prognostic factors, although the significant time points were different among the stud- ies.4-6 In addition, c‐KIT mutation was found to be the most prevalent mutation in t(8;21) AML and has been demon- strated to be related to poor outcomes by many studies.7-9 However, the specificity and sensitivity of the current risk stratification are not perfect. Whether other parameters could complement the use of molecular markers needs to be evaluated in t(8;21) AML. Relapse was a major challenge for the long‐term outcome of AML patients and may be caused by the persistence of LSCs. LSCs have been reported to be quiescent, well pro- tected within the bone marrow niche, resistant to chemother- apy, and responsible for recurrent disease.30 Some reports have demonstrated that the frequency of LSCs in patients with AML might be prognostic.31,32 Evidence for the iden- tification of LSCs involves a demonstration of the capacity 5466 YANG et al AUTHOR CONTRIBUTIONS 12. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3(7):730‐737. LY wrote manuscript; WMC performed the PCR analysis; LY, FTD, YHZ YZW, and YC performed flow cytometry; QJ, XHZ, KYL, and XJH collected clinical data; YZQ de- signed the study and revised the manuscript. All authors gave final approval. 13. Blatt K, Menzl I, Eisenwort G, et al. Phenotyping and target ex- pression profiling of CD34(+)/CD38(−) and CD34(+)/CD38(+) Stem‐ and Progenitor cells in Acute Lymphoblastic Leukemia. 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Conditioning including antithymocyte globulin followed by unmanipulated HLA‐mismatched/haploiden- tical blood and marrow transplantation can achieve comparable outcomes with HLA‐identical sibling transplantation. Blood. 2006;107(8):3065‐3073. 18. Black WJ, Stagos D, Marchitti SA, et al. Human aldehyde dehy- drogenase genes: alternatively spliced transcriptional variants and their suggested nomenclature. Pharmacogenet Genomics. 2009;19(11):893‐902. 30. Yan B, Chen Q, Shimada K, et al. Histone deacetylase inhibitor tar- gets CD123/CD47‐positive cells and reverse chemoresistance phe- notype in acute myeloid leukemia. Leukemia. 2018;33(4):931‐944. 19. Ginestier C, Hur MH, Charafe‐Jauffret E, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predic- tor of poor clinical outcome. Cell Stem Cell. 2007;1(5):555‐567. 31. Pearce DJ, Taussig D, Zibara K, et al. AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML. Blood. 2006;107(3):1166‐1173. 20. Sullivan JP, Spinola M, Dodge M, et al. Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling. Can Res. 2010;70(23):9937‐9948. 21. Landen CN, Goodman B, Katre AA, et al. Targeting aldehyde de- hydrogenase cancer stem cells in ovarian cancer. Mol Cancer Ther. 2010;9(12):3186‐3199. 32. van Rhenen A, Feller N, Kelder A, et al. High stem cell fre- quency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res. 2005;11(18):6520‐6527. 22. Pearce DJ, Taussig D, Simpson C, et al. Characterization of cells with a high aldehyde dehydrogenase activity from cord blood and acute myeloid leukemia samples. Stem Cells. 2005;23(6):752‐760. 33. Gerber JM, Zeidner JF, Morse S, et al. Association of acute my- eloid leukemia's most immature phenotype with risk groups and outcomes. Haematologica. 2016;101(5):607‐616. 23. Storms RW, Green PD, Safford KM, et al. Distinct hematopoietic progenitor compartments are delineated by the expression of alde- hyde dehydrogenase and CD34. Blood. 2005;106(1):95‐102. 24. Ran D, Schubert M, Pietsch L, et al. Aldehyde dehydrogenase activity among primary leukemia cells is associated with stem cell features and correlates with adverse clinical outcomes. Exp Hematol. 2009;37(12):1423‐1434. ORCID Qian Jiang https://orcid.org/0000-0001-7131-0522 Xiao‐Hui Zhang https://orcid.org/0000-0003-0245-6792 Kai‐Yan Liu https://orcid.org/0000-0002-6751-7827 Ya‐Zhen Qin https://orcid.org/0000-0002-1548-0946 15. Taussig DC, Vargaftig J, Miraki‐Moud F, et al. Leukemia‐ini- tiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(‐) fraction. Blood. 2010;115(10):1976‐1984. YANG et al 5467 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article. 25. Cheung A, Wan T, Leung J, et al. Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting poten- tial. Leukemia. 2007;21(7):1423‐1430. How to cite this article: Yang L, Chen W‐M, Dao F‐T, et al. High aldehyde dehydrogenase activity at diagnosis predicts relapse in patients with t(8;21) acute myeloid leukemia. Cancer Med. 2019;8:5459– 5467. https://doi.org/10.1002/cam4.2422 How to cite this article: Yang L, Chen W‐M, Dao F‐T, et al. High aldehyde dehydrogenase activity at diagnosis predicts relapse in patients with t(8;21) acute myeloid leukemia. Cancer Med. 2019;8:5459– 5467. https://doi.org/10.1002/cam4.2422 How to cite this article: Yang L, Chen W‐M, Dao F‐T, et al. High aldehyde dehydrogenase activity at diagnosis predicts relapse in patients with t(8;21) acute myeloid leukemia. Cancer Med. 2019;8:5459– 5467. https://doi.org/10.1002/cam4.2422 26. Ran D, Schubert M, Taubert I, et al. Heterogeneity of leukemia stem cell candidates at diagnosis of acute myeloid leukemia and their clinical significance. Exp Hematol. 2012;40(2):155‐165. e151. 27. Hoang VT, Buss EC, Wang W, et al. The rarity of ALDH(+) cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients. Int J Cancer. 2015;137(3):525‐536.
https://openalex.org/W3166577797	https://www.frontiersin.org/articles/10.3389/fvets.2021.668679/pdf	English	null	Using a Bayesian Network Predictive Model to Understand Vulnerability of Australian Sheep Producers to a Foot and Mouth Disease Outbreak	Frontiers in veterinary science	2,021	cc-by	9,546	ORIGINAL RESEARCH published: 11 June 2021 doi: 10.3389/fvets.2021.668679 Using a Bayesian Network Predictive Model to Understand Vulnerability of Australian Sheep Producers to a Foot and Mouth Disease Outbreak Jennifer Manyweathers 1,2, Yiheyis Maru 3, Lynne Hayes 2, Barton Loechel 4, Heleen Kruger 5, Aditi Mankad 4, Gang Xie 6, Rob Woodgate 1,2 and Marta Hernandez-Jover 1,2 1 Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW, Australia, 2 School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia, 3 Commonwealth Scientiﬁc and Industrial Research Organisation Land and Water, Canberra, ACT, Australia, 4 Commonwealth Scientiﬁc and Industrial Research Organisation, Brisbane, QLD, Australia, 5 Australian Bureau of Agricultural and Resource Economics and Sciences (ABARES), Canberra, ACT, Australia, 6 Quantitative Consulting Unit, Charles Sturt University, Wagga Wagga, NSW, Australia Edited by: The most Reviewed by: Georgina Limon, Pirbright Institute, United Kingdom Pedro Larrañaga, Polytechnic University of Madrid, Spain Correspondence: Jennifer Manyweathers jmanyweathers@csu.edu.au Specialty section: This article was submitted to Veterinary Epidemiology and Economics, a section of the journal Frontiers in Veterinary Science Received: 16 February 2021 Accepted: 22 April 2021 Published: 11 June 2021 Specialty section: This article was submitted to Veterinary Epidemiology and Economics, Specialty section: This article was submitted to Veterinary Epidemiology and Economics, a section of the journal Frontiers in Veterinary Science Received: 16 February 2021 Received: 16 February 2021 Accepted: 22 April 2021 Published: 11 June 2021 Edited by: Edited by: Alejandra Victoria Capozzo, Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET), Argentina Edited by: Alejandra Victoria Capozzo, Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET), Argentina To maintain and strengthen Australia’s competitive international advantage in sheep meat and wool markets, the biosecurity systems that support these industries need to be robust and effective. These systems, strengthened by jurisdictional and livestock industry investments, can also be enhanced by a deeper understanding of individual producer risk of exposure to animal diseases and capacity to respond to these risks. This observational study developed a Vulnerability framework, built from current data from Australian sheep producers around behaviors and beliefs that may impact on their likelihood of Exposure and Response Capacity (willingness and ability to respond) to an emergency animal disease (EAD). Using foot and mouth disease (FMD) as a model, a cross-sectional survey gathered information on sheep producers’ demographics, and their practices and beliefs around animal health management and biosecurity. Using the Vulnerability framework, a Bayesian Network (BN) model was developed as a ﬁrst attempt to develop a decision making tool to inform risk based surveillance resource allocation. Populated by the data from 448 completed questionnaires, the BN model was analyzed to investigate relationships between variables and develop producer Vulnerability proﬁles. Respondents reported high levels of implementation of biosecurity practices that impact the likelihood of exposure to an EAD, such as the use of appropriate animal movement documentation (75.4%) and isolation of incoming stock (64.9%). However, adoption of other practices relating to feral animal control and biosecurity protocols for visitors were limited. Respondents reported a high uptake of Response Capacity practices, including identifying themselves as responsible for observing (94.6%), reporting unusual signs of disease in their animals (91.0%) and daily/weekly inspection of animals (90.0%). The BN analysis identiﬁed six Vulnerability typologies, with three levels of Exposure (high, moderate, low) and two levels of Response Capacity (high, low), as described by producer demographics and practices. INTRODUCTION approach is a comprehensive understanding of the factors that may inﬂuence both likelihood of exposure to FMD and the response capacity (willingness and ability) to respond to an outbreak. At a national level, East et al. (5) used a multicriteria analysis and found that current surveillance eﬀorts are eﬀectively targeting the highest risk areas for an FMD incursion. Subsequent research has highlighted further opportunities for improving FMD outbreak preparedness by decreasing the time between introduction and diagnosis (19). Garner et al. (20) also showed an inverse correlation between the likelihood of successful eradication of FMD and the time taken for initial diagnosis. The Australian sheep industry has long been recognized for its signiﬁcant contribution to the global sheep meat and wool industries (1). Protecting the sheep meat and wool industries in the face of increasing global risks associated with signiﬁcant disease outbreaks such as foot and mouth disease (FMD) (2–4) and maintaining Australia’s clean and green reputation should remain a high priority. The current animal health surveillance system for notiﬁable diseases includes targeted programs but also relies on general surveillance primarily underpinned by producers notifying their private or government veterinarian of unusual signs of disease. By strengthening the capacity of rural communities and the producers themselves to rapidly identify and contain any possible future emergency animal disease (EAD) outbreaks will enhance preparedness of the existing surveillance systems (2, 5–7). To reduce the time taken between infection and diagnosis, understanding biosecurity beliefs and practices of producers and those regularly in contact with livestock is paramount (12, 21). This includes the ability to recognize unusual signs of disease in livestock and to take appropriate steps to get a diagnosis (22). When modeling the spread of FMD from a single index farm, one of the most inﬂuential factors was found to be the ability of the producer to detect unusual signs leading to identiﬁcation of FMD (23). Therefore, any risk-based approach to surveillance needs to be informed by stakeholder engagement, including producers’ beliefs and practices around their role in general surveillance, deﬁned in this study as monitoring for, recognizing, and reporting unusual signs of disease in their animals (22, 24–27). Historically, Australian Government resources have facilitated locally based veterinarians and supported personnel spending time on-farm. In keeping with global trends (8–10), these resources are decreasing, resulting in a weakening of local relationships and diminishing the strong surveillance networks that previously existed (2, 11–13). Citation: Manyweathers J, Maru Y, Hayes L, Loechel B, Kruger H, Mankad A, Xie G, Woodgate R and Hernandez-Jover M (2021) Using a Bayesian Network Predictive Model to Understand Vulnerability of Australian Sheep Producers to a Foot and Mouth Disease Outbreak. Front. Vet. Sci. 8:668679. doi: 10.3389/fvets.2021.668679 June 2021 \| Volume 8 \| Article 668679 Frontiers in Veterinary Science \| www.frontiersin.org 1 Manyweathers et al. BN Model of Producer Vulnerability inﬂuential Exposure variables on producer Vulnerability included adoption levels of visitor biosecurity and visitor access protocols. Findings from this study can guide decisions around resource allocation to improve Australia’s readiness for EAD incursion and strengthen the country’s biosecurity system. Keywords: Bayesian network model, foot and mouth disease, biosecurity, vulnerability, Australian sheep producers, surveillance, partnership Keywords: Bayesian network model, foot and mouth disease, biosecurity, vulnerability, Australian sheep producers, surveillance, partnership INTRODUCTION The “shared responsibility” approach to surveillance adopted by the Australian Government (4) has great potential. However, in practice, it has not been met with a consistent level of engagement by stakeholders (10). This has resulted in a perception among producers that the government is devolving itself of responsibility for surveillance and decreasing the priority of livestock industries more generally, further undermining the surveillance system (2, 14). To inform risk-based approaches to strengthening surveillance strategies, an EAD risk characterization of livestock producers based on FMD vulnerability was developed from a cross-sectional survey that collected producer information from the FMD-susceptible livestock industries in Australia. The survey data were then used to populate a Bayesian Network (BN) model for analysis of producer vulnerability (28). This paper focuses on Australian sheep producers’ beliefs and practices that may inﬂuence their likelihood of exposure and capacity to respond to an FMD outbreak. The results pertaining to the Australian beef and goat industries have been reported elsewhere (22, 29). Any weakening of partnerships within the surveillance system is of particular concern within the sheep industry, which has traditionally had lower levels of engagement with animal health professionals (15, 16). Learnings from the FMD outbreak across the United Kingdom suggest that sheep are likely to play a signiﬁcant role in any undetected occurrence and spread of FMD in the event of an outbreak because of the variable and transient nature of ovine FMD symptoms (6, 17, 18). As part of maintaining and strengthening protective biosecurity and surveillance strategies, and strengthening partnerships, understanding the diversity of the sheep industry and its unique risk proﬁle is vital. Frontiers in Veterinary Science \| www.frontiersin.org Questionnaire Distribution The questionnaire was distributed online and via post between August 2017 and June 2018, with ﬁnal distribution routes guided by available support from industry and government agencies. Multiple farming system groups from the Riverina region in New South Wales (NSW), along with Local Lands Services (NSW government agency), distributed the questionnaire via direct email link to members, with accompanying coverage in newsletters and on Twitter and Facebook. The Livestock Biosecurity Network distributed the questionnaire link in their newsletters and also discussed the study during biosecurity workshops in NSW. Interested producers were followed up and provided with the link. In addition, a link to the questionnaire was distributed by the Graham Centre for Agricultural Innovation, a research center of Charles Sturt University, and NSW Department of Primary Industries, as well as the Victorian Farmers Federation, via their newsletters. In Western Australia (WA), a random sample of 750 sheep producers with a registered Property Identiﬁcation Code (PIC) was emailed a link to the questionnaire. The questionnaire, which consisted of 61 questions, gathered information in relation to practices, perceptions, and attitudes around the risk of and response to a FMD outbreak. The following four main areas were included in the questionnaire: Demographics and husbandry practices (24 questions), Biosecurity Sheep producers who completed the questionnaire were invited to enter a draw for 20 × $50 retail vouchers. In addition, they also had the opportunity to enter a draw for two smart tablets across participants from all industries. FIGURE 1 \| Classiﬁcation matrix of vulnerability as the intersection of exposure and response capacity: light gray = low vulnerability; medium gray = moderate vulnerability; black = high vulnerability [(22), adapted from Nelson et al. (30)]. MATERIALS AND METHODS A cross-sectional study was designed, and a questionnaire was developed to gather quantitative data to build a vulnerability- based typology of the Australian sheep industry, using FMD as a model. The methodological approach used for this study was the same of that used in Manyweathers et al. (22, 29). All research activities were approved by the Human Research Ethics Committee at Charles Sturt University (H400201720). Risk-based approaches to surveillance for FMD could reduce the probability of threats occurring and assuage consequences of a possible FMD outbreak in Australia. These approaches can assist the identiﬁcation of potential routes of entry and establishment of FMD and eﬀective allocation of resources for surveillance and response (2, 8). The foundation of such an June 2021 \| Volume 8 \| Article 668679 2 Manyweathers et al. BN Model of Producer Vulnerability Questionnaire Design practices and beliefs (8), Animal health management practices (23), and Networks and trust (10). The questionnaire was developed with reference to existing epidemiological, behavioral, and social science research and aimed at the examination and characterization of sheep producers’ vulnerability to an FMD outbreak. Further details of the design and development of the questionnaire are provided by Manyweathers et al. (22). In brief, a vulnerability matrix (Figure 1) guided questionnaire development, with questions examining producers’ likelihood of exposure to FMD and their response capacity (willingness and ability to inspect animals, detect and report disease) to an FMD outbreak. The questionnaire was developed by a multidisciplinary team, including social and behavioral science researchers, veterinarians, systems science, and biosecurity researchers, through an iterative process. The questionnaire was then piloted prior to distribution by one sheep industry representative, one veterinarian, and two sheep producers, to improve validity and clarity. Bayesian Network Analysis was not distinguishable, while the elicited “exposure” levels with three categories were more distinctive. In our study, the information contained in the observed variables that deﬁne the “response capacity” was not strong enough to distinguish three categories. Based on the principle of parsimony for modeling, we decided to specify/model “response capacity” with two categories. Hence, the ﬁnalized conceptual model was modiﬁed with the vulnerability deﬁnition matrix to be a 2 × 3 one (two categories for response capacity and three categories for exposure). The vulnerability deﬁnition matrix implemented in our completed BN model is therefore a result of a compromise between our disciplinary understanding/intention and the technical possibility/limitation due to the information contained in the observed variables (35). A Bayesian network model is a probabilistic graphical tool that allows for modeling of biological, social, and physical systems that operate under uncertainty (28, 31) and is suitable for a large number of diﬀerent data types and hidden variables being connected through complex relationships (32). Formally, a BN model is a graphical representation, i.e., a directed acyclic graph (DAG), of a joint probability distribution of a set of random variables in which each variable is represented by a node and the dependence relationship is represented by a link/edge for two associated variables (28, 33). Essentially, a BN model follows a machine learning approach for data analysis. Although the theoretical foundation and computational algorithms underlying BNs are utilized in subjects such as computer science, mathematics, and statistics, the applications of BN models are very intuitive and relatively straightforward because of the availability of many well-tested BN application software packages (28, 31). In this study, we used the most popular commercial BN software Netica (version 6.05) (34) to characterize Australian sheep producers based on their enterprises’ vulnerability to an FMD outbreak. This approach has also been used by beef and goat producers (22, 29). Observed variables were imported into the BN model. The category levels in each observed variable were deﬁned a priori in the information table related to Figure 2. Because we had a very well-deﬁned conceptual model, the BN model structure was manually speciﬁed by deﬁning each hidden variables with various local naive Bayes models to explore the suitability of identifying and classifying the possible meaningful categorical groups with each hidden variable in the model. Bayesian Network Analysis This was done initially for “response capacity” and “exposure.” Based on the principle of parsimony, the number of category levels for each hidden variable were determined arbitrarily. Every BN model has two components in its model speciﬁcation. The qualitative component of a BN speciﬁes the network structure through a set of (conditional) dependence and independence statements among a set of random variables, informational precedence, and preference relations; the quantitative component of a BN determines the conditional probability tables (CPTs) that quantiﬁes the strengths of dependence relations using probability theory and preference relations using utility theory (28). In this study, the BN model development processes started with the conceptual model as detailed in Figures 1, 2. The data collected from sheep farmers included 41 observed variables. According to our disciplinary theory, these observed variables were the building blocks for deﬁning various composite or hidden variables with which the level of “vulnerability” could be determined. Table 1 speciﬁes which hidden variable was deﬁned/characterized by the observed variables and the layers of the hidden variables. The hierarchical structure of the hidden variables and how they related to those observed variables are shown in Figure 2. The top level hidden variable “vulnerability” was deﬁned by “response capacity” and “exposure.” In turn, “response capacity” was further deﬁned by three sublevel hidden variables, and the “exposure” was deﬁned directly by seven observed variables; those demographics variables were all observed variables and should aﬀect both “response capacity” and “exposure.” Note that, due to space limitation, there were a large number of observed variables behind the three sublevel hidden variables that are not displayed in Figure 2 with detailed information provided in Table 1. The purpose of the FMD outbreak risk management decision- making system was to determine the vulnerability level as characterized by the producers’ demographics variables. This was implemented by integrating the two individual naive Bayesian net (one for deﬁning the response capacity and one for deﬁning the exposure) into one BN model with each of the 12 demographics variables connected as a child node to both response capacity and exposure nodes. Essentially, this was equivalent to the merger of two naive Bayesian nets with those demographics variables as the common linkage. Data Analysis Descriptive Analysis Data from the online and postal questionnaires were collated in Excel (PC/Windows XP, 2007) and checked for data entry errors. Descriptive statistics were used to obtain an overview of participant demographic and husbandry characteristics, practices, and attitudes (IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.). FIGURE 1 \| Classiﬁcation matrix of vulnerability as the intersection of exposure and response capacity: light gray = low vulnerability; medium gray = moderate vulnerability; black = high vulnerability [(22), adapted from Nelson et al. (30)]. FIGURE 2 \| Bayesian network conceptual model for examining Australian sheep producers’ vulnerability to foot and mouth disease (FMD). RE 2 \| Bayesian network conceptual model for examining Australian sheep producers’ vulnerability to foot and mouth disease (FMD) Frontiers in Veterinary Science \| www.frontiersin.org June 2021 \| Volume 8 \| Article 668679 3 BN Model of Producer Vulnerability Manyweathers et al. Frontiers in Veterinary Science \| www.frontiersin.org Bayesian Network Analysis To complete the BN model, the vulnerability node was added into the model that is categorically deﬁned by “response capacity” and “exposure.” As shown in the Supplementary Material, this is a static representation of the interactive model. The model contains 55 variables/nodes, of which 41 variables are observed data and 14 are hidden variables. Hidden variables are distributed in four diﬀerent layers according to our a priori knowledge regarding the relationship between the observed variables and the categories of exposure and response capacity and detailed in Table 1. Since a BN model represents the joint distribution of all variables included in the model, any one (or more than one) variable(s) may be selected as a target variable (equivalent to the “response” variable in a regression model). Various inferential analyses can be performed by assuming diﬀerent scenarios in terms of the “ﬁndings” of other variables. In this study, the primary statistical inference analysis was undertaken on the resulting BN model to investigate how, given one or more than observed variables/nodes, other variables/nodes changed. The focus of this analysis was to explore the interrelationships between vulnerability and producers’ demographic variables and to identify key characteristics driving exposure, response Initially, the conceptual model speciﬁed that the categorical matrix for deﬁning vulnerability was based on a 3 × 3 matrix with vulnerability being categorized by nine possible combinations between the levels of response capacity and exposure. However, the initial completed BN model showed that the hidden variable “response capacity” with three categories June 2021 \| Volume 8 \| Article 668679 4 BN Model of Producer Vulnerability Manyweathers et al. TABLE 1 \| A list of questions considered for assessing the likelihood of exposure and response capacity of Australian sheep producers to an foot and mouth disease (FMD) outbreak and the classiﬁcations of response. Questions Exposure related questions Classiﬁcation of responses High likelihood Moderate likelihood Low likelihood Do you Employ overseas workers? Yes Occasionally No Isolate new stock? Never, rarely Occasionally Most of the time, always Restrict access? Never, rarely Occasionally Most of the time, always Require visitor biosecurity practices? Never, rarely Occasionally Most of the time, always Take action to control feral animals? Never, rarely Yes Most of the time, always Have neighbors with FMD Susceptible species? Yes Yes No Have FMD susceptible feral species on your property? (Continued) Bayesian Network Analysis Attitudes Toward Foot and Mouth Disease Attitudes of producers toward FMD were investigated by the cross-sectional study. Overall concern about FMD was moderate, with a quarter of respondents (25.3%) reporting no concern and 22.6% reporting extreme levels of concern. The majority of respondents perceived little or no likelihood of an FMD outbreak occurring on their own property (92.8%) or region (80.3%). However, only 35.7% of producers thought there was little or no likelihood of an FMD outbreak somewhere in Australia. There was general agreement that an FMD outbreak would be extremely serious at all levels: on their property (78.3%), the region (73.6%), and the country (65.8%). capacity, and overall vulnerability. For example, by ﬁnding “evidence” in terms of the demographic variables (i.e., by assuming diﬀerent farmers’ proﬁles), the completed BN model allows us to investigate the vulnerability status and the nuances of its determining factors of response capacity and exposure. The BN model also allows us to examine the farmers proﬁles by assuming various vulnerability status (namely, diﬀerent combinations of response capacity, and exposure levels). Although a BN model is not a solution to the universal problem of lack of representativeness of data regarding the study population, it is a logically consistent and systematic way to perform the what-if analysis regarding the FMD outbreak risk management. capacity, and overall vulnerability. For example, by ﬁnding “evidence” in terms of the demographic variables (i.e., by assuming diﬀerent farmers’ proﬁles), the completed BN model allows us to investigate the vulnerability status and the nuances of its determining factors of response capacity and exposure. The BN model also allows us to examine the farmers proﬁles by assuming various vulnerability status (namely, diﬀerent combinations of response capacity, and exposure levels). Although a BN model is not a solution to the universal problem of lack of representativeness of data regarding the study population, it is a logically consistent and systematic way to perform the what-if analysis regarding the FMD outbreak risk management. The study also asked producers about their level of conﬁdence in identifying clinical signs of FMD in their animals, with only 12.8% reporting high levels of conﬁdence and a third reporting little to no conﬁdence. The next step was to identify the relative inﬂuence of relevant variables on exposure, response capacity, and vulnerability using the Netica’s built-in sensitivity analysis algorithm (28, 34). Bayesian Network Analysis Yes No Response-capacity-related questions Classiﬁcation of responses High capacity Low capacity How frequently do you undertake the following activities? Visual inspection Once a day, once a week Once a month, few times a year, once a year or less, never Visual and physical inspection Once a day, once a week Once a month, few times a year, once a year or less, never Inspection of unwell animals Once a day, once a week Once a month, few times a year, once a year or less, never Who do you think is responsible for Inspecting animals for unusual signs Me, staff Private or gov vet, stock agent, neighbors, industry group Recognizing unusual signs of disease Me, staff Private or gov vet, stock agent, neighbors, industry group Reporting unusual signs Me, staff Private or gov vet, stock agent, neighbors, industry group In the last 12 months, how often have you Used an NVD/health statement when buying animals Always, most of the time Occasionally, rarely, never Inspected stock before buying them Always, most of the time Occasionally, rarely, never How conﬁdent are you that you could identify FMD in your sheep Extremely, very, moderately Slightly, Not at all Rank ﬁrst three actions when you see unusual signs of disease Call private vet 1st, 2nd 3rd action Not in top 3 actions Call gov vet 1st, 2nd 3rd action Not in top 3 actions Watch and wait Not in top 3 actions 1st, 2nd 3rd action Do nothing Not in top 3 actions 1st, 2nd, 3rd action Call hotline 1st, 2nd, 3rd action Not in top 3 actions In a single event, what number of animals showing unusual signs/dead would you be concerned about Number showing unusual signs <10 10–50, more than 50 Number animals dead <5, 5–10 11–50, more than 50 How often have you Reported unusual signs Always, most of the time Occasionally, rarely, never (C ti d) How often have you (Continued) Frontiers in Veterinary Science \| www.frontiersin.org June 2021 \| Volume 8 \| Article 668679 Manyweathers et al. BN Model of Producer Vulnerability TABLE 1 \| Continued Questions Response-capacity-related questions Classiﬁcation of responses High capacity Low capacity Do you use Private vets Yes No Govt vets Yes No Do you trust Private vets Completely, very, moderately A little, not at all Govt vets Completely, very, moderately A little, not at all BN Model of Producer Vulnerability Manyweathers et al. Exposure-Related Practices Among participating producers, there was good implementation of biosecurity practices in relation to incoming animals, with producers reporting that they always inspected new stock for disease (84.1%), used animal movement documentation (75.4%), inspected stock before purchase (71.3%), and isolated new animals (64.9%). However, implementation of other biosecurity measures, mainly in relation to feral animal control and visitors, was limited. Approximately a third of respondents reported regularly restricting access for visitors (32.3%) and having speciﬁc control plans for feral animals (29.6%). In addition, 53.6% of producers did not require visitors to follow biosecurity practices when visiting their property. Bayesian Network Analysis Responses to the observed variables were qualitatively categorized based on the potential contribution of the practice to the risk of exposure or response capacity of the producer and their enterprise in relation to FMD. The qualitative descriptors were determined based on biosecurity and EAD management literature and historical epidemiological evidence from previous FMD outbreaks (2, 17, 23). Based on the sensitivity analysis results, some variables in the initial model were removed from the ﬁnal model due to their lack of inﬂuence (22, 29). RESULTS Demographics and Husbandry Practice Overall, postal and online responses were obtained from 497 sheep producers from ﬁve Australian States and Territories. A total of 448 responses were complete and included in the analysis. Table 2 provides a summary of the demographic and husbandry characteristics for participating producers. Most producers were over 50 years of age and third-generation farmers with more than 20 years of experience in sheep farming. Most properties were located in NSW and Victoria, with approximately half of the respondents running a mixed livestock enterprise. Response-Capacity-Related Practices Practices used to deﬁne producer Response Capacity were mainly those related to inspection of animals, recognizing unusual signs of disease and reporting of these unusual signs to competent authorities or appropriate stakeholders. The majority of respondents identiﬁed themselves as responsible for observing (94.6%), recognizing (79.3%), and reporting (91.0%) unusual signs of disease in their animals. In relation to routine inspection of animals, most producers June 2021 \| Volume 8 \| Article 668679 Frontiers in Veterinary Science \| www.frontiersin.org 6 BN Model of Producer Vulnerability Manyweathers et al. TABLE 2 \| Demographic and husbandry characteristics of sheep producers participating in a cross-sectional study in 2017–2018. Characteristic N (%) respondents State VIC 206 (46) NSW 197 (44) WA 21 (5) QLD 9 (2) SA 8 (2) NT – ACT – TAS – NA 7 Age 18–25 7 (2) 26–35 51 (11) 36–50 122 (27) 51–65 181 (40) 66–80 81 (18) Over 80 6 (1) Farming background First generation 108 (24.3) Second generation 63 (14.2) Third generation 274 (61.6) NA 3 Years farming <5 51 (11.5) 5–10 50 (11.3) 11–20 64 (14.4) More than 20 279 (62.8) NA 4 Production system Sheep and other livestock 191 (50.1) Sheep and cropping 119 (31.2) Sheep only 66 (17.3) Sheep and other 5 (1.3) NA 67 Property size (ha) Mean 2120.3 Min–max 1.5–125,000.0 Median 500.0 5–95% 7.0–7570.5 Number of ewes Mean 1,571 Min–max 3–13,500 Median 800 5–95% 9–7,000 checking their animals for disease prior to moving them oﬀ farm. Movement documentation when selling stock were used by 86.9% of respondents. In relation to recognizing and reporting unusual signs of disease, nearly half of respondents reported that they usually (most of the times or always) report unusual signs of disease (49.0%), with 59.4% reported knowing who to call if they found unusual signs of disease in their animals. Most producers reported keeping records of animal health (96.9%) and stock movements (73.2%). Producers were also asked about their ﬁrst three actions in the event of unusual signs of disease in their sheep, with calling a private veterinarian being the most commonly selected option (Table 3). However, a signiﬁcant proportion of producers (31.1%) chose “Watch and wait” as one of the top three options. Furthermore, the questionnaire asked producers about their knowledge on subsidies to ﬁnancially support the veterinary costs of reporting and diagnosis, with the majority (61.8%) being unaware of the existence of these subsidies. Sensitivity Analysis The sensitivity of the observed and hidden variables to aﬀect the determination of the vulnerability status was estimated through the Netica’s built-in sensitivity analysis procedure. The results are presented in Table 4 and can be interpreted as follows. The mutual information quantiﬁes the “amount of information” obtained about the target variable “vulnerability” through observing the other random variables. Using sensitivity analysis, property size, number of ewes, visitor biosecurity, primary income, years of farming, state, restricted access, and education were identiﬁed as those variables with the greatest inﬂuence on respondent vulnerability. Bayesian Network Analysis Sheep producers participating in the study were categorized into diﬀerent typologies of vulnerability to an FMD outbreak using the BN model and analysis. As a result of the analysis, six typologies were derived. The typologies are based on the likelihood of producers holding certain beliefs and adopting certain practices related to exposure and response capacity. The six typologies are summarized in Figure 3 and shown in full in the Supplementary Information. Response-Capacity-Related Practices Attitudes toward reporting were further investigated when producers were asked about their agreement on the eﬀectiveness of reporting in preventing the spread of animal diseases, with the majority agreeing or strongly agreeing with the statement (68.3%). DISCUSSION A BN model, populated by data from a cross-section survey of Australian sheep producers, has been used to enhance understanding of the vulnerability of producers to an FMD outbreak. This study found that sheep producers could be categorized into six risk-based typologies, based on two response capacity variables (high, low) and three exposure variables (low, moderate, high). This has resulted in a deeper understanding of A BN model, populated by data from a cross-section survey of Australian sheep producers, has been used to enhance understanding of the vulnerability of producers to an FMD outbreak. This study found that sheep producers could be categorized into six risk-based typologies, based on two response capacity variables (high, low) and three exposure variables (low, moderate, high). This has resulted in a deeper understanding of reported visually inspecting their animals daily or weekly (90.0%) and almost half of respondents also reported a daily or weekly physical inspection of the animals (44.1%). Approximately a third of producers (27.7%) reported checking unwell animals every day. In addition, most producers (75.4%) reported June 2021 \| Volume 8 \| Article 668679 Frontiers in Veterinary Science \| www.frontiersin.org 7 BN Model of Producer Vulnerability Manyweathers et al. TABLE 3 \| Ranking of actions in response to seeing unusual signs of disease in your sheep. Response n (%) First action Second action Third action Not top 3 Watch and wait 63 (15.3) 33 (8.0) 32 (7.8) 283 (68.8) Do nothing – 2 (0.5) 10 (2.4) 398 (97.0) Call private vet 106 (25.9) 83 (20.2) 83 (20.2) 138 (33.7) Call gov vet 61 (14.8) 64 (15.6) 57 (13.9) 229 (55.6) Call disease hotline 9 (2.2) 16 (3.9) 29 (7.1) 356 (86.7) These categories were selected from 11 response options, based on their impact on response capacity to a suspect FMD outbreak. TABLE 3 \| Ranking of actions in response to seeing unusual signs of disease in your sheep. Frontiers in Veterinary Science \| www.frontiersin.org role that private veterinarians might play in an early response to unusual signs of animal disease, two-thirds of respondents reported that they would contact their private veterinarian (66.3%), with just less than half reporting that they would contact their government veterinarian (44.4%). When examining the role of trust in the predicted vulnerability proﬁles, the likelihood of sheep producers having high levels of trust in both private and government veterinarians decreases slightly as vulnerability increases. This is in contrast to the reported early response of Australian beef producers to unusual signs of disease (22). The majority of those beef producers (83.6%) reported that calling their private veterinarian would be one of their top three actions, with 43.8% contacting their government veterinarian. However, the likelihood of producers having high levels of trust both private and government veterinarians increased as vulnerability increased. These variations again reﬂect the need for a wide systems-based approach that can examine the actions and beliefs from multiple stakeholders within the industries and government agencies (14). The importance of including social and psychological research ﬁndings into biosecurity projects to examine institutional and personal barriers and drivers that impact Australia’s vulnerability to an EAD should not be underestimated. While trust in veterinary services is recognized as important for adoption of biosecurity messages (16, 38), these results, including the low sensitivity of the model to producer trust in veterinarians (Table 4), need clariﬁcation. Further research is required for a deeper understanding of the role that trust plays in the producer–veterinarian relationship. However, the low sensitivity to trust found by the BN model signals the need to prioritize relationship building, possibly over regulation, and to consider strengthening relationships when allocating future surveillance resources, including location and selection of government veterinarians (14, 16, 39, 40) and training of private veterinarians (41, 42). oﬃcers and government veterinarians. The latter cohort with predicted high vulnerability are likely to have less experience and not come from a farming background. Their requirements for biosecurity information, animal health management skills, and partnerships with regulatory bodies will be diﬀerent. Therefore, access to more speciﬁc information about producers based on their vulnerability proﬁle may encourage more tailored communication and extension activities and will also inform more focused research in the future into adoption of and beliefs around biosecurity practices. The need for such a perspective is highlighted by considering the variation across sheep producers with predicted highest vulnerability to a FMD outbreak. According to the predictions of the BN model, these producers are moderate to large mixed cropping enterprises based in NSW (low response capacity, moderate exposure) and small, mixed livestock producers in Victoria (low response capacity, high exposure). The former cohort would most likely have animals of less individual worth and are older, more experienced farmers, with longer exposure to changes in agricultural governance over time (16). This may contribute to a high sense of self-eﬃcacy and to a disconnection with agents of regulation, such as biosecurity the sheep industry in Australia and current biosecurity practices and exposure risks. Overall, the risk vulnerability characteristics identiﬁed in the present study and in other similar studies strongly suggest that a one-size-ﬁts-all approach to extension and implementation of surveillance activities across and within livestock industries may not be appropriate. Rather, more research into context- based drivers of, and barriers to, uptake of protective surveillance behavior at individual producer or risk-based cohort level is likely required. A wider perspective on institutional constraints to adoption of such behaviors is also required, reﬂected in the levels of trust in government veterinary agents. June 2021 \| Volume 8 \| Article 668679 8 BN Model of Producer Vulnerability Manyweathers et al. TABLE 4 \| Bayesian network sensitivity analysis. Node Mutual information Percent Vulnerability 2.51039 100 Exposure level 1.51051 60.2 Response capacity 0.99988 39.8 Inspection 0.99984 39.8 Number of ewes 0.84204 33.5 Property size ha 0.81601 32.5 Recognizing 0.70731 28.2 Attitude** 0.50905 20.3 State 0.41989 16.7 Years farming 0.41085 16.4 Primary income 0.35666 14.2 Restrict access 0.30847 12.3 Age 0.29445 11.7 Visitor biosecurity 0.27619 11.0 Mutual information (i.e., “entropy reduction”)—a measure of the dependence between two random variables, the changes in uncertainty of X due to knowing Y (36). BN hidden variables. **Vulnerability was 100% explained by itself and the “Exposure Level” has the highest inﬂuence on deﬁning vulnerability with 60.2% mutual information. Note that the second highest inﬂuential variable was “Response capacity” (with 39.8% mutual information) and the two mutual information adding up to 100% (60.2 + 39.8 = 100) because the vulnerability status was deterministically deﬁned by two sublevel hidden variables: response capacity and exposure level, as detailed in Figure 1. Frontiers in Veterinary Science \| www.frontiersin.org BN Model of Producer Vulnerability embedded in the existing documentation as a prompt or a further breakdown of the desirable practices in order to remove barriers to uptake. embedded in the existing documentation as a prompt or a further breakdown of the desirable practices in order to remove barriers to uptake. While these results are useful in providing insights into vulnerability characteristics of sheep producers, the limited representativeness of the sample in reﬂecting the total population of Australian sheep producers is a constraint of this study. For example, Western Australian sheep producers were underrepresented in the data collected, as were South Australian sheep producers. The south western coast of Western Australia was identiﬁed by East et al. (5) as an area of risk for introduction, establishment, and spread of FMD. This lack of representativeness can be a common limitation of self- report data. More targeted and tightly enforced data collection methods with subsequent analysis using the BN model could facilitate wider consideration of the Australian sheep industry’s vulnerability to an incursion of FMD, but this approach is also not without its limitations. However, the beneﬁts of using the BN approach means that the exploratory ﬁndings from this model are still useful to inform potential policy considerations and future directions of research, despite not being intended to be generalizable. New data can also be added at any stage and the model updated. The sensitivity analysis of the BN model indicates that the input factors that most strongly inﬂuence sheep producer vulnerability are the exposure variables of restricting visitor access and enforcing visitor biosecurity practices. This ﬁnding also highlights the importance of deeper examination into the barriers to adoption of these practices and consideration of how these practices are communicated about (22, 45, 46). Results from the BN analysis suggests that exposure variables have more inﬂuence over a producer’s vulnerability than response capacity. Future research, including evaluation of the models’ predictive capacity using external data from independent on- farm vulnerability assessments will explore the robustness of the assumptions made in the development of the vulnerability framework. This work will also explore the usefulness of the concepts of exposure and response capacity to inform strengthening of Australia’s preparedness for an EAD outbreak. g g p p Vulnerability of the Australian sheep industry can be analyzed across geographic regions using the BN model to reﬂect on existing jurisdictional surveillance systems. Previous research found that the eastern and southern regions of Australia have a higher likelihood of entry, establishment, and spread of FMD (5). The BN analysis found that sheep producers with the highest vulnerability to an FMD outbreak are likely to be located in NSW (88.6% low response capacity, moderate exposure) and Victoria (88.4% low response capacity, high exposure). The overlap between these ﬁndings and that of East et al. (5) should inform development and evaluation of future risk-based surveillance strategies. East et al. (5) concluded that there is limited opportunity for improving current surveillance strategies based on geographic risk-based approaches because current surveillance activities are already focused in the areas of greatest risk. Our study strengthens these ﬁndings and allows for a deeper examination of actual on-farm practices. This approach can inform how existing surveillance strategies may be enhanced by focusing on the activities themselves. The model also predicts the likelihood of sheep producers in the study always/most of the time enforcing visitor biosecurity practices, showing increases from Victorian producers (12.3%), to NSW producers (17.3%), and Queensland producers (24.8%). The same gradient is observed when considering restricting visitor access: Victoria (27.3%), New South Wales (35.9%), and Queensland (44.6%). This comparison may provide an opportunity to examine the eﬃcacy of any existing extension activities that focuses on these practices and facilitate interjurisdictional sharing and consultation around communication strategies and extension approaches. Future work may also include examination of vulnerability of diﬀerent enterprises on the same farm, when producers farm more than one species. Another factor to consider is the capacity of the model to capture the impact on vulnerability, of how diﬀerent species of livestock react to FMD, with sheep clinical signs being less apparent that cattle and pigs (18) and therefore more likely to go unnoticed for longer. A further point to consider when interpreting these results is that the link to the questionnaire was distributed through emails, newsletters, social media, etc. Thus, there may be sampling bias in utilizing a convenience sample (47), and response rate cannot be determined. The limitations of a BN methodological approach to examine Australian sheep producer vulnerability are deﬁned primarily by the number and nature of the variables. The selection of variables is not exhaustive, and their inclusion needs to be considered in light of historical and current epidemiological data. The BN model approach cannot only be used to understand vulnerability but also to reﬂect on the distribution and utilization of current surveillance resources. When considering the disease hotline as part of the early response system available to livestock producers, sheep producers who were part of this study were very unlikely to use the disease hotline if they saw anything unusual in their animals, regardless of location or vulnerability level. This reported low uptake is also replicated in the study of beef producer vulnerability (22) and goat producers (29) and does suggest that adoption of this resource as a reporting and support tool might beneﬁt from further examination of barriers to adoption and subsequent strengthening of its capability and usefulness to producers. The BN model predicts that vulnerability of sheep producers increases as property size and ewe numbers decrease. This is supported by past research around smallholders and smaller producers. Hernandez-Jover et al. (37) found that smallholders with sheep were associated with a decreased engagement around surveillance activities such as frequency of animal inspection and use of veterinary services. This ﬁnding is in contrast to a BN model analysis looking at the vulnerability of Australian beef producers to an FMD outbreak (22), where the model indicated that larger beef producers are more likely to be categorized as highly vulnerable. The ﬁndings from the sheep produce study conﬁrm the need for greater investment in relationships and network building with smallholders, to encourage interactions between private/government veterinarians and landholders with small numbers of sheep. This is particularly signiﬁcant given the mild and transient nature of FMD signs in sheep. Recent research around adoption of biosecurity behaviors by livestock producers has highlighted the importance of focusing on speciﬁc behaviors when considering behavior change and adoption of new practices (43, 44). In this study, nearly three-fourths of respondents reported using the appropriate movement documentation when buying or selling stock. This suggests that there is an opportunity to use animal movement documentation as a gateway to behavior adoption of other desirable biosecurity practices. This might include a checklist When considering the top three actions that producers would undertake in response to unusual signs of disease, the majority of sheep producers reported that they would not watch and wait (68.8%) or do nothing (97.0%). When reﬂecting on the Frontiers in Veterinary Science \| www.frontiersin.org June 2021 \| Volume 8 \| Article 668679 9 Manyweathers et al. The advantages of the BN approach is its capacity to incorporate new data and the resultant ongoing model validation process. Continuing research in the use of BN models and increasing software capacity ensures that the limitations around variables is becoming less signiﬁcant. Evaluation of the model is being undertaken with input of new producer data to test the model’s predictions against on- farm assessments. This process will also progress consideration of the usefulness of the concept of vulnerability as a tool to strengthen Australian sheep producers’ preparedness for a possible FMD outbreak. Frontiers in Veterinary Science \| www.frontiersin.org FUNDING This project was supported by Meat and Livestock Australia, through funding from the Australian Government Department of Agriculture, Water and the Environment as part of its Rural R&D for Proﬁt program and by producer levies from Australian FMD-susceptible livestock (cattle, sheep, goats, and pigs) industries and Charles Sturt University (CSU), leveraging signiﬁcant in-kind support from the research partners. The research partners for this project are the Commonwealth Science and Industrial Research Organisation (CSIRO), CSU through the Graham Centre for Agricultural Innovation, the Bureau of Meteorology (BOM) and the Australian Department of Agriculture, Water and the Environment, supported by Animal Health Australia (AHA). The project commenced in July 2016 and concluded in December 2020. The BN model approach has aﬀorded a nuanced, holistic perspective through which to consider sheep producer vulnerability to a FMD outbreak and the development of a tool with the potential to support risk-based allocation of resources for animal disease surveillance in Australia. 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/s. SUPPLEMENTARY MATERIAL The studies involving human participants were reviewed and approved by Charles Sturt University Human Research Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2021.668679/full#supplementary-material CONCLUSION The need to conﬁne response capacity to two categories also needs reﬂection. This may indicate that the concept of response capacity needs to be clariﬁed to avoid missing more nuanced data. It may also be that the questions used to collect the response capacity data may need revision. As part of the evaluation process, these issues will be addressed. The present study used a BN model to interrogate questionnaire data that reﬂects producer practices and beliefs around surveillance and biosecurity and develop vulnerability typologies of Australian sheep producers. Examination of June 2021 \| Volume 8 \| Article 668679 Frontiers in Veterinary Science \| www.frontiersin.org 10 BN Model of Producer Vulnerability Manyweathers et al. AUTHOR CONTRIBUTIONS the typologies and the practices and beliefs of producers increases understanding around how to enhance the capacity of biosecurity and surveillance resources and identify opportunities for improving Australia’s preparedness for any future EAD incursion. JM, YM, LH, BL, HK, AM, RW, and MH-J made signiﬁcant contributions to the development of the questionnaire. JM, LH, and MH-J were responsible for the distribution of the questionnaire and the descriptive data analysis. GX developed the BN model, with contributions from JM, LH, and MH-J to the framework. BN analysis was undertaken by JM. JM was responsible for the manuscript with signiﬁcant contributions from YM, LH, BL, HK, AM, GX, RW, and MH-J. All authors contributed to the article and approved the submitted version. The results from this study highlight that more work is needed to understand drivers of and barriers to sheep producer uptake of biosecurity messages, so that risk management and communication strategies are appropriate to the enterprise and delivered in a way that facilitates adoption. This may include revising current biosecurity protocols for the Australian sheep industry to ensure that barriers to adoption are addressed and embedding biosecurity messages in existing tools such as animal movement documentation that appear to be more readily adopted. REFERENCES 8. Stärk DKC, Gertraud R, Jorge H, Lea K, Klemens F, Morris RS, et al. Concepts for risk-based surveillance in the ﬁeld of veterinary medicine and veterinary public health: Review of current approaches. BMC Health Serv Res. (2006) 6:20–20. doi: 10.1186/1472-6963-6-20 1. Department of Agriculture Water Resources. Farm Survey Data. (2019). ABARES. 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Towards Piloting Producer-Led Partnerships for Surveillance: Learning June 2021 \| Volume 8 \| Article 668679 Frontiers in Veterinary Science \| www.frontiersin.org 11 Manyweathers et al. BN Model of Producer Vulnerability 33. Pearl J. Probabilistic Reasoning in Intelligent Systems. San Mateo, CA: Morgan Kaufmann (1988). From the Current State of Animal Health Surveillance and Partnership Inititaives. Canberra, ACT: CSIRO (2017). 15. Taylor M, Dhand NK, Lee A. Farm Biosecurity Attitudes and Practices: Factors Inﬂuencing the Sheep Industry. Wagga Wagga: NSW: Meat and Livestock Australia (2011). 34. Norsys Software Corp. Netica [version 6.5]. (2018). Available online at: https:// www.norsys.com/ 35. Darwiche A. Modeling and Reasoning With Bayesian Networks. Cambridge: Cambridge University Press (2009). doi: 10.1017/CBO9780511811357 16. Palmer S, Fozdar F, Sully M. The eﬀect of trust on west australian farmers’ responses to infectious livestock diseases. Soc Rural. 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East IJ, Martin PAJ, LangstaﬀI, Iglesias RM, Sergeant ESG, Garner MG. Assessing the delay to detection and the size of the outbreak at the time of detection of incursions of foot and mouth disease in Australia. Prev Vet Med. (2016) 123:1–11. doi: 10.1016/j.prevetmed.2015.12.005 39. Cairns G, de Andrade M, MacDonald L. Reputation, relationships, risk communication, and the role of trust in the prevention and control of communicable disease: a review. J Health Commun. (2013) 18:1550–65. doi: 10.1080/10810730.2013.840696 20. Garner MG, Bombarderi N, Cozens M, Conway ML, Wright T, Paskin R, et al. REFERENCES Bard AM, David M, Emma R, Anne H, Helen Rebecca W, Kristen Reyher K. To change or not to change? Veterinarian and farmer perceptions of relational factors inﬂuencing the enactment of veterinary advice on dairy farms in the United Kingdom. J Dairy Sci. (2019) 102:10379–94. doi: 10.3168/jds.2019-16364 26. Schembri N, Hernandez-Jover M, Toribio JALML, Holyoake PK. On- farm characteristics and biosecurity protocols for small-scale swine producers in eastern Australia. Prev Vet Med. (2015) 118:104–16. doi: 10.1016/j.prevetmed.2014.11.008 27. Manyweathers J, Field H, Jordan D, Longnecker N, Agho K, Smith C, et al. Risk Mitigation of Emerging Zoonoses: Hendra Virus and Non-Vaccinating Horse Owners. Transb Emerg Dis. (2017) 64:1898–1911. doi: 10.1111/tbed. 12588 46. Svensson C, Lind N, Reyher KK, Bard AM, Emanuelson U. Trust, feasibility, and priorities inﬂuence Swedish dairy farmers’ adherence and nonadherence to veterinary advice. J Dairy Sci. (2019) 102:10360–68. doi: 10.3168/jds.2019-16470 28. KjærulﬀU, Madsen AL. Bayesian networks and inﬂuence diagrams: a guide to construction and analysis. J Am Stat Assoc. (2008) 12:1273. 47. Dillman DA. Internet, Phone, Mail, and Mixed-Mode Surveys: The Tailored Design Method. Hoboken, NJ: Wiley (2014). 29. Manyweathers J, Maru Y, Hayes L, Loechel B, Kruger H, Mankad A. et al. The goat industry in Australia: using Bayesian network analysis to understand vulnerability to a foot and mouth disease outbreak. Prev Vet Med. (2021) 187:105236. doi: 10.1016/j.prevetmed.2020.105236 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. 30. Nelson R, Kokic P, Crimp S, Martin P, Meinke H, Howden SM, et al. The vulnerability of Australian rural communities to climate variability and change: Part II—Integrating impacts with adaptive capacity. Environ Sci Policy. (2010) 13:18–27. doi: 10.1016/j.envsci.2009.09.007 Copyright © 2021 Manyweathers, Maru, Hayes, Loechel, Kruger, Mankad, Xie, Woodgate and Hernandez-Jover. 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. REFERENCES Copyright © 2021 Manyweathers, Maru, Hayes, Loechel, Kruger, Mankad, Xie, Woodgate and Hernandez-Jover. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 31. Korb KB, Nicholason AE. Bayesian Artiﬁcal Intelligence. New York, NY: CRC Press, Taylor and Francis group (2011). 32. Australian Government. A Beginners Guide to Bayesian Network Modelling for Integrated Catchment Management. Canberra, ACT: Landscape Logic (2009). June 2021 \| Volume 8 \| Article 668679 Frontiers in Veterinary Science \| www.frontiersin.org 12
https://openalex.org/W4328124527	https://www.frontiersin.org/articles/10.3389/fenrg.2023.1130459/pdf	English	null	Experimental investigation on microlayer behavior and bubble growth based on laser interferometric method	Frontiers in energy research	2,023	cc-by	9,879	KEYWORDS laser interferometry, microlayer dynamics, pool boiling, visualization, pressure effect TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fenrg.2023.1130459 TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fenrg.2023.1130459 TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fenrg.2023.1130459 OPEN ACCESS OPEN ACCESS EDITED BY Wei Ding, Helmholtz Association of German Research Centres (HZ), Germany REVIEWED BY Wan Sun, Chongqing University, China Xuehui Wang, Imperial College London, United Kingdom CORRESPONDENCE Jinbiao Xiong, xiongjinbiao@sjtu.edu.cn SPECIALTY SECTION This article was submitted to Nuclear Energy, a section of the journal Frontiers in Energy Research RECEIVED 23 December 2022 ACCEPTED 20 February 2023 PUBLISHED 16 March 2023 CITATION Wang J, Wang H and Xiong J (2023), Experimental investigation on microlayer EDITED BY Wei Ding, Helmholtz Association of German Research Centres (HZ), Germany Ji Wang, Hongbin Wang and Jinbiao Xiong School of Nuclear Science and Engineering, Shanghai Jiao Tong University, Shanghai, China High-speed laser interferometry is synchronized with a high-speed camera to visualize the dynamic microlayer behavior during bubble growth in a pool boiling under pressures from 0.1 to 0.3 MPa. An Indium–Tin-Oxide (ITO) ﬁlm coated on sapphire is employed as the heating unit to provide the nominal surface heat ﬂuxes in the range from 90 to 150 kW/m2. Based on the instantaneous microlayer thickness and photographed bubble images, microlayer formation and depletion and their relationship with bubble growth are analyzed. Appreciable effects of pressure on microlayer dynamics and bubble growth have been observed. At higher pressure, the microlayer existence time decreases and consequently, the contribution of the microlayer evaporation becomes less important. At elevated pressure, the effects of liquid subcooling and surface heat ﬂux on bubble growth become more pronounced. The dimensionless instantaneous maximum microlayer thickness, δ max/ ]t √ , shows exponential dependence on the ratio rd/rb,1 which increases linearly with time before the microlayer depletion. A correlation is proposed to predict the instantaneous maximum microlayer thickness synthesizing the two relations. The local heat ﬂux will be overestimated and the wall temperature proﬁle is contrary to the experimental observation when the ﬂow inside the microlayer is negligible. During the bubble growth period, only part of the microlayer is evaporated and the internal ﬂow inside cannot be neglected. Wang J, Wang H and Xiong J (2023), Experimental investigation on microlayer behavior and bubble growth based on laser interferometric method. Front. Energy Res. 11:1130459. doi: 10.3389/fenrg.2023.1130459 COPYRIGHT © 2023 Wang, Wang and Xiong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). OPEN ACCESS 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. frontiersin.org COPYRIGHT © 2023 Wang, Wang and Xiong. 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. 1 Introduction More recently, Narayan and Srivastava (2021) synchronized laser interferometry and rainbow schlieren deﬂectometry to measure the microlayer thickness and thermal ﬁeld around a nucleated bubble in a saturated water pool. They concluded that the contribution of microlayer evaporation to bubble volume is less than 15%. Demiray and Kim (2004) investigated the FC-72 nucleating bubble growth over a constant temperature microheater array at two different subcoolings and concluded that the microlayer and contact line heat transfer were not signiﬁcant. Kenning et al. (2001); Kenning and Bustnes (2007) employed liquid crystal thermography to measure the surface temperature and found that the microlayer beneath sliding bubbles can be as thick as 40–70 μm on the inclined surface in saturated water. They found that heat transfer through a microlayer contributed less than 35% of the heat required for the initial rapid growth of a bubble nucleated on the plate. Myers et al. (2005) investigated the transient temperature variation on a 10 × 10 array of constant heat ﬂux heaters with nucleate boiling of FC-72 and stated that the microlayer evaporation contributes to no more than 23% of the total heat transferred from the surface. Yabuki and Nakabeppu (2014) employed the micro-electro-mechanical system (MEMS) sensor to measure the instantaneous wall temperature beneath a growing water bubble and found that about 50% of bubble growth resulted from the microlayer evaporation in the saturated pool boiling. Gerardi et al. (2010) employed a high-speed infrared camera to visualize the temperature variation induced by nucleating water bubbles on an Indium–Tin-Oxide (ITO) heater and analyzed the inﬂuence of microlayer on bubble growth. In the above-mentioned experiment, the microlayer beneath the nucleated bubble under atmospheric pressure or even lower pressure has been investigated. The pressure effects on microlayer dynamics have not been well investigated (2020, 2021) (Kossolapov et al., 2020; Kossolapov et al., 2021). During the actual reactor operation, the pressure reaches 15.5 MPa and it is difﬁcult to carry out visual experiments at such high pressure. The method of the dimensionless parameter is used to reduce the high-pressure working condition to low pressure for the experiment. The similarity criterion is the dimensionless number, such as Re, Bo. It can not only ensure the accuracy of the results but also reduce the difﬁculty of the experiment. The bubble size in wall boiling and the microlayer contribution to the bubble growth are both greatly affected by the physical properties, especially the pressure. 1 Introduction During nucleate boiling, a thin liquid layer, i.e., the microlayer, is generated beneath the bubble during the early stage of growth, as shown in Figure 1. Generation and extinction of the microlayer has been investigated as an important mechanism contributing to nucleated bubble growth and boiling heat transfer (Zhao et al., 2002; Colombo and Fairweather, 2015). Early experimental investigation on the microlayer was indirect and mainly based on the measurement of instantaneous surface temperature. Based on the observation of the occasional drop in boiling surface temperature, Moore and Mesler (1961) proposed the hypothesis of the microlayer. Based on the deduction of the measured instantaneous wall temperature, Cooper and Lloyd (1969) obtained the microlayer thickness (6–30 μm) in toluene and the contribution of microlayer evaporation to bubble growth. They concluded that the inﬂuence of the microlayer evaporation was overwhelming in the saturated pool boiling and became less important in the case of high subcooling. Cooper and Lloyd (1969) Frontiers in Energy Research 01 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 studied the microlayer formation and evaporation for nucleate boiling in water and ethanol under atmospheric pressure. They found that the microlayer thickness in ethanol is 1.6 times of that for water. FIGURE 1 Structure of the microlayer. Gao et al. (2013) employed a He-Ne laser to generate interference fringe for the microlayer beneath a growing ethanol bubble on an ITO heater under atmospheric pressure and obtained a dynamic change of microlayer volume and micro-contact angle. Chen et al. (2017) studied the microlayer beneath a water bubble nucleated on a glass heated with a nitrogen jet from the opposite side. They found a crest-like structure at the edge of the microlayer in the late stage. Based on the same methodology, Utaka et al. (2018) found that the microlayer evaporation contributed to the total evaporation, approximately 39% for ethanol and 14%–44% for water. also derived the ﬁrst correlation of the initial microlayer thickness based on the theoretical analysis and measured microlayer thickness. Jung and Kim (2018); Jung and Kim (2019) employed laser interferometry for microlayer thickness measurement and a high- speed infrared camera for surface temperature measurement. They found that the heat ﬂux through the microlayer can be reasonably derived based on heat conduction and that the initial microlayer thickness in the outer region decreases because of microlayer evaporation. 2 Experiment methodology Sharp (1964) pioneered the interferometry measurement of microlayer thickness and obtained the interference fringe of the microlayer beneath a nucleating bubble using monochromatic and white light. Later, Voufsinos and Judd (1975) studied the growth and evaporation of the microlayer under a bubble forming on a glass heater surface with laser interferometry and high-speed photography. They found that microlayer evaporation contributed to 25% of the total nucleate boiling heat transfer rate. Utilizing laser interferometry, Koffman and Plesset (1983) Frontiers in Energy Research 1 Introduction In order to reveal the growth mechanism of bubbles under different pressures, the laser interferometry and high- speed camera are synchronized to simultaneously visualize the microlayer dynamics and bubble growth behavior in a water pool boiling at different pressures in this paper. The effects of pressure, subcooling, and surface heat ﬂux on the microlayer formation, depletion, and bubble growth will be discussed. The laser extinction method and laser interferometry method have been developed to directly measure the microlayer thickness. Utaka et al. (2014) measured the local microlayer thickness beneath the nucleated bubble in water and ethanol utilizing the laser extinction method in which the microlayer thickness was obtained based on the analysis of attenuated laser signal. They stated that the contribution of microlayer evaporation to bubble growth (15%–70%) increases linearly with the bubble inception wall superheat. frontiersin.org 2.1 Experiment setup As shown in Figure 2, the experimental apparatus is a cubic stainless vessel with an inner dimension of 200 mm × 200 mm × 200 mm. Four vertical 450 W Joule heating rods are utilized to adjust the subcooling of the water pool. Eight thermocouples are located at four elevations to measure water temperature. A pressure gauge is 02 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 FIGURE 2 Schematic of the experimental system. FIGURE 3 Schematic diagram of the heating unit. FIGURE 2 Schematic of the experimental system FIGURE 2 Schematic of the experimental system. Schematic of the experimental system. FIGURE 3 Schematic diagram of the heating unit. installed on the top of the vessel to monitor the pressure in the vessel. The uncertainty of the thermocouple is ± 0.5°C. Illumination and photograph windows are fabricated on the opposite vertical walls of the vessel. The illumination light source is a LED panel. The bubble growth process is photographed using a Phantom V710 high-speed camera (HSC) with a frame rate of 7,500 Hz at the full resolution, i.e., 800 × 1280 px2. The bubble diameter or radius is determined based on the pixel analysis of high-speed images. Blurring leads to an uncertainty of 5 pixels while positioning the bubble surface in the bubble images, which is equivalent to the uncertainty of 0.1 mm in the bubble radius. two terminals of the heating element. The substrate is 30 mm in diameter and 1.1 mm in thickness. The transparent heating unit is installed on a Teﬂon platform which is mounted over the hole on the bottom of the stainless vessel. A high-accuracy DC power unit is employed to adjust the heating power of the ITO ﬁlm. The heating power supplied to the ITO heater is determined based on the current and voltage. The relative error of current and voltage measurement is ±0.1 A and ±0.01V, respectively. Assuming the uniform current density through the narrow band of ITO ﬁlm, the nominal heat ﬂux is deﬁned and utilized in this paper. Before the experiment, the deionized water is degassed via argon injection and pre-boiling. The subcooling of the liquid is measured by two K-type thermocouples, which are arranged closer to the heating surface. 2.1 Experiment setup In order to facilitate visualization of interference fringe from the bottom of the heating surface, the transparent indium tin oxide (ITO) ﬁlm, 650 nm in thickness, is deposited onto a cylindrical sapphire substrate, as shown in Figure 3. The deposited ITO ﬁlm is etched into the H shape to assure the high heat ﬂux appears in the center of the plate. Consequently, early bubble nucleation occurred over the narrow band of the ITO ﬁlm. The static contact angle of the ITO surface is about 101.5 under the working condition of the atmospheric pressure of 20°C, as shown in Figure 3C. A gold ﬁlm, 100 nm in thickness, is sputtered onto the ITO ﬁlm to serve as the Frontiers in Energy Research frontiersin.org 2.2 Laser interferometry The principle of laser interferometry for microlayer thickness measurement is shown in Figure 4. Reﬂection of incident laser occurs at the ITO top surface and the liquid microlayer surface. The two reﬂected laser beams interfere with each other. The phase 03 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 difference of the laser beams is determined by the microlayer thickness. Hence, variation of microlayer thickness in the order of laser wavelength can be deduced based on the interval of interference fringe. At the center of dark fringes, the two reﬂected laser beams are in opposite phases with the corresponding microlayer thickness δ0 λ 2n m −1 2 m 1, 2, 3 . . . (1) where λ is the laser wavelength in the air; n is the reﬂective index of the microlayer, n = 1.32 is utilized in this study; m is the order of the dark fringe from center to the periphery, m = 0 is deﬁned for the central dark spot which corresponds to the dry patch. At bright fringes, the two laser beams are in phase with the corresponding microlayer thickness δ0 λ 2n m m 1, 2, 3 . . . (2) where m is the order of the bright fringe from the center to the periphery, and m = 1 is deﬁned for the ﬁrst inner fringe. The CAVILUX pulsed diode laser with the wavelength λ = 640 nm is employed for illumination. As shown in Figure 2, the laser head is installed beneath the heating unit to illuminate from the bottom. In the experiment, the laser pulse duration is 100 ns at a FIGURE 4 Principle of ﬁlm thickness measurement with laser interferometry. FIGURE 5 Synchronization time sequence. FIGURE 6 An example of fringe image processing. FIGURE 4 Principle of ﬁlm thickness measurement with laser interferometry. FIGURE 4 Principle of ﬁlm thickness measurement with laser interferometry. FIGURE 4 Principle of ﬁlm thickness measurement with laser interferometry. FIGURE 5 Synchronization time sequence. FIGURE 6 An example of fringe image processing. FIGURE 5 Synchronization time sequence. difference of the laser beams is determined by the microlayer thickness. Hence, variation of microlayer thickness in the order of laser wavelength can be deduced based on the interval of interference fringe. frontiersin.org Frontiers in Energy Research 3.1 Synchronized visualization In order to enhance the accuracy of microlayer thickness measurement, the background noise is subtracted from the original bottom HSC images. The centers of dark and bright fringes are localized in the enhanced images based on the grey scale. With the position of dark and bright fringes, the distribution of microlayer thickness can be determined based on Eqs 1, 2. Figure 6A shows the fringes of one frame of the post-processed images. Correspondingly, the proﬁles of bright fringes indicating the microlayer thickness along AB →are illustrated in Figure 6bB. The measurement is carried out under eight test conditions, as shown in Table 1, which allows for the investigation of pressure, subcooling, and surface heat ﬂux effect. The test pressure ranges from 0.1 to 0.3 MPa, while the subcooling ranges from 2°C to 7°C. The microlayer depletion time, tml, i.e., the time period between microlayer inception and extinction, and the bubble growth time, tg, i.e., the time period between bubble nucleation and departure from the heating surface are also seen in Table 1. The increase in pressure signiﬁcantly reduces the microlayer depletion time and the bubble growth time, as well as the time ratio tml/tg. Small tml/tg in high- pressure cases implies that the evaporation of microlayer contributes to less portions of bubble growth. The effects of subcooling and surface heat ﬂux are minor compared to that of pressure. The relative error of the microlayer thickness results mainly from the error of laser wavelength and the error in fringe position determination which was a result of image processing. The wavelength error of the CAVILUX pulsed diode laser is ±10 nm. According to Eqs 1, 2, the relative error of microlayer thickness equals that of laser wavelength, i.e., ±1.56%. The relative error in positioning the dark and bright fringe center is in the order of ±1 pixel in the image of the bottom HSC, which is equivalent to ±3 μm. Shown in Figure 7 are the synchronized images of microlayer interference fringe and bubble side view obtained in case No. 1. As can be seen in Figure 7A, the inception of bubble nucleation is accompanied by the formation of the microlayer. At the same time, a dry spot or patch, i.e., the bright spot, appears in the center of the interference fringe. 2.2 Laser interferometry At the center of dark fringes, the two reﬂected laser beams are in opposite phases with the corresponding microlayer thickness δ0 λ 2n m −1 2 m 1, 2, 3 . . . (1) (1) where λ is the laser wavelength in the air; n is the reﬂective index of the microlayer, n = 1.32 is utilized in this study; m is the order of the dark fringe from center to the periphery, m = 0 is deﬁned for the central dark spot which corresponds to the dry patch. At bright fringes, the two laser beams are in phase with the corresponding microlayer thickness FIGURE 6 An example of fringe image processing. FIGURE 6 δ0 λ 2n m m 1, 2, 3 . . . (2) (2) The CAVILUX pulsed diode laser with the wavelength λ = 640 nm is employed for illumination. As shown in Figure 2, the laser head is installed beneath the heating unit to illuminate from the bottom. In the experiment, the laser pulse duration is 100 ns at a where m is the order of the bright fringe from the center to the periphery, and m = 1 is deﬁned for the ﬁrst inner fringe. frontiersin.org 04 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 TABLE 1 Parameter conﬁguration of the test matrix. g Case no. Pressure (MPa) Subcooling (°C) Nominal heat ﬂux (kW/m2) tml (ms) tg (ms) tml/tg rml, max (mm) 1 0.1 2 119.3 11.07 24 0.461 1.302 2 2 147.2 11.60 23.06 0.503 1.329 3 7 147.2 10.80 22.74 0.475 1.386 4 0.2 2 91.3 2.8 10.91 0.257 0.3 5 5 91.3 1.87 12.0 0.156 0.274 6 5 147.2 2.13 11.6 0.184 0.336 7 0.3 5 91.3 1.33 5.85 0.227 0.213 8 5 147.2 0.53 2.79 0.190 0.129 departure morphology. Its inﬂuence on the deformed bubble is approximately 8% by analyzing the shape of the center bubble. frequency of 10 kHz. A beam splitter is placed between the laser head and the heating unit. The bottom-view high-speed camera (Phantom V710) with a microscopic lens is installed horizontally. The magniﬁcation factor of the microscopic lens is 7, which allows for high-resolution measurement of interference fringe. A synchronizer is utilized to synchronize the laser and the two HSCs. The synchronization time sequence is shown in Figure 5. Frontiers in Energy Research 3.1 Synchronized visualization In the early stage of bubble growth, the bubble is semi-elliptical, and the interference fringes are concentric rings expanding rapidly in the radial direction, as shown in Figure 7B. As the fringes expand outwards, the spacing between neighboring fringes becomes non-uniform, i.e., the inner fringe spacing is dense, while the outer fringes distribute coarsely. Good concentricity of inner fringes indicates the effect of force balance at the triple-phase line. The outer fringes distort appreciably. Such distortion is a result of the asymmetricity of bubble growth which is weak and can hardly be observed from the side-view image. The dry spot grows relatively slowly. Finally, all the microlayer-covered surface becomes dry as the microlayer depletes. The dry spot remains at the maximum radius for a certain period during which the bubble starts to rise away from the surface. In the ﬁnal stage, the dry patch shrinks as the bubble departs from the surface. The defects on the edges of the etching pattern are natural nucleation points. Although the growth process of a single bubble is selected to be studied under the condition of low heat ﬂux, without bubble overlap there can also be some interaction between the center bubble and the edge bubble. When the center bubble grows, the edge can generate bubbles at the same time. The edge bubble size is small since it mostly grows at the edge of the ITO and the heat ﬂux is lower. Through the comparison of the images, it is found that in the initial stage of the growth of the central bubble, the edge bubble does not inﬂuence it due to the small volume of both of them. In the late stage of the growth of the central bubble, its volume is larger and it will interact with the small edge bubbles and the shape will be deformed. However, the interaction process often occurs in the center bubble departure period, and the microlayer has been evaporated totally. That is to say the small edge bubbles have no effect on the microlayer behavior but have some effect on the bubble Frontiers in Energy Research 05 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 ubble radius, dry spot radius, and contact vely compared for the test cases at p = facilitate the comparison, in the abscissa of Figure 8, the time scale is normalized by the microlayer depletion time, i.e., tml, which is given in Table 1. Frontiers in Energy Research 3.1 Synchronized visualization FIGURE 8 FIGURE 10 The proﬁle of ratio rd/rb,1 under different pressures. FIGURE 8 The evolution of dry spot radius, contact radius, and bubble radius. FIGURE 8 The evolution of dry spot radius, contact radius, and bubble radius. TABLE 2 Coefﬁcients in correlations of the dry spot radius and the microlayer thickness. FIGURE 9 The proﬁle of ratio rc/rb,1 under different pressures. Pressure (MPa) c1 c2 c3 c4 0.1 0.35 0.13 0.34 2.45 0.2 0.28 0.19 0.225 1.96 0.3 0.27 0.24 0.16 1.89 microlayer depletes. It indicates that the depletion of the microlayer is highly correlated with bubble growth. As shown in Figure 9, the ratio rc/rb,1 decreases linearly with t/tml. The proﬁle at the early stage is discrete, while in the second half, the values tend to be the same for different pressures. At the same time, the increase of the dry spot radius is also approximately linear, and the proﬁles are affected by the pressure, as shown in Figure 10, which can be correlated with rd rb,1 c1 t tml + c2 (3) (3) The coefﬁcients c1 and c2 show dependence on pressure and are seen in Table 2. The coefﬁcients c1 and c2 show dependence on pressure and are seen in Table 2. radius do not show signiﬁcant discrepancy for case No. 1 through 3, while a slight effect of bulk subcooling and surface heat ﬂux can be observed in Figure 8. In the early stage of bubble growth, drb,1/dt is large and the bubble grows rapidly. Large growth rate leads to a signiﬁcant bubble growth force which pushes the bubble towards the heating surface. Consequently, the initial bubble is approximately hemispherical, as shown in the snapshot at t = 1.33 ms in Figure 7. Another indication of the hemispherical bubble shape is the coincidence of the bubble radius and the contact radius when t/tml <0.1, as can be seen in Figure 8. As the bubble growth continues, the expansion of the bubble contact area soon falls behind the bubble growth. Consequently, the bubble center starts to rise away vertically. At t/tml ≈0.5, the contact radius reaches its maximum and stays constant before decreasing when the bubble starts to depart. The bubble radius reaches its maximum at t/tml ≈1.0, i.e., when the radius do not show signiﬁcant discrepancy for case No. Frontiers in Energy Research 3.1 Synchronized visualization We can see a signiﬁcant effect of pressure on tml. The contact and dry spot eft) and bubble side views (right) in case No. 1. eft) and bubble side views (right) in case No. 1. FIGURE 7 Interference fringes (left) and bubble side views (right) in case No. 1. FIGURE 7 Interference fringes (left) and bubble side views (right) in case No. 1. of Figure 8, the time scale is normalized by the microlayer depletion time, i.e., tml, which is given in Table 1. We can see a signiﬁcant effect of pressure on tml. The contact and dry spot of Figure 8, the time scale is normalized by the microlayer depletion time, i.e., tml, which is given in Table 1. We can see a signiﬁcant effect of pressure on tml. The contact and dry spot In Figure 8, the bubble radius, dry spot radius, and contact radius are quantitatively compared for the test cases at p = 0.1 MPa. In order to facilitate the comparison, in the abscissa Frontiers in Energy Research 06 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 microlayer depletes. It indicates that the depletion of the microlayer is highly correlated with bubble growth. As shown in Figure 9, the ratio rc/rb,1 decreases linearly with t/tml. The proﬁle at the early stage is discrete, while in the second half, the values tend to be the same for different pressures. At the same time, the increase of the dry spot radius is also approximately linear, and the proﬁles are affected by the pressure, as shown in Figure 10, which can be correlated with rd rb,1 c1 t tml + c2 (3) The coefﬁcients c1 and c2 show dependence on pressure and are FIGURE 8 The evolution of dry spot radius, contact radius, and bubble radius. FIGURE 9 The proﬁle of ratio rc/rb,1 under different pressures. FIGURE 10 The proﬁle of ratio rd/rb,1 under different pressures. TABLE 2 Coefﬁcients in correlations of the dry spot radius and the microlayer thickness. Pressure (MPa) c1 c2 c3 c4 0.1 0.35 0.13 0.34 2.45 0.2 0.28 0.19 0.225 1.96 0.3 0.27 0.24 0.16 1.89 FIGURE 10 The proﬁle of ratio rd/rb,1 under different pressures. FIGURE 10 The proﬁle of ratio rd/rb,1 under different pressures. FIGURE 8 The evolution of dry spot radius, contact radius, and bubble radius. FIGURE 8 The evolution of dry spot radius, contact radius, and bubble radius. frontiersin.org 3.2 Microlayer dynamic behavior FIGURE 11 Microlayer thickness in the growth period in case No. 1 The instantaneous proﬁles of microlayer thickness obtained in case No.1 are shown in Figure 11. The maximum microlayer thickness, δ max ≈5.7μm, appears at t ≈1.2 ms. Until t = 2.8 ms, the maximum thickness is retained as the microlayer expands outwards. A similar phenomenon has also been reported by Chen et al. (2017). Since the dry spot expands slower than the microlayer, the slope of the microlayer reduces, especially near the outer edge. After the contact radius, rc, reaches its maximum, the merging of neighboring interference fringes is observed in the peripheral microlayer region. A similar fringe merging phenomenon has been reported by Chen et al. (2017) who proposed that fringe merging was a consequence of bulged microlayer surface. When the bulge shrinks, the interference fringes move toward the bulge peak, which results in fringe merging. Hence, the decrease in the microlayer thickness with the radius is expected at the peripheral zone, when the fringe merging is observed, e.g., t = 6.4 ms and 8 ms in Figure 11. FIGURE 11 Microlayer thickness in the growth period in case No. 1 FIGURE 11 Microlayer thickness in the growth period in case No. 1 The initial microlayer thickness, δ0(r), deﬁned as the initially measured microlayer thickness at radial position r has been employed to characterize the shape of the microlayer by Cooper and Lloyd (1969). The initial microlayer thickness obtained in the present experiment, as well as that measured by Chen et al. (2017) and Jung and Kim (2018) in water and that measured by Gao et al. (2013), Utaka et al. (2018), and Liu et al. (2019) in ethanol, are plotted in Figure 12, where the initial microlayer thickness is normalized with its maximum value and the radial position is normalized with the radial position where δ0, max ﬁrst occurs, i.e., rml, max. As can be seen in Figure 11, in case No.1 δ0, max 5.7μm, rml, max 1.3mm occurs at t = 1.2 ms. We can observe a fairly good similarity of the initial microlayer thickness proﬁle in the radial direction by different authors, when rml/rml, max < 1. 3.1 Synchronized visualization 1 through 3, while a slight effect of bulk subcooling and surface heat ﬂux can be observed in Figure 8. In the early stage of bubble growth, drb,1/dt is large and the bubble grows rapidly. Large growth rate leads to a signiﬁcant bubble growth force which pushes the bubble towards the heating surface. Consequently, the initial bubble is approximately hemispherical, as shown in the snapshot at t = 1.33 ms in Figure 7. Another indication of the hemispherical bubble shape is the coincidence of the bubble radius and the contact radius when t/tml <0.1, as can be seen in Figure 8. As the bubble growth continues, the expansion of the bubble contact area soon falls behind the bubble growth. Consequently, the bubble center starts to rise away vertically. At t/tml ≈0.5, the contact radius reaches its maximum and stays constant before decreasing when the bubble starts to depart. The bubble radius reaches its maximum at t/tml ≈1.0, i.e., when the Similar observations were reported by Duan et al. (2013) for saturated water pool boiling with surface heat ﬂux of 28.7 kW/m2, by Jung and Kim (2014) for water pool boiling (ΔTsub = 3°C) with surface heat ﬂux of 53 kW/m2, and by Liu et al. (2019) who investigated the ethanol boiling (ΔTsub = 5°C) over a surface with heat ﬂux of 50.4 kW/ m2. Jung and Kim (2014) showed a smaller and earlier occurrence of maximum contact radius than that of our experiments, which results from the appreciably lower surface heat ﬂux than in our experiment. Another interesting difference is that in the present, in Duan et al.’s (2013) and Jung and Kim’s (2014) experiments, the dry spot or contact radius is stable for a certain time before shrinking, while an immediate shrinking of the dry spot was observed by Liu et al. (2019). An acute dynamic contact angle of the heating surface was reported by Liu et al., 2019, which can be attributed to immediate surface rewetting and, consequently, quick dry spot shrinking. Frontiers in Energy Research 07 frontiersin.org frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 3.2 Microlayer dynamic behavior (2019) Ethanol 50.4 5 Laser interferometry — FIGURE 13 Comparison of experimental data with the models. FIGURE 14 Relation between maximum thickness and ratio rd/rb,1. TABLE 3 Previous experimental studies for the initial microlayer thickness. Author Fluid Pressure (MPa) Heat ﬂuxes (kW/m2) Subcooling (°C) Measurement method Correlations Koffman and Plesset (1983) Water 0.1 26.5–204 5.7–21.7 Laser interferometry δ0 0.00188r0.6 Yabuki and Nakabeppu (2014) Water 30–39 0 MEMS sensor δ0 0.00434r0.69 Utaka et al.(2014) Water 102–135 0 Laser extinction Ethanol: δ0 10.2 × 10−3rL Ethanol 50–103 Water: δ0 4.46 × 10−3rL Gao et al.(2013 Ethanol 32.4 6.1 Laser interferometry δ(Rb,t) υt √ 0.051 t td + 0.2 (td represents dry spot occurrence time) Jung and Kim (2018) Water 53–209 0–3 Laser interferometry δ0 0.53 × 2]t ( 8 a3+1)(1−n)+ 2 a3 (1 n−1)(n−2)+a20.66n+ 4σ a3ρc4 n2 t4n−3 Chen et al. (2017 Water 41–86 0 Laser interferometry — Liu et al. (2019) Ethanol 50.4 5 Laser interferometry — FIGURE 13 Comparison of experimental data with the models. FIGURE 14 Relation between maximum thickness and ratio rd/rb,1. FIGURE 13 Comparison of experimental data with the models. technologies and covered different ﬂuids, i.e., water and ethanol, heat ﬂuxes, and subcooling, but all of these were carried out at atmospheric pressure. Various initial microlayer thickness models were proposed. The comparison between the initial thickness distribution in the present study and these models is shown in Figure 13. In these models’ prediction results, the result of Koffman and Plesset (1983) are all smaller than those of others. For the present data, when r < 1.25 mm, the data are in good agreement with the predicted results of Yabuki and Nakabeppu (2014) and Utaka et al. (2014). However, when 1.25 < r < 2.0, the experimental values are lower than the predicted values. Besides, when r > 1.8 mm, the experimental values even decrease. This is due to the rapid growth of the bubble and the small amount of evaporation inside in the initial stage. As the bubble size increases, internal evaporation becomes stronger. Due to the depletion of the microlayer by evaporation, the initial thickness is lower than predicted by the linear model in the late period. Yabuki and Nakabeppu (2014) also suggested that the increase rate of the initial microlayer thickness is gradually slowed down, which is consistent with the observations of this experiment. 3.2 Microlayer dynamic behavior During the very early phase of bubble nucleation, the formation of the microlayer is as a result of the inertial growth of the bubble, and hence the microlayer shape is mainly affected by the ﬂuid property, e.g., the surface tension and the viscosity. The proﬁle of the initial microlayer thickness can be correlated as FIGURE 12 Normalized initial microlayer thickness in the pool boiling at atmospheric pressure. FIGURE 12 Normalized initial microlayer thickness in the pool boiling at atmospheric pressure. δ0 δ0, max −0.9 rml rml, max 2 + 1.9 rml rml, max , rml rml, max < 1 (4) (4) When rml/rml, max > 1, the above similarity is not valid anymore, which indicates that other factors, e.g., the surface wettability, the heat ﬂux, and the liquid subcooling, prevail. As shown in Figure 12A, we can see that at atmospheric pressure, the proﬁle of the normalized initial microlayer thickness is in a consistent form and our data correlated well with other experimental data collected from the studies shown in Table 3. However, the data of ethanol have little difference from water. The maximum radius is smaller and the initial thickness decreases more after reaching the maximum value. For water, the initial thickness distribution is consistent, despite the different working conditions of each experiment. FIGURE 12 Normalized initial microlayer thickness in the pool boiling at atmospheric pressure. Previous experimental studies for the initial microlayer thickness are summarized in Table 3. The studies used different 08 Frontiers in Energy Research frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 TABLE 3 Previous experimental studies for the initial microlayer thickness. Author Fluid Pressure (MPa) Heat ﬂuxes (kW/m2) Subcooling (°C) Measurement method Correlations Koffman and Plesset (1983) Water 0.1 26.5–204 5.7–21.7 Laser interferometry δ0 0.00188r0.6 Yabuki and Nakabeppu (2014) Water 30–39 0 MEMS sensor δ0 0.00434r0.69 Utaka et al.(2014) Water 102–135 0 Laser extinction Ethanol: δ0 10.2 × 10−3rL Ethanol 50–103 Water: δ0 4.46 × 10−3rL Gao et al.(2013 Ethanol 32.4 6.1 Laser interferometry δ(Rb,t) υt √ 0.051 t td + 0.2 (td represents dry spot occurrence time) Jung and Kim (2018) Water 53–209 0–3 Laser interferometry δ0 0.53 × 2]t ( 8 a3+1)(1−n)+ 2 a3 (1 n−1)(n−2)+a20.66n+ 4σ a3ρc4 n2 t4n−3 Chen et al. (2017 Water 41–86 0 Laser interferometry — Liu et al. Frontiers in Energy Research 3.2 Microlayer dynamic behavior However, the model of Yabuki and Nakabeppu (2014) still overestimates the initial microlayer thickness. δ max(t), the instantaneous maximum microlayer thickness is normalized by ]t √, which is usually employed to characterize the hydrodynamic formation of the microlayer (Cooper and Lloyd, 1969). In Figure 14, the ratio δ max/ ]t √ is plotted against the ratio of rd/rb,1 for the test cases under different pressures. The data set obtained under different pressure show good consistency and can be correlated with δ max νt √ 0.85 exp −7 · rd rb,1 (5) (5) Substituting Eq. 3 into Eq. 5, the maximum thickness can be correlated as δ max νt √ c3e−c4 t tml (6) (6) frontiersin.org Frontiers in Energy Research 09 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 The coefﬁcients c3 and c4 varies with pressure and are shown in Table 2. In order to verify the accuracy of the above correlation, we selected the experimental data sets of Gao et al. (2013); Jung and Kim (2014); Jung and Kim (2018), and Liu et al. (2019) for comparison, as shown in Figure 15. Since these experiments are all carried out at atmospheric pressure, the ﬁrst correlation is used to analyze the accuracy of the prediction. The working ﬂuid of Gao et al. (2013) and Liu et al. (2019) is ethanol, while water is used by Jung and Kim (2014); Jung and Kim (2018). The predicted results are in good agreement with Gao and Liu, but a relatively large error appears in Jung’s data prediction. However, the prediction error of most of the data falls within 35%. In order to intuitively study the inﬂuence of pressure on the the microlayer thickness is normalized with its maximum value, and the radial position is normalized with the maximum contact radius, e.g., rc,max. Due to a relatively short microlayer depletion time at the pressure 0.3 MPa, the ﬁrst appearance of the interference fringe is t/tml ≈0.1. Hence, the initial proﬁle of the microlayer is not shown for the case of 0.3 MPa. At different pressures, the normalized microlayer thickness distribution is consistent in general. The FIGURE 15 Comparison between the experimental data and the prediction with Eq. 6. FIGURE 16 Comparison of microlayer under different pressures. FIGURE 17 Axis ratio rb,1/rb,2 under different pressures. FIGURE 15 Comparison between the experimental data and the prediction with Eq. 6. frontiersin.org 3.2 Microlayer dynamic behavior FIGURE 19 Proﬁle of microlayer thickness depletion in cases No. 1 and 4 FIGURE 18 The relation between microlayer evaporation and bubble growth. microlayer is independent of the pressure. However, in the early stage, the distribution is linear, that is, the microlayer thickness is directly proportional to the bubble radius. microlayer thickness. The evaporated microlayer volume during the time interval between frame i and i+1, (dV)i, can be calculated with Frontiers in Energy Research 3.2 Microlayer dynamic behavior FIGURE 17 Axis ratio rb,1/rb,2 under different pressures. FIGURE 15 Comparison between the experimental data and the prediction with Eq. 6. FIGURE 15 Comparison between the experimental data and the prediction with Eq. 6. FIGURE 16 Comparison of microlayer under different pressures. FIGURE 16 Comparison of microlayer under different pressures. The coefﬁcients c3 and c4 varies with pressure and are shown in Table 2. In order to verify the accuracy of the above correlation, we selected the experimental data sets of Gao et al. (2013); Jung and Kim (2014); Jung and Kim (2018), and Liu et al. (2019) for comparison, as shown in Figure 15. Since these experiments are all carried out at atmospheric pressure, the ﬁrst correlation is used to analyze the accuracy of the prediction. The working ﬂuid of Gao et al. (2013) and Liu et al. (2019) is ethanol, while water is used by Jung and Kim (2014); Jung and Kim (2018). The predicted results are in good agreement with Gao and Liu, but a relatively large error appears in Jung’s data prediction. However, the prediction error of most of the data falls within 35%. the microlayer thickness is normalized with its maximum value, and the radial position is normalized with the maximum contact radius, e.g., rc,max. Due to a relatively short microlayer depletion time at the pressure 0.3 MPa, the ﬁrst appearance of the interference fringe is t/tml ≈0.1. Hence, the initial proﬁle of the microlayer is not shown for the case of 0.3 MPa. At different pressures, the normalized microlayer thickness distribution is consistent in general. The root of each group was located at the same position, which indicates that the dimensionless evaporation rate of the In order to intuitively study the inﬂuence of pressure on the microlayer thickness, the distribution of the dimensionless thickness under different pressure is compared and shown in Figure 16, where 10 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 microlayer is independent of the pressure. However, in the early stage the distribution is linear that is the microlayer thickness is FIGURE 18 The relation between microlayer evaporation and bubble growth. FIGURE 19 Proﬁle of microlayer thickness depletion in cases No. 1 and 4 FIGURE 18 The relation between microlayer evaporation and bubble growth. FIGURE 19 Proﬁle of microlayer thickness depletion in cases No. 1 and 4 FIGURE 18 The relation between microlayer evaporation and bubble growth. 3.3 Bubble growth FIGURE 20 FIGURE 20 Derived local heat ﬂux assuming that evaporation is the only mechanism for microlayer depletion. Derived local heat ﬂux assuming that evaporation is the only mechanism for microlayer depletion. In Figure 19, the instantaneous microlayer thickness at selected radial positions is shown for cases No. 1 and 4. It is found that at the same radial position, the microlayer depletes linearly. When moving outwards, the magnitude of dδ/dt decreases. Assuming the microlayer depletion only results from evaporation, the corresponding local heat ﬂux can be calculated with In Figure 18, the bubble volume calculated with Eq. 9 and the bubble volume corresponding to the microlayer evaporation estimated with Eq. 8 are compared for the test cases at the pressure of 0.1 and 0.2 MPa. Besides, the appreciable effect of pressure on the bubble size and the contribution ratio of microlayer evaporation to bubble growth is sensitive to pressure. At atmospheric pressure, the microlayer shows an overwhelming impact on bubble growth. As we can see, at the very beginning of bubble nucleation, the estimated bubble size with Eq. 8 is larger than the bubble size measured, i.e., Vb,ml > Vb,hsc. The overestimation is not due to the condensation at the bubble top which is not taken into consideration here. It should be noted that the bubble size is small in the early stage and the tip of the bubble is within the superheated liquid layer. Hence, Vb,ml > Vb,hsc implies that there can be other mechanisms, e.g., outward ﬂow in the microlayer contributing to microlayer depletion. As can be seen from Figure 18B, the evaporation of the microlayer becomes less important for bubble growth at elevated pressure. The low signiﬁcance of the microlayer at a higher pressure can be attributed to a smaller bubble growth rate, as will be discussed later. Another factor leading to such low signiﬁcance is the small tml/tg at a higher pressure, as shown in Table 1. q r ( ) ρlhf g dδ r ( ) dt (10) (10) where hfg is the latent heat of vaporization. In Figure 20, the derived local heat ﬂuxes in case No. 1 are plotted. The maximum derived heat ﬂux (3.91 MW/m2) locates near the center of the dry spot. Jung and Kim (2014) derived the surface heat ﬂux based on heat conduction analysis with the measured surface temperature in water pool boiling. 3.3 Bubble growth dV ( )i 2π rc,i rd,i δi r ( )rdr − rc,i rd,i+1 δi+1 r ( )rdr (7) 3/6/2023 In order to investigate the bubble shape during bubble growth, the ratio rb,1/rb,2 is plotted in Figure 17 for the test cases under different pressure from the nucleation inception to bubble departure. We can see that for all the test cases the ratio rb,1/rb,2 does not show appreciable variation during bubble growth. We should clarify that rb,2 is measured based on the upper part of the bubble. As the bubble approaches the departure state, the lower part of the bubble is of an inverted cone shape. Hence, the ratio rb,1/rb,2 indicates the upper-part shape of the bubble. At the atmospheric pressure, rb,1/rb,2 ≈1.2. At a higher pressure, i.e., 0.2 MPa and 0.3 MPa, the ratio approaches unity, i.e., the bubble is approximately spherical, i.e., rb,1/rb,2 ≈1. (7) where rd,i and rd,i+1 are the dry spot radius at frames i and i+1, rc,i is the contact radius at frame i, δi(r), and δi+1(r) are the microlayer thickness at radial position r in the frame i and i+1. Hence, the contributed bubble volume corresponding to microlayer evaporation can be calculated with Vb,ml tj ρl ρv j i0 dV ( )i (8) (8) In order to evaluate the instantaneous bubble volume, the coordinates of the bubble edge (xe, ye) is obtained based on pixel analysis. Here, we deﬁne the origin of the x-y plane at the center of the dry spot. Based on the assumption of axisymmetric shape, the volume can be calculated with The mechanism of microlayer depletion results from the evaporation and hydrodynamic ﬂow of the microlayer. If we assume the frozen state of the microlayer, evaporation is the sole mechanism for microlayer depletion; the local microlayer evaporation rate can be estimated based on the reduction of the Vb,hsc tj ye, max tj ( ) 0 πxe tj 2dye (9) (9) frontiersin.org 11 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 In Figure 19, the instantaneous microlayer thickness at FIGURE 20 Derived local heat ﬂux assuming that evaporation is the only mechanism for microlayer depletion. FIGURE 21 Evaluation of bubble growth models. FIGURE 21 Evaluation of bubble growth models. FIGURE 20 Derived local heat ﬂux assuming that evaporation is the only mechanism for microlayer depletion. FIGURE 21 Evaluation of bubble growth models. Frontiers in Energy Research Author contributions JW: Investigation, methodology, data curation, software, and writing—original draft. HW: Investigation, validation, and formal analysis. JX: Supervision, investigation, resources, writing-original draft, writing-review and editing, and funding acquisition. 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. • Pressure shows a signiﬁcant inﬂuence on microlayer formation and depletion, as well as bubble growth. At higher pressure, the duration with the microlayer is comparably short and, consequently, the contribution of microlayer evaporation becomes less important. Compared with heat ﬂux, the subcooling shows a more appreciable effect on bubble growth. The effect of liquid subcooling and surface heat ﬂux on bubble growth is more pronounced at elevated pressure. • Pressure shows a signiﬁcant inﬂuence on microlayer formation and depletion, as well as bubble growth. At higher pressure, the duration with the microlayer is comparably short and, consequently, the contribution of microlayer evaporation becomes less important. Compared with heat ﬂux, the subcooling shows a more appreciable effect on bubble growth. The effect of liquid subcooling and surface heat ﬂux on bubble growth is more pronounced at elevated pressure. 3.3 Bubble growth drb dt ml drb dt sup 2 c π √Pr−0.5 Jaml Jasup (15) (15) With Eqs 13, 15, the above equation can be further written as With Eqs 13, 15, the above equation can be further written as drb dt ml drb dt sup 4 c π √Pr−0.5 (16) (16) 4 Conclusion The laser interferometry and high-speed camera are synchronously employed to measure the microlayer behavior and bubble growth in pool boiling under pressure from 0.1 to 0.3 MPa. Based on the comprehensive analysis of the formation and depletion of the microlayer and the bubble growth, the following conclusions are reached. Data availability statement Increasing pressure reduces the Prandtl number and, consequently, increases the ratio of the microlayer and superheated layer contribution to bubble growth. Based on the Cooper-Lloyd model and the Zuber model, the microlayer evaporation contributes to 68.1% and 70.1% of bubble growth, at 0.1 and 0.2 MPa, respectively, which is not consistent with what is shown in Figure 18. Another fact demanding caution in bubble growth modeling is that the microlayer evaporation contributes to bubble growth in the early phase, while the superheated layer evaporation becomes important in a later phase. The current models do not seem capable to account for such a fact. Hence, more extensive investigation on microlayer evaporation under diverse conditions is still desired to achieve mechanistic prediction of bubble growth. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Acknowledgments The authors are grateful for the ﬁnancial support of the National Natural Science Foundation of China (Grant No. 52076132). 3.3 Bubble growth The Jakob number in the superheated layer is deﬁned by • The analysis of microlayer depletion shows that the dimensionless instantaneous maximum microlayer thickness, δ max/ ]t √, shows exponential dependence on the ratio rd/rb,1, which increases linearly with time before microlayer depletion. A correlation is proposed to predict the instantaneous maximum microlayer thickness synthesizing the two relations. • The analysis of microlayer depletion shows that the dimensionless instantaneous maximum microlayer thickness, δ max/ ]t √, shows exponential dependence on the ratio rd/rb,1, which increases linearly with time before microlayer depletion. A correlation is proposed to predict the instantaneous maximum microlayer thickness synthesizing the two relations. Jasup ρlCp,lΔTw 2ρghf g (14) (14) According to Gerardi et al. (2010), Duan et al. (2013), Yabuki and Nakabeppu (2014), and Sato and Niceno (2015), it is fair to take ΔTw 10 °C. Integrating Eqs 12, 14, we can estimate the bubble volume as a function of time. From Figure 21, we can see that with the Cooper-Lloyd model and the Zuber model a rough estimation on bubble growth rate can be given. According to the two models, the ratio of microlayer and superheated layer contribution to bubble growth is • The local microlayer thickness decreases linearly with time. The assumption of the negligible hydrodynamic ﬂow of the microlayer leads to an overestimation of surface heat ﬂux based on the sole depletion mechanism, i.e., evaporation. • The local microlayer thickness decreases linearly with time. The assumption of the negligible hydrodynamic ﬂow of the microlayer leads to an overestimation of surface heat ﬂux based on the sole depletion mechanism, i.e., evaporation. The hydrodynamic ﬂow of the microlayer cannot be neglected and not all the microlayer is evaporated for the bubble growth. The hydrodynamic ﬂow of the microlayer cannot be neglected and not all the microlayer is evaporated for the bubble growth. • Combining the Cooper-Lloyd model and the Zuber model, the measured bubble growth can be roughly predicted, though the contribution portion of the microlayer and superheated layer to bubble growth predicted by the models is not consistent with the experimental observation. 3.3 Bubble growth One of Jung and Kim’s data sets is also shown in Figure 20. We can see that the derivation based on Eq. (10) can signiﬁcantly overestimate the instantaneous heat ﬂux, which implies that the hydrodynamic ﬂow is a signiﬁcant mechanism of microlayer depletion. The equivalent bubble radius is calculated based on the bubble volume and shown in Figure 21 for the cases at the pressure of 0.1 and 0.2 MPa. The cases at the pressure of 0.3 MPa are not included here due to comparably large relative uncertainty. The bubble growth rate resulting from microlayer evaporation is formulated by Cooper and Lloyd (1969). The dominance of heat ﬂux on bubble growth is observed during the initial phase of bubble growth for all pressures. For example, similar bubble growth is observed in cases No. 1 and 2 and No. 4 and 5, when t/tml < 0.2. In the later phase, the effects of subcooling on the bubble growth manifest. In case No. 3, a slight decrease in bubble volume is observed after microlayer depletion, which indicates that condensation plays a more signiﬁcant role in the late period of bubble growth. In contrast, bubble growth always continues at a higher pressure. As a consequence of the signiﬁcant inﬂuence of microlayer evaporation on bubble growth, bubble growth is less sensitive to heat ﬂux and subcooling at low pressure. At elevated pressure, low subcooling and high heat ﬂux appreciably promote bubble growth. Both factors affect the thermal boundary layer near the heating surface and, consequently, change the evaporation in the superheated sublayer. drb dt ml 1 cPrl −0.5Jaml λl ρlCp,l 0.5 t−0.5 (11) (11) where c = 0.8 is taken and the microlayer Jakob number is deﬁned by Jaml ρlCp,lΔTw ρghf g (12) (12) Forster and Zuber (1954) model was proposed to consider bubble growth by the evaporation of superheated ﬂuid around the bubble. The corresponding growth rate is deﬁned by drb dt sup π √ 2 Jasup λl ρlCp,l 0.5 t−0.5 (13) (13) frontiersin.org Frontiers in Energy Research 12 Wang et al. Wang et al. 10.3389/fenrg.2023.1130459 10.3389/fenrg.2023.1130459 In the superheated layer, the characteristic temperature is taken as (Tw + Tsat)/2. Frontiers in Energy Research frontiersin.org Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Frontiers in Energy Research 13 frontiersin.org Wang et al. 10.3389/fenrg.2023.1130459 10.3389/fenrg.2023.1130459 References Chen, Z., Haginiwa, A., and Utaka, Y. (2017). Detailed structure of microlayer in nucleate pool boiling for water measured by laser interferometric method. Int. J. Heat Mass Transf. 108, 1285–1291. doi:10.1016/j.ijheatmasstransfer.2017.01.003 Kossolapov, A., Chavagnat, F., Nop, R., Dorville, N., Phillips, B., Buongiorno, J., et al. (2020). The boiling crisis of water under exponentially escalating heat inputs in subcooled ﬂow boiling at atmospheric pressure. 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Int. J. Heat Mass Transf. 48 (12), 2429–2442. doi:10.1016/j.ijheatmasstransfer.2004.12.050 Forster, H. K., and Zuber, N. (1954). Growth of a vapor bubble in a superheated liquid. J. Appl. Phys. 25 (4), 474–478. doi:10.1063/1.1721664 Gao, M., Zhang, L., Cheng, P., and Quan, X. (2013). An investigation of microlayer beneath nucleation bubble by laser interferometric method. Int. J. Heat Mass Transf. 57 (1), 183–189. doi:10.1016/j.ijheatmasstransfer.2012.10.017 Narayan, L. S., and Srivastava, A. (2021). References On the identiﬁcation and mapping of three distinct stages of single vapor bubble growth with the corresponding microlayer dynamics. Int. J. Multiph. Flow 142, 103722. doi:10.1016/j.ijmultiphaseﬂow.2021.103722 Gerardi, C., Buongiorno, J., Hu, L. w., and McKrell, T. (2010). Study of bubble growth in water pool boiling through synchronized, infrared thermometry and high-speed video. Int. J. 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Heat transfer mechanisms in isolated bubble boiling of water observed with MEMS sensor. Int. J. Heat Mass Transf. 76, 286–297. doi:10.1016/j.ijheatmasstransfer.2014.04.012 Kenning, D. B. R., Kono, T., and Wienecke, M. (2001). Investigation of boiling heat transfer by liquid crystal thermography. Exp. Therm. Fluid Sci. 25, 219–229. doi:10. 1016/s0894-1777(01)00070-x Zhao, Y.-H., Masuoka, T., and Tsuruta, T. (2002). Uniﬁed theoretical prediction of fully developed nucleate boiling and critical heat ﬂux based on a dynamic microlayer model. Int. J. Heat Mass Transf. 45 (15), 3189–3197. doi:10.1016/s0017-9310(02) 00022-4 Koffman, L. D, P. M. References S., and Plesset, M. S. (1983). Experimental Observations of the Microlayer in vapor bubble Growth on a heated solid. J. Heat Transf. 105 (3), 625–632. doi:10.1115/1.3245631 14 Frontiers in Energy Research frontiersin.org 14 Wang et al. 10.3389/fenrg.2023.1130459 Subscripts 0 initial b bubble b,1 the horizontal radius b,2 the vertical radius c contact d dry spot f ﬂuid g gas i,j time max the maximum value mic-gas the microlayer evaporation amount ml microlayer sup superheat total the total amount w wall ml microlayer sup superheat Nomenclature δ0 initial microlayer thickness θ angle λ wavelength of laser ρ the density of ﬂuid or gas δ microlayer thickness υ kinematic viscosity of the liquid c1, c2, c3, c4 coefﬁcients vary with pressure cp speciﬁc heat D diameter f frequency of camera Ja jakob number m fringe order n refractive index Pr prandtl number r radius rL distance between bubble site and measurement position rml,max distance from bubble inception site when microlayer ﬁrst reached its maximum value t time tg bubble growth time tml microlayer depletion time V volume xe bubble edge ye bubble edge △T the wall superheat Greek symbols hf g latent heat of vaporization Greek symbols total the total amount w wall hf g latent heat of vaporization 15 frontiersin.org Frontiers in Energy Research 15
https://openalex.org/W2793407684	https://vtechworks.lib.vt.edu/bitstream/10919/89758/1/journal.pone.0193259.pdf	English	null	A dual boundary classifier for predicting acute hypotensive episodes in critical care	PloS one	2,018	cc-by	10,711	A dual boundary classifier for predicting acute hypotensive episodes in critical care Sakyajit Bhattacharya1, Vijay Huddar2, Vaibhav Rajan3, Chandan K. Reddy4 1 TCS Innovation Labs, Kolkata, India, 2 Amazon, Bangalore, India, 3 School of Computing, National University of Singapore, Singapore, Singapore, 4 Department of Computer Science, Virginia Tech, Arlington, United States of America vaibhav.rajan@nus.edu.sg * vaibhav.rajan@nus.edu.sg a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Feng Luo, Clemson University, UNITED STATES Received: May 8, 2017 Accepted: February 7, 2018 Published: February 23, 2018 Received: May 8, 2017 Accepted: February 7, 2018 Published: February 23, 2018 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 RESEARCH ARTICLE Abstract An Acute Hypotensive Episode (AHE) is the sudden onset of a sustained period of low blood pressure and is one among the most critical conditions in Intensive Care Units (ICU). With- out timely medical care, it can lead to an irreversible organ damage and death. By identifying patients at risk for AHE early, adequate medical intervention can save lives and improve patient outcomes. In this paper, we design a novel dual–boundary classification based approach for identifying patients at risk for AHE. Our algorithm uses only simple summary statistics of past Blood Pressure measurements and can be used in an online environment facilitating real–time updates and prediction. We perform extensive experiments with more than 4,500 patient records and demonstrate that our method outperforms the previous best approaches of AHE prediction. Our method can identify AHE patients two hours in advance of the onset, giving sufficient time for appropriate clinical intervention with nearly 80% sensi- tivity and at 95% specificity, thus having very few false positives. OPEN ACCESS Citation: Bhattacharya S, Huddar V, Rajan V, Reddy CK (2018) A dual boundary classifier for predicting acute hypotensive episodes in critical care. PLoS ONE 13(2): e0193259. https://doi.org/ 10.1371/journal.pone.0193259 Citation: Bhattacharya S, Huddar V, Rajan V, Reddy CK (2018) A dual boundary classifier for predicting acute hypotensive episodes in critical care. PLoS ONE 13(2): e0193259. https://doi.org/ 10.1371/journal.pone.0193259 Editor: Feng Luo, Clemson University, UNITED STATES Predicting acute hypotensive episodes in critical care Automated systems for predicting AHE and other complications in ICU patients are becoming increasingly important given the enormous difficulties of manually monitoring the abundance of data collected for each patient and the shortage of qualified clinical staff in ICUs [5, 6]. There have been many previous attempts to build automated systems that can identify patients at risk for AHE in advance (see section 2.2 for a brief survey). Funding: This work was supported by the US National Science Foundation grants IIS-1646881 and IIS-1527827 (CR) and Xerox Research Center India (VR). Competing interests: Our affiliations to TCS Innovation Labs, Amazon, National University of Singapore and Virginia Tech do not alter our adherence to PLOS ONE policies on sharing data and materials. This paper presents a novel supervised classification based system for identifying patients at risk for AHE. The algorithm uses only simple statistical summaries of past vital measurements (such as blood pressure and heart rate) of patients and can be employed in an online manner, facilitating real–time updates in the model and prediction. The novelty of the algorithm comes from learning to separate patient cases into three groups: (1) easy to recognize as AHE cases (2) easy to recognize as non–AHE cases and (3) ambiguous cases. During prediction, the algo- rithm deals with each of the cases in a different manner. Our contributions in this paper are: Our contributions in this paper are: • A new supervised binary classification algorithm for identifying patients at risk for Acute Hypotensive Episodes. Our algorithm can be used both in offline and online modes. As more data from new patients arrive, the classifier updates itself in an online manner using only the newly available data (i.e. without re–training itself with the entire dataset). • Extensive experiments on data from patients in critical care show that our algorithm achieves significantly higher accuracy, sensitivity and specificity compared to existing algo- rithms. Performance improvement is observed for predictions made up to 2 hours in advance, thus giving clinicians sufficient time for adequate medical intervention. A preliminary version of this paper appeared in [7] where the dual boundary approach was first presented. A comparative analysis therein shows the superiority of our approach over state-of-the-art methods on a dataset of 1,700 patient records. 1 Introduction Copyright: © 2018 Bhattacharya 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. An Acute Hypotensive Episode (AHE) is the sudden onset of a period of sustained low blood pressure [1]. If left untreated, it can rapidly deteriorate a patient’s health and cause a number of complications including death. Fig 1 shows the episode in a graph of a patient’s Mean Arte- rial Blood Pressure (MAP) measurements. Many different conditions may cause AHE; these include sepsis, myocardial infarction, car- diac arrhythmia, pulmonary embolism, hemorrhage, dehydration, anaphylaxis, effects of med- ication, or any of a wide variety of other causes of hypovolemia, insufficient cardiac output, or vasodilatory shock [1]. In many cases, AHE is a precursor to or predictor of other complica- tions [2–4]. Determining the most appropriate medical intervention for such patients depends on first ascertaining the cause of the hypotensive episode: often due to lack of sufficient time, intervention begins by first selecting a suboptimal treatment to prevent immediate damage and to have sufficient time to assess the patient thoroughly [1]. Early identification of patients at risk for AHE helps not only in preventing the episodes but also in finding the best possible treatment for the patient [1]. Data Availability Statement: All data used is part of MIMIC II clinical database from Physionet that is publicly available. To obtain access, users must sign and abide by a data-use agreement directly with Physionet. (https://physionet.org/mimic2/ mimic2_access.shtml). Access to this data is permitted only after registration at Physionet and completion of a course on the use of human subjects and patient privacy. More details can be found on: (https://mimic.physionet.org/ gettingstarted/access/). PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 1 / 17 2 Background In this section we first provide a short description of blood pressure, hypotension and acute hypotensive episodes. This is followed by a brief survey of clinical studies on hypotensive epi- sodes and related work in data mining on prediction of acute hypotensive episodes. In this paper, we provide a com- plete treatment of the most general online version of our algorithm (that was only briefly intro- duced earlier). We also analyze the behavior of the algorithm and reasons for its superior performance over baseline methods in detail. New experiments with additional baselines and data (vitals in addition to blood pressure) are presented. All experimental results in this paper are on a larger dataset of 4,593 patient records from the publicly available MIMIC II ICU data- base. To our knowledge, the only previously published work on dual boundary classifier is a Fig 1. Definition of acute hypotensive episode: A period of 30 minutes where at least 90% of Mean Arterial Pressure measurements are no greater than 60 mmHg. https://doi.org/10.1371/journal.pone.0193259.g001 s://doi.org/10.1371/journal.pone.0193259 February 23, 2018 2 / 17 Fig 1. Definition of acute hypotensive episode: A period of 30 minutes where at least 90% of Mean Arterial Pressure me greater than 60 mmHg. Fig 1. Definition of acute hypotensive episode: A period of 30 minutes where at least 90% of Mean Arterial Pressure measurements are no greater than 60 mmHg. Fig 1. Definition of acute hypotensive episode: A period of 30 minutes where at least 90% of Mean Arterial Pressure measurements are no greater than 60 mmHg. https://doi org/10 1371/journal pone 0193259 g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 2 / 17 Predicting acute hypotensive episodes in critical care preliminary version of this manuscript, as described above. Multiple decision boundaries may be obtained in margin-based classifiers, like SVM, and the difference between that and our algorithm is explained in section 4.1. The rest of the paper is organized as follows. We begin with a brief background on AHE and a survey of related work in AHE prediction. Section 3 gives a formal definition of the problem. This is followed by a detailed discussion on our new classification algorithm in sec- tion 4. Details of the data and experimental protocol are presented in section 5 and section 5.5 describes the results obtained. We conclude our discussion in section 6. 2.1 Acute hypotensive episodes Arterial Blood Pressure (ABP) is the pressure exerted by blood on the vessels. Along with body temperature, respiratory rate and pulse rate, blood pressure is a vital sign that is routinely monitored by healthcare providers by measuring the pressure on the arterial walls. Blood pressure values are measured often using a sphygmomanometer, in millimeters of mercury (mmHg). In each heartbeat blood pressure varies between the highest and the lowest pressure in the vessels, which are called systolic and diastolic pressures, respectively. ABP mea- surements are written as X/Y mmHg (e.g. 120/80) where X denotes the systolic pressure and Y denotes the diastolic pressure. The Mean Arterial Pressure (MAP) is the pressure generated as blood is pumped out of the left ventricle of the heart into the arteries. It can be approximated using the systolic (X) and diastolic (Y) pressures as follows [8]: MAP ¼ 2 3 X 1 3 Y: The normal range for blood pressure is considered to be between 90/60 and 130/80 mmHg [8]. Thus hypotension is the condition when the blood pressure is lower than 90/60 mmHg. Following [1], we define an Acute Hypotensive Episode as a period of 30 minutes during which at least 90% of the MAP measurements are no greater than 60 mmHg. Other guidelines have also been published [9, 10]. Commercial bedside monitoring systems provide alerts when the blood pressure falls below a predetermined level. But, to the best of our knowledge, there is no system that can predict AHE in advance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 2.2 Related work Many clinical studies have examined the role of hypotension in complications like acute coro- nary syndromes [11], acute kidney injury [12] and sepsis [13, 14]. Hypotension is also found to be an important mortality predictor for patients with cardiovascular abnormalities [15, 16]. Hypotension is a precursor to septic shock, the second most common cause of death in ICU patients in the United States [17], and a study shows that mortality in such cases depends criti- cally on the duration of hypotension before treatment [18]. Existing intervention approaches to AHE are reactive, that is, after the onset of AHE. These interventions include administration of vasopressors, fluid resuscitation and other treatments depending on the case [10, 19]. Instead if patients at risk for AHE are identified in advance, the intervention most appropriate to the patient can be determined and administered. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 3 / 17 Predicting acute hypotensive episodes in critical care AHE prediction. In the MIMIC II critical care database [20], out of 2320 patients, AHE was found in 41% of the patients for whom arterial blood pressure was recorded. The mortal- ity rate of these patients was found to be more than twice that of the entire MIMIC II popula- tion. Recognizing the importance of AHE, automated AHE prediction was put forth as a challenge in the 2009 Computers in Cardiology competition [1]. Vital signs data for 110 patients from the MIMIC II database [20] were provided, divided into training and test data- sets containing 60 and 50 patient records respectively. The results of the top three performers of the challenge are published in [21], [ 22] and [23], respectively. The best result was obtained by Henriques et al [21] using a neural network based classifier. A similar method, called Che- byshev’s neural network, applied on the challenge dataset by Zhou et al [24], gave similar results. Simple mean based predictions such as those of Chen [22] who used the last 5 minutes of average diastolic blood pressure as a predictor of AHE and of Mneimneh et al [23] who used the last 20 minutes of MAP measurements had comparable performance. A hidden markov model based approach is proposed by Singh et al [25] and the use of contrast pattern mining for AHE prediction is discussed by Ghosh et al [26]. Both these techniques have per- formance similar to that of mean based predictions. 2.2 Related work Note that these methods were tested on a small sample size (110 patients) and for predicting AHE only 30 minutes in advance. A detailed analysis of the problem as well as these methods is given in Marzyeh Ghassemi’s thesis [27] wherein the neural network based method is tested on 1,168 patient records using four vital signs’ data—heart rate, respiration rate, oxygenation levels and MAP. Sun et al [28] design a method based on particle swarm optimization and k-means to extract features from MAP measurements which are then classified using SVM. Donald et al [29] developed a Bayes- ian Artificial Neural Network trained on demographic and physiological data of nearly 2,000 patients to predict hypotension in brain injury subjects with observation windows of 15 and 30 minutes before the injury. Lee et al [30], used heart rate, MAP and clinical data of 1,311 patient records from the MIMIC II database. Their algorithm uses 102 statistical, wavelet-based and clinical features and predicts the event 1 hour in advance using a neural network based classifier. Some other studies have also addressed the problem of predicting AHE but have not been tested extensively. A Binomial Sign Test based classifier was tested by Crespo et al [31] on the BP measurements of 7 patients using 5 time series features extracted from MAP waveforms. Their method predicts the event only 20 seconds in advance. Ghaffari et al [32] develop a method of detecting AHE using ECG and MAP waveforms and test it on 15 subjects from the MIMIC II database. Lehman et al [33] use a combination of Gaussian Mixture Model based clustering and K-Nearest Neighbours Classifier on 227 patient records from the MIMIC II database using both Heart Rate and MAP measurements. Rocha et al [34] use correlation anal- ysis and neural networks on MAP measurements to build a predictive model for AHE. The best predictive accuracy when only vital measurements are used (among existing methods) is achieved by the mean-based prediction of Chen [22] which was shown to be outperformed by our classifier in [7]. Fig 2 shows a schematic of the three windows. Note that each patient is potentially at risk of a future acute hypotensive episode at each point in time. What we have, from historical data, is a single indicator of an occurrence of AHE at a particular timepoint (the risk at previous timepoints can only be estimated, not known with certainty). Our aim is to predict this as early as possible. The observation window indicates the amount of data we use for training. The test window indicates the amount of data we use during prediction. The windows can be measured with respect to either time duration (that we use) or number of measurements. For conducting our experiments, we vary the length of these windows (i.e. the time duration of the measurements considered for training and prediction) independently and study the performance of the classi- fiers. Also note that by testing the performance on expanding test windows, we effectively test the performance at regular intervals for the same patient. We also study the classifier perfor- mance in an online setting, where we update the classifier with new measurements obtained. In a real-life deployment, the classifier can predict, with each new blood pressure measure- ment, whether a patient is at risk (A) or not at risk (NA) and when the prediction is A, an alert is raised for the clinical staff. So the labels can also be viewed as an indicator for alert at the patient level. determining the class label of the patient. There are three parameters that can be varied during the prediction, which we will refer to as the [P,O,T] parameters of an algorithm: Prediction Window (P): Duration of time (in minutes) before which predictions are made, i.e., we predict whether or not a patient will have AHE w minutes in the future; we call w the prediction window width. Prediction Window (P): Duration of time (in minutes) before which predictions are made, i.e., we predict whether or not a patient will have AHE w minutes in the future; we call w the prediction window width. Observation Window (O): Duration of time (in minutes) before the prediction window during which MAP measurements are considered for training the classifier. Observation Window (O): Duration of time (in minutes) before the prediction window during which MAP measurements are considered for training the classifier. Test Window (T): Duration of time (in minutes) before the prediction window during which MAP measurements are considered for predicting AHE in a patient. Test Window (T): Duration of time (in minutes) before the prediction window during which MAP measurements are considered for predicting AHE in a patient. 3 Problem definition An Acute Hypotensive Episode (AHE) is defined as a period of 30 minutes during which at least 90% of the MAP measurements are no greater than 60 mmHg [1]. We cast the problem of predicting AHE in a patient as a supervised binary classification problem. Using data from previous ICU patients—both with and without AHE events—a binary classifier is trained, where the classes are denoted by A (those likely to have AHE) and NA (those unlikely to have AHE). The classifier can then be used to predict AHE in a previously unseen patient by PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 4 / 17 Predicting acute hypotensive episodes in critical care determining the class label of the patient. There are three parameters that can be varied during the prediction, which we will refer to as the [P,O,T] parameters of an algorithm: 4 DBC: A novel classification method In this section, we describe our new algorithm for classifying patients into classes 1 (e.g. class A, those at risk for developing AHE) and 2 (e.g. class NA, those not at risk for developing AHE) based on their blood pressure signals. Our method uses only the mean (μ1, μ2 for classes Fig 2. [O,P,T] parameters for a predictive algorithm. Note that the Test and Observation windows can vary in duration independently of each other. htt //d i /10 1371/j l 0193259 002 Fig 2. [O,P,T] parameters for a predictive algorithm. Note that the Test and Observation windows can vary in duration independently of each other https://doi.org/10.1371/journal.pone.0193259.g002 5 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 Predicting acute hypotensive episodes in critical care 1 and 2 respectively) and standard deviation (σ1, σ2, for classes 1 and 2 respectively) of the vital measurements within a given observation window as features. Our classifier, called Dual Boundary Classifier (DBC) uses a combination of margin-based and distance-based classifica- tion techniques. We first describe the intuition behind the algorithm and explain how it differs from standard classifiers like SVM and KNN. 4.1 Intuition behind the classifier We observe in the training data that the MAP measurements in class A in the hour before the Acute Hypotensive event is generally lower than the MAP measurements in class NA. Hence, a margin-based classifier is expected to work well. As seen in the experiments, SVM has high specificity but its sensitivity is very low. To minimize both false positives and false negatives (type-I and type-II errors, respectively), where true positive denotes correct identification in class A, our new method builds an inter- val-based margin (in other words, two decision boundaries, using a parameter κ). Our interval is based on the mean and standard deviation values in classes A and NA, observed in the train- ing data: (μ1 + κσ1, μ2 −κσ2). Any value (in the test set) falling below the lower bound of the interval is considered to be in class A and a value above the upper bound in class NA. We choose the bounds (by choosing the right κ) such that the errors are minimized, i.e., the sum of the number of class A values falling above the lower bound (n1 + n2) and the number of class NA values falling below the upper bound (n3 + n4) is minimized. In addition we also min- imize the number of values in the uncertainty region. Fig 3 shows a schematic. Test samples falling in the region of class A or class NA are classified in a straightforward manner. For those test samples that lie in the uncertainty region, we use a distance-based Fig 3. Schematic of our novel classifier. The feature space is divided into three regions based on the values of μ1, μ2, σ1, σ2, κ0. A test sample in the AHE region is classified into class A, Non-AHE region is classified into class NA. A distance-based approach is used for test samples in the Uncertainty region. The arrows for a point in the uncertainty region (shown only for one of the points), show that distances are calculated with respect points outside the uncertainty region only. Fig 3. Schematic of our novel classifier. The feature space is divided into three regions based on the values of μ1, μ2, σ1, σ2, κ0. A test sample in the AHE region is classified into class A, Non-AHE region is classified into class NA. A distance-based approach is used for test samples in the Uncertainty region. https://doi.org/10.1371/journal.pone.0193259.g003 Predicting acute hypotensive episodes in critical care approach to classify the sample by comparing the distance of the test case to (training data) points outside the uncertainty region. Comparison with other classifiers. Note that our approach is not an ensemble-based technique since we do not use a combination of classifiers on any test datapoint. Our classifier is a discriminative classifier which applies two different classification rules and both rules differ in methodology from standard classifiers. One is a boundary based rule (similar to, but not the same as, SVM) for data points outside the uncertainty region, and another is a nearest neigh- bor rule (similar to, but not the same as, KNN) for datapoints in the uncertainty region. Thus the selection of the rule is also determined by our classifier in a data-driven manner. These dif- ferences distinguish DBC from standard classifiers. Comparison with margin-based classifiers. The two boundaries in DBC are not determined using support vectors (as in SVM) but using the mean and standard deviation values of training data from both classes. In the optimization setup for training, DBC minimizes the misclassification rate and the number of points in the uncertainty region which is not the same as maximizing the margin. Indeed, our formulation yields a mixed integer linear program (shown in [7]) whereas the classical SVM formulation results in a quadratic program. Comparison with distance-based classifiers. DBC uses a distance-based approach only for the uncertainty region points in testing and note that we determine the distance only with respect to the points outside the AHE region—which makes it different from a KNN rule. For exam- ple, if a test data point lies in the uncertainty region and the closest point is also within the uncertainty region, KNN will consider it whereas DBC will not. To further illustrate the difference, note that a combination of SVM and KNN would apply both classifiers on each test point. But DBC first determines whether the test point lies in the uncertainty region. If it is in the uncertainty region, it applies a distance-based rule— checking distance only with respect to training points outside the uncertainty region. If it is not in the uncertainty region, then the boundary determines the class and the boundaries are learnt from the training data in a manner that is different from that of margin-based classifiers. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 4.1 Intuition behind the classifier The arrows for a point in the uncertainty region (shown only for one of the points), show that distances are calculated with respect points outside the uncertainty region only. https://doi.org/10.1371/journal.pone.0193259.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 6 / 17 4.2 Online classification algorithm PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 7 / 17 Predicting acute hypotensive episodes in critical care • Compute the cumulative means and variance (for all measurements of vital v up to the nth observation window), m ðnÞ Av ; n ðnÞ Av as follows [35], where x is each measurement in y ðnÞ Av : • Compute the cumulative means and variance (for all measurements of vital v up to the nth observation window), m ðnÞ Av ; n ðnÞ Av as follows [35], where x is each measurement in y ðnÞ Av : • Compute the cumulative means and variance (for all measurements of vital v up to the nth observation window), m ðnÞ Av ; n ðnÞ Av as follows [35], where x is each measurement in y ðnÞ Av : m ðnÞ Av ¼ m ðn1Þ Av þ x m ðn1Þ Av n n ðnÞ Av ¼ ðn 1Þn ðn1Þ Av þ ðx m ðn1Þ Av Þðx m ðnÞ Av ÞÞ n Similarly, we compute cumulative mean and variance m ðnÞ NAv; n ðnÞ NAv for class NA. • Let nAv ¼ jy ðnÞ Av j and nNAv ¼ jy ðnÞ Av j, the cardinalities of the two vectors. This is updated in each round: nAv ¼ jy ðn1Þ Av j þ jy ðnÞ Av j nNAv ¼ jy ðn1Þ NAv j þ jy ðnÞ NAvj: • Let κv be a value such that m ðnÞ Av þ kvs ðnÞ Av < m ðnÞ NAv kvs ðnÞ NAv. Let nv1: Number of y ðnÞ Av values greater than m ðnÞ NAv kvs ðnÞ NAv. nv2: Number of y ðnÞ Av values in the range ðm ðnÞ Av þ kvs ðnÞ Av ; m ðnÞ NAv kvs ðnÞ NAvÞ. nv3: Number of y ðnÞ NAv values lesser than m ðnÞ Av þ kvs ðnÞ Av . nv4: Number of y ðnÞ NAv values in the range ðm ðnÞ Av þ kvs ðnÞ Av ; m ðnÞ NAv kvs ðnÞ NAvÞ. nv4: Number of y ðnÞ NAv values in the range ðm ðnÞ Av þ kvs ðnÞ Av ; m ðnÞ NAv kvs ðnÞ NAvÞ. • Find the value of κv that minimizes nv1 + nv2 + nv3 + nv4. Let that value be k v. 4.2 Online classification algorithm We describe the DBC algorithm in the more general online mode. The offline mode is a special case with a single round that uses all available historical data for training. An implementation, in R, is available from the authors upon request. The online algorithm proceeds in rounds. In each round, we use the measurements made in the new observation window to update the model. We denote the round with superscript (n). Measurements can be made for each of the vitals (subscript v): MAP, heart rate (HR), res- piration rate (RR), Oxygen Saturation (OSAT) and temperature (TEMP). Training the classifier. Training the classifier consists of the following steps: • Denote by y ðnÞ Av the vector where each coordinate has the mean of the measurements of vital v in the nth observation window for patients in class A. Let y ðnÞ NAv be the corresponding vector for class NA. Denote by y ðnÞ Av (y ðnÞ NAv) all the measurements of vital v for patients in class A (NA) until round n. • Let m ðnÞ Av ¼ meanðy ðnÞ Av Þ, m ðnÞ NAv ¼ meanðy ðnÞ NAvÞ, s ðnÞ Av ¼ stdevðy ðnÞ Av Þ, s ðnÞ NAv ¼ stdevðy ðnÞ Av Þ, n ðnÞ Av ¼ varianceðy ðnÞ Av Þ, n ðnÞ NAv ¼ varianceðy ðnÞ Av Þ, are the corresponding means, standard devia- tions and variance. 4.2 Online classification algorithm A simple heu- ristic for selecting k v can be employed in practice: • Select a range of values for κv and compute nv1 + nv2 + nv3 + nv4 for each value in the range. • Select the value of κv with the (local) minimum nv1 + nv2 + nv3 + nv4. Alternatively, a mixed integer linear program can be used as shown in [7] but that can become computationally expensive especially in an online setting. Thus a trained classifier at the end of the nth round consists of a 7-tuple: ½nAv; nNAv; m ðnÞ Av ; s ðnÞ Av ; m ðnÞ NAv; s ðnÞ NAv; k v for each vital v. Thus a trained classifier at the end of the nth round consists of a 7-tuple: Classifying test data. We compute the mean of vital measurements v over the period of the Test Window, denoted by zv. For each vital, we obtain a classification label lv as follows: • If zv < m ðnÞ Av þ k vs ðnÞ Av , assign the patient to class A. • If zv < m ðnÞ Av þ k vs ðnÞ Av , assign the patient to class A. • If zv < m ðnÞ Av þ k vs ðnÞ Av , assign the patient to class A. • If zv < m ðnÞ Av þ k vs ðnÞ Av , assign the patient to class A. • If zv > m ðnÞ NAv k vs ðnÞ NAv, assign the patient to class NA. • If zv > m ðnÞ NAv k vs ðnÞ NAv, assign the patient to class NA. • If zv > m ðnÞ NAv k vs ðnÞ NAv, assign the patient to class NA. • If zv falls in the range ½m ðnÞ Av þ k vs ðnÞ Av ; m ðnÞ NAv k vs ðnÞ NAv, compute the mean squared deviation of zv from all the points in y ðnÞ Av and y ðnÞ NAv (from the training data), denoted by dAv and dNAv, respectively. If dAv > dNAv assign the patient to class NA, otherwise assign the patient to class A. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 4.2 Online classification algorithm • If zv falls in the range ½m ðnÞ Av þ k vs ðnÞ Av ; m ðnÞ NAv k vs ðnÞ NAv, compute the mean squared deviation of zv from all the points in y ðnÞ Av and y ðnÞ NAv (from the training data), denoted by dAv and dNAv, respectively. If dAv > dNAv assign the patient to class NA, otherwise assign the patient to class A. Note that this does not require us to store all the patient data used during the training phrase. Note that this does not require us to store all the patient data used during the training phrase. The computation can be simplified as follows. The mean squared deviation of zv from all the Note that this does not require us to store all the patient data used during the training phrase. The computation can be simplified as follows The mean squared deviation of zv from all the q p g g p The computation can be simplified as follows. The mean squared deviation of zv from all the The computation can be simplified as follows. The mean squared deviation of zv from all t PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 8 / 17 Predicting acute hypotensive episodes in critical care points in y ðnÞ Av is PnAv i¼1 ðzv xivÞ 2 where xiv is the i-th coordinate of y ðnÞ Av . The above expression can be written as points in y ðnÞ Av is PnAv i¼1 ðzv xivÞ 2 where xiv is the i-th coordinate of y ðnÞ Av . The above expression can be written as dAv ¼ X nAv i¼1 ðzv m ðnÞ Av þ m ðnÞ Av xivÞ 2 ¼ nAv½ðzv m ðnÞ Av Þ 2 þ n ðnÞ NAv and similarly for the distance of zv from y ðnÞ NAv. Thus the computation only requires the values of the total number of datapoints: nAv, nNAv and the mean and variance computed during training: m ðnÞ Av ; n ðnÞ Av ; m ðnÞ NAv; n ðnÞ NAv. The final classification label is set by a majority voting rule: ∑v lv where ties are broken by using the vote for MAP. 5.1 ICU data MIMIC II [20] is a publicly available database, part of Physionet [36], containing physiological signals and clinical data of more than 5000 ICU patients. Vital signs of most of these ICU patients were recorded, sampled either every minute or every second. We consider only the following vital measurements for our study: Mean Arterial Blood Pressure (MAP), Heart Rate (HR), Pulse Rate (PUL), Respiratory Rate (RR) and Oxygen Saturation (OSAT). Each patient record consists of a time series signal xt, t = 1,. . .,Ni where Ni is the total num- ber of vital measurements for the ith patient, and xt is the MAP measurement at time t, time being reckoned from the start of the measurements in the ICU at an interval of 1 minute. Whenever the sampling is per second, we use the mean of all measurements in a minute. An Acute Hypotensive Event (AHE) is defined as an interval in the record, [xt, xt+30], in which at least 27 of the MAP measurements are no greater than 60. Not all the records are usable since many of them have missing or erroneous data. From the available cleaned data in MIMIC II, we use 4,593 records of ICU patients out of which 1,307 are found to contain AHE events (class A) and 3,286 do not contain AHE events (class NA). We considered data only until the first AHE event in each patient record for training and pre- diction. During training we discard records that contain less than 6 hours of MAP measure- ments and those that contain Acute Hypotensive Events in the first 5 hours of recorded data. 4.3 Time complexity The time complexity of training (for each round, and each vital) is dominated by the step of finding κv. Let K be the number of choices for κv considered in our heuristic. The training time complexity is OðK þ nAv þ nNAvÞ and testing each test window zv containing nz vital mea- surements takes OðnzÞ time (to compute the mean). PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 5.2 Experimental setup We test the performance of the algorithms for the following choices of 6 observation windows (O), 24 prediction windows (P) and 6 test windows (T): O 2 f20; 40; 60; 80; 100; 120g; P 2 f5; 10; 15; . . . ; 110; 115; 120g; T 2 f20; 40; 60; 80; 100; 120g: 9 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 Predicting acute hypotensive episodes in critical care In the online mode, for each of the 864 [O,P,T] settings, first approximately 20% (= 910) of the patient records are chosen for initial training. The ratio of AHE and Non-AHE patients in this initial training set is 260:650 which approximately maintains the same ratio found in the original data (1307:3286). After that we incrementally predict and update the classifier, using a single observation window for each patient. In this manner, prediction is done for the remain- ing 3,683 (= 4593-910) patients containing 1,047 AHE and 2,636 non-AHE cases. In the offline mode, we perform 5-fold cross validation over all the patient records, for each of the 864 [O,P, T] settings. 5.3 Baseline comparison methods As mentioned earlier, among the previous methods for AHE prediction that use only vital measurements, the best predictive accuracy is achieved by the mean-based prediction of Chen [22] which we also confirm with experiments on our larger dataset of 1,700 patient records in our previous work [7]. We use this classifier, denoted by MU, as a baseline along with the other standard classifiers—Support Vector Machines (SVM) with RBF kernel, Random Forest (RF), K–Nearest Neighbors (KNN) and Adaboost (ADA)—using the same mean–based fea- tures. Implementations in R are used for these classifiers: packages ‘e1071’, ‘Random Forest’, ‘Class’ and ‘Ada’ respectively. Tunable parameters for the baseline classifiers are obtained in the following way. A portion of the training data is held out for validation. A grid search on the parameter space is performed and for each classifier, the parameters that give the best per- formance on the validation set are chosen. Prediction results are shown only for the best parameters thus selected. 5.4 Evaluation metrics We measure the performance of all the tested methods by their sensitivity, specificity and clas- sification accuracy. Sensitivity is defined as the percentage of class A test samples accurately classified into class A. Specificity is defined as the percentage of class NA test samples accu- rately classified into class NA. Classification accuracy of a classifier is the percentage of total test samples correctly classified. Note that there is no discriminating threshold in our method that creates a trade-off between sensitivity and specificity (unlike many other classifiers). Hence the sensitivity and specificity do not change with either any internal parameter or time window. We show the sensitivity (true positive rate) and specificity (true negative rate) separately, where ‘positive’ denotes correct prediction of AHE. P-values are computed using a one-sided two-sample t-test. We compare the mean value of the performance measure (sensitivity, specificity and accuracy) for a baseline (μ0) against the corresponding mean value for DBC (μ1). We compare the null hypothesis H0 : μ1 = μ0 against the alternative hypothesis Ha : μ1 > μ0 at level of significance 5%. Predicting acute hypotensive episodes in critical care f performance of algorithms—Our (DBC), KNN, RF, SVM, ADA and MU—Averaged over all the 864 [O,P,T] online mode and over 5-fold cross validation in the offline mode. Table 1. Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, RF, SVM, ADA and MU—Averaged over all the 864 [O,P,T] window settings: Averaged for 3,683 patients in the online mode and over 5-fold cross validation in the offline mode. Algorithm DBC KNN RF SVM ADA MU Online Sensitivity 0.77 (0.04) 0.31 (0.05) 0.30 (0.11) 0.17 (0.05) 0.24 (0.05) 0.39 (0.06) Specificity 0.92 (0.02) 0.47 (0.03) 0.37 (0.03) 0.82 (0.04) 0.60 (0.21) 0.81 (0.05) Accuracy 0.85 (0.01) 0.40 (0.03) 0.34 (0.06) 0.78 (0.07) 0.41 (0.07) 0.72 (0.07) Offline Sensitivity 0.83 (0.01) 0.34 (0.02) 0.43 (0.03) 0.19 (0.03) 0.39 (0.11) 0.59 (0.03) Specificity 0.90 (0.01) 0.56 (0.04) 0.34 (0.03) 0.78 (0.03) 0.57 (0.09) 0.79 (0.02) Accuracy 0.87 (0.01) 0.46 (0.03) 0.37 (0.03) 0.74 (0.04) 0.44 (0.08) 0.69 (0.02) https://doi.org/10.1371/journal.pone.0193259.t001 Table 1. Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, RF, SVM, window settings: Averaged for 3,683 patients in the online mode and over 5-fold cross validation in the offline m other algorithms (p-value <0.05) in terms of sensitivity, specificity and overall accuracy, in both online and offline modes. We show the boxplots for the sensitivity, specificity and accuracy obtained at a prediction window of 120 minutes (2 hours) in both offline and online modes, averaged over 144 [O,T] settings in Fig 4. Note that in these 144 experiments, even the lowest accuracy achieved by our algorithm is higher than the highest accuracy of any other algorithm. Results for other values of prediction window are similar. Algorithm analysis. Algorithm DBC is a hybrid approach that combines margin-based and distance-based classification methods. As described in section 4, by learning two bound- aries DBC finds an uncertainty region between the boundaries and uses two different classifi- cation rules depending on whether a test case falls within or outside the uncertainty region. We now evaluate the benefit of this approach by comparing the performance of DBC with a margin-based classifier without the uncertainty region and a purely distance-based approach that does not use the dual boundaries of DBC—that is same as KNN. Table 2 shows the results over all the 864 [O,P,T] window settings: averaged for 3683 patients in the online mode and over 5-fold cross validation in the offline mode as described above. The goal of the hybrid approach of DBC is to learn the two boundaries such that we have very good classification performance outside the uncertainty region. This hypothesis is con- firmed in our experiments. Table 3 shows the classification performance of DBC (for the off- line case, as shown in Tables 2 and 3 above) in each of the three regions of the feature space (see Fig 3): AHE, Non-AHE and Uncertainty Region. We observe that the performance of DBC is indeed much higher outside the uncertainty region. 5.5 Performance results The prediction window is the most important parameter here since it determines how early we can predict the possibility of AHE in a patient. The values of O and T parameters can be set by the user based on the sensitivity-specificity requirements or availability of data. We first present the results of online and offline classification using only MAP measure- ments first. The results, shown in Table 1 are averages over all the 864 [O,P,T] window set- tings: averaged for 3683 patients in the online mode and over 5-fold cross validation in the offline mode as described above. We observe that our algorithm significantly outperforms all PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 10 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 5.6 Addition of vitals We study the performance of all the classifiers when additional vitals—HR, PUL, RR, and OSAT—are used. Table 4 shows the performance averaged over 5-fold cross validation, over all 864 choices of [O,P,T] window settings. The uppermost rows show the results of using (1) only MAP measurements. The other rows (downwards) show the results of using (2) MAP and HR, (3) MAP, HR and PUL, (4) MAP, HR, PUL and RR and (5) MAP, HR, PUL, RR and OSAT respectively. As additional vitals are used, the accuracy of our classifier reduces and the best results are obtained when only MAP measurements are used. With more vitals, SVM achieves com- parable or higher than that of DBC. However, in the case of even SVM, the highest accuracy is obtained with the use of MAP measurements only. Overall, among all the different combi- nations of vitals tested, the best results are obtained by our algorithm when only MAP measurements are used. These results suggest that MAP measurements provide the most dis- criminatory feature to identify patients who are at risk of AHE. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 11 / 17 Predicting acute hypotensive episodes in critical care -Nearest Neighbors (KNN), Random Forest (RF), Support Vector prediction window of 120 minutes, averaged over 144 choices of [O, cross validation (offline) Fig 4. Accuracy, sensitivity and specificity of our algorithm (DBC), K-Nearest Neighbors (KNN), Random Forest (RF), Support Vector Machine (SVM), AdaBoost (ADA) and Mean based method (MU) at prediction window of 120 minutes, averaged over 144 choices of [O, T] settings and over (LEFT:) 3683 patients (online)/ (RIGHT:) 5–fold cross validation (offline). https://doi org/10 1371/journal pone 0193259 g004 Fig 4. Accuracy, sensitivity and specificity of our algorithm (DBC), K-Nearest Neighbors (KNN), Random Forest (RF), Support Vector Machine (SVM), AdaBoost (ADA) and Mean based method (MU) at prediction window of 120 minutes, averaged over 144 choices of [O, T] settings and over (LEFT:) 3683 patients (online)/ (RIGHT:) 5–fold cross validation (offline). https //doi o g/10 1371/jo nal pone 0193259 g004 5.7 Discussion Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, RF, SVM, ADA and MU—Averaged over all the 864 [O,P,T] window settings using 5-fold cross validation. The topmost rows show the results of using (1) only MAP measurements. The remaining rows show the results of using (2) MAP and HR, (3) MAP, HR and PUL, (4) MAP, HR, PUL and RR and (5) MAP, HR, PUL, RR and OSAT respectively. Best results in each row are indicated in bold. Table 4. Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, RF, SVM, ADA and MU—Averaged over all the 864 [O,P,T] window settings using 5-fold cross validation. The topmost rows show the results of using (1) only MAP measurements. The remaining rows show the results of using (2) MAP and HR, (3) MAP, HR and PUL, (4) MAP, HR, PUL and RR and (5) MAP, HR, PUL, RR and OSAT respectively. Best results in each row are indicated in bold. s) of performance of algorithms—Our (DBC), KNN, RF, SVM, ADA and MU—Averaged over all the 864 [O,P,T] he topmost rows show the results of using (1) only MAP measurements. The remaining rows show the results of using P, HR, PUL and RR and (5) MAP, HR, PUL, RR and OSAT respectively. Best results in each row are indicated in bold. 5.7 Discussion 5.7 Discussion Why does our classification algorithm work well?. The answer lies in the data. We first observe that mean MAP measurements over an observation window is a good predictor of Table 2. Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, Linear SVM —Averaged over all the 864 [O,P,T] window settings: Averaged for 3,683 patients in the online mode and over 5-fold cross validation in the offline mode. Algorithm DBC KNN Linear SVM Online Sensitivity 0.77 (0.04) 0.31 (0.05) 0.11 (0.05) Specificity 0.92 (0.02) 0.47 (0.03) 0.83 (0.05) Accuracy 0.85 (0.01) 0.40 (0.03) 0.73 (0.05) Offline Sensitivity 0.83 (0.01) 0.34 (0.02) 0.17 (0.03) Specificity 0.90 (0.01) 0.56 (0.04) 0.85 (0.02) Accuracy 0.87 (0.01) 0.46 (0.03) 0.78 (0.04) https://doi.org/10.1371/journal.pone.0193259.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 12 / 17 Why does our classification algorithm work well?. The answer lies in the data. We first observe that mean MAP measurements over an observation window is a good predictor of g p Table 2. Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, Linear SVM —Averaged over all the 864 [O,P,T] window settings: Averaged for 3,683 patients in the online mode and over 5-fold cross validation in the offline mode. Algorithm DBC KNN Linear SVM Online Sensitivity 0.77 (0.04) 0.31 (0.05) 0.11 (0.05) Specificity 0.92 (0.02) 0.47 (0.03) 0.83 (0.05) Accuracy 0.85 (0.01) 0.40 (0.03) 0.73 (0.05) Offline Sensitivity 0.83 (0.01) 0.34 (0.02) 0.17 (0.03) Specificity 0.90 (0.01) 0.56 (0.04) 0.85 (0.02) Accuracy 0.87 (0.01) 0.46 (0.03) 0.78 (0.04) https://doi.org/10.1371/journal.pone.0193259.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 12 / 17 Table 2. Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, Linear SVM —Averaged over all the 864 [O,P,T] window settings: Averaged for 3,683 patients in the online mode and over 5-fold cross validation in the offline mode. Algorithm DBC KNN Linear SVM Online Sensitivity 0.77 (0.04) 0.31 (0.05) 0.11 (0.05) Specificity 0.92 (0.02) 0.47 (0.03) 0.83 (0.05) Accuracy 0.85 (0.01) 0.40 (0.03) 0.73 (0.05) Offline Sensitivity 0.83 (0.01) 0.34 (0.02) 0.17 (0.03) Specificity 0.90 (0.01) 0.56 (0.04) 0.85 (0.02) Accuracy 0.87 (0.01) 0.46 (0.03) 0.78 (0.04) https://doi.org/10.1371/journal.pone.0193259.t002 Table 2. Mean (standard deviation in parentheses) of performance of algorithms—Our (DBC), KNN, Linear SVM —Averaged over all the 864 [O,P,T] window settings: Averaged for 3,683 patients in the online mode and over 5-fold cross validation in the offline mode. https://doi.org/10.1371/journal.pone.0193259.t004 https://doi.org/10.1371/journal.pone.0193259.t004 s://doi.org/10.1371/journal.pone.0193259.t004 5.7 Discussion 12 / 17 Predicting acute hypotensive episodes in critical care Table 3. Mean performance of algorithm DBC in the three regions learned—Averaged over all the 864 [O,P,T] window settings: Over 5-fold cross validation in the offline mode. AHE Non-AHE Uncertainty Sensitivity 0.93 0.96 0.73 Specificity 0.97 0.99 0.82 Accuracy 0.96 0.98 0.79 https://doi.org/10.1371/journal.pone.0193259.t003 Table 3. Mean performance of algorithm DBC in the three regions learned—Averaged over all the 864 [O,P,T] window settings: Over 5-fold cross validation in the offline mode. https://doi.org/10.1371/journal.pone.0193259.t003 AHE. This has been validated by previous work as well [22]. Our experiments also show that addition of mean values of other vitals in our framework does not improve the predictive accuracy. AHE. This has been validated by previous work as well [22]. Our experiments also show that addition of mean values of other vitals in our framework does not improve the predictive accuracy. However, not all AHE patients show very low mean in their observation windows. Fig 5 shows the mean MAP measurements of the patients in our dataset with different markers indi- cating division into train and test sets, as well as AHE and non-AHE data. The boundary obtained by a linear SVM classifier and the dual boundaries obtained by our classifier on the training data are also shown. While it is true that most of the AHE datapoints (triangle markers) lie below the SVM boundary, with low mean MAP, there are several above the boundary as well. Our algorithm gives special consideration to some of the datapoints that are between our two boundaries— the region we call the uncertainty region. These datapoints are difficult to classify into AHE or non–AHE by using a single boundary. Hence we use a distance based strategy to classify these datapoints. The data in the uncertainty region mostly belong to patients whose MAP measurements were observed to fluctuate considerably in the observation window. For example, in some cases, the BP remains in the normal range initially and becomes low and again comes back to the normal range after some time. In such cases, the mean is often close to the decision bound- ary and a single boundary classifier tends to misclassify them. These were the cases that were better classified by finding the distance from the mean of the training data in each class. Table 4. 5.7 Discussion ( ) ( ) ( ) ( ) p y Vitals Algorithm DBC KNN RF SVM ADA MU MAP Sensitivity 0.83 (0.01) 0.34 (0.02) 0.43 (0.03) 0.19 (0.03) 0.39 (0.11) 0.59 (0.03) Specificity 0.90 (0.01) 0.56 (0.04) 0.34 (0.03) 0.78 (0.03) 0.57 (0.09) 0.79 (0.02) Accuracy 0.87 (0.01) 0.46 (0.03) 0.37 (0.03) 0.74 (0.04) 0.44 (0.08) 0.69 (0.02) + HR Sensitivity 0.78 (0.01) 0.36 (0.05) 0.22 (0.04) 0.19 (0.03) 0.38 (0.10) 0.56 (0.1) Specificity 0.69 (0.01) 0.62 (0.05) 0.22 (0.04) 0.82 (0.04) 0.62 (0.11) 0.54 (0.1) Accuracy 0.72 (0.01) 0.51 (0.05) 0.22 (0.04) 0.61 (0.04) 0.44 (0.09) 0.55 (0.1) + PULSE Sensitivity 0.55 (0.005) 0.45 (0.1) 0.22 (0.04) 0.25 (0.06) 0.45 (0.12) 0.29 (0.06) Specificity 0.54 (0.005) 0.44 (0.1) 0.21 (0.05) 0.82 (0.06) 0.61 (0.13) 0.45 (0.04) Accuracy 0.54 (0.003) 0.44 (0.1) 0.22 (0.04) 0.66 (0.05) 0.49 (0.10) 0.37 (0.04) + RR Sensitivity 0.61 (0.01) 0.5 (0.05) 0.39 (0.05) 0.15 (0.05) 0.20 (0.05) 0.29 (0.06) Specificity 0.54 (0.01) 0.44 (0.05) 0.55 (0.02) 0.84 (0.06) 0.63 (0.12) 0.65 (0.05) Accuracy 0.57 (0.005) 0.47 (0.05) 0.47 (0.02) 0.51 (0.1) 0.33 (0.08) 0.52 (0.1) + OSAT Sensitivity 0.46 (0.02) 0.53 (0.03) 0.37 (0.05) 0.14 (0.04) 0.21 (0.04) 0.26 (0.04) Specificity 0.72 (0.02) 0.44 (0.02) 0.53 (0.04) 0.88 (0.04) 0.67 (0.17) 0.68 (0.04) Accuracy 0.61 (0.01) 0.49 (0.02) 0.45 (0.03) 0.7 (0.19) 0.42 (0.11) 0.45 (0.06) PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 13 / 17 Predicting acute hypotensive episodes in critical care Fig 5. Mean MAP measurements (along Y-axis) of AHE and non-AHE patients in train and test data. Each point belongs to a single patient stretched out along the X-axis. Circles: Non-AHE, Triangles: AHE, Blue: Train data, Red: Test data, Red solid line: Classification boundary learned by SVM, Greed dashed lines: Dual classification boundaries learned by our classifier. https://doi.org/10.1371/journal.pone.0193259.g005 Fig 5. Mean MAP measurements (along Y-axis) of AHE and non-AHE patients in train and test data. Each point belongs to a single patient stretched out along the X-axis. Circles: Non-AHE, Triangles: AHE, Blue: Train data, Red: Test data, Red solid line: Classification boundary learned by SVM, Greed dashed lines: Dual classification boundaries learned by our classifier. https://doi.org/10.1371/journal.pone.0193259.g005 https://doi.org/10.1371/journal.pone.0193259.g005 Design choices. Our hybrid approach utilizes a combination of margin-based and dis- tance-based classification methods. A detailed analysis of the differences between our approach and these approaches is presented in section 4. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 5.7 Discussion It would be useful to study predictive models for AHE for clinically meaningful groups based on patient characteristics, potentially leading PLOS ONE \| https://doi.org/10.1371/journal.pone.0193259 February 23, 2018 14 / 17 Predicting acute hypotensive episodes in critical care to hierarchical models. MIMIC II data is limited to a single tertiary hospital and multi-center studies are needed to further validate the findings of this study. 6 Conclusion We present a new method to identify patients at risk of Acute Hypotensive Episodes (AHE) in critical care. Our method uses a first-of-its-kind approach of using a dual–boundary classifier whose design is motivated by characteristics of the Mean Arterial Pressure (MAP) measure- ments observed in critical care patients. We perform extensive experiments using data of more than 4,500 patients from the MIMIC II database in more than 850 different experimental set- tings. Our algorithm, that can be used in both online and offline manner, is compared with the best known classifiers for AHE prediction and is shown to significantly outperform them in predictive accuracy. In particular, we achieve nearly 80% sensitivity and 95% specificity while predicting 2 hours in advance of the onset of AHE. Project administration: Vaibhav Rajan. Software: Sakyajit Bhattacharya. Supervision: Vaibhav Rajan, Chandan K. Reddy. Writing – original draft: Vaibhav Rajan. Writing – review & editing: Vaibhav Rajan, Chandan K. Reddy. Writing – review & editing: Vaibhav Rajan, Chandan K. Reddy. 5.7 Discussion Previous work in AHE prediction (described in section 2.2) have used these and other standard classifiers. Our work has focussed more on algorithmic development and lesser on feature design. Our choice of mean– based feature was primarily motivated by its performance demonstrated in previous studies [22]. An advantage of using only mean and standard deviation of all measurements, is that there is no restriction on the number of measurements or duration in which these measure- ments are taken for the observation or test windows in our classifier. This is useful since the number of measurements and length of stay varies considerably across patients. This also avoids the problems of missing values and irregular sampling of vitals within and across patient records that often needs to be addressed if other features are used. There is a growing body of literature on feature design from clinical time series (e.g. [37]) and further studies on the effectiveness of other features would be useful. Limitations and future work. Features from other clinical data have been used for AHE prediction (e.g. [30]) but a large-scale evaluation has not been done. In our study, we evaluate the performance of classification on addition of mean values of other vitals as features. We find no improvement over the performance achieved when only mean MAP values are used. This conclusion, although not surprising since we are predicting a condition of sustained low blood pressure, is limited to the feature design and classification framework used in our exper- iments. For example, we train our classifier for each vital separately and use a majority voting scheme to determine the final label, but many other approaches are possible using the DBC classifier itself to combine data from different vitals. Performance of DBC on features that combine measurements from vitals have also not been tested. The DBC classifier was designed specifically for AHE prediction. However it could be appli- cable in other contexts with similar data characteristics with respect to the vital measurements. This remains to be explored. Acute Hypotensive Episodes can be caused by a variety of clinical conditions. This study, similar to many previous works on predictive models for AHE prediction, remains oblivious to the underlying causes and patient diversity. References References 1. Moody GB, Lehman L. Predicting acute hypotensive episodes: The 10th annual physioNet/computers in cardiology challenge. In: Computers in Cardiology, 2009. IEEE; 2009. p. 541–544. 2. Heidenreich PA, Foster E, Cohen NH. Prediction of outcome for critically ill patients with unexplained hypotension. Critical care medicine. 1996; 24(11):1835–1840. https://doi.org/10.1097/00003246- 199611000-00013 PMID: 8917034 3. 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Acute cardiogenic pulmonary edema treated with mechanical ventilation: factors determining in-hospital mortality. Chest. 1991; 99(5):1220–1226. https:// doi.org/10.1378/chest.99.5.1220 PMID: 2019182 5. Huddar V, Desiraju BK, Rajan V, Bhattacharya S, Roy S, Reddy CK. Predicting Complications in Critical Care Using Heterogeneous Clinical Data. IEEE Access. 2016; 4:7988–8001. https://doi.org/10.1109/ ACCESS.2016.2618775 6. Reddy CK, Aggarwal CC. Healthcare data analytics. Chapman and Hall/CRC; 2015. 7. Bhattacharya S, Rajan V, Huddar V. A novel classification method for predicting acute hypotensive epi- sodes in critical care. In: Proceedings of the 5th ACM Conference on Bioinformatics, Computational Biology, and Health Informatics. ACM; 2014. p. 43–52. 8. Klabunde RE. Cardiovascular Physiology Concepts;. http://www.cvphysiology.com/Blood% 20Pressure/BP006.htm. Author Contributions Conceptualization: Sakyajit Bhattacharya, Vaibhav Rajan, Chandan K. Reddy. Data curation: Vijay Huddar. 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https://openalex.org/W2082486277	https://europepmc.org/articles/pmc3117794?pdf=render	English	null	Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the Differentiation Potential of Spinal Progenitors into Neuronal Cells	PloS one	2,011	cc-by	8,945	Abstract Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively. Citation: Nakamura M, Tsuji O, Bregman BS, Toyama Y, Okano H (2011) Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the Differentiation Potential of Spinal Progenitors into Neuronal Cells. PLoS ONE 6(6): e20717. doi:10.1371/journal.pone.0020717 Editor: Cesario V Borlongan, University of South Florida, United States of America Received March 9, 2011; Accepted May 8, 2011; Published June 17, 2011 Copyright: 2011 Nakamura et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by grants from Grants-in-Aid for Scientific Research from JSPS and the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), the project for realization of regenerative medicine and support for the core institutes for iPS cell research from MEXT; Research Fellowships for Young Scientists from the Japan Society for the Promotion of Science; Keio Gijuku Academic Development Funds; and by a Grant-in-aid for the Global COE program from MEXT to Keio University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: masa@sc.itc.keio.ac.jp Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the Differentiation Potential of Spinal Progenitors into Neuronal Cells Masaya Nakamura1, Osahiko Tsuji1, Barbara S. Bregman2, Yoshiaki Toyama1, Hideyuki Okano3 1 Department of Orthopaedic Surgery, Keio University, Tokyo, Japan, 2 Department of Neuroscience, Georgetown University Medical Center, Washington, D. C., United States of America, 3 Department of Physiology, Keio University, Tokyo, Japan Citation: Nakamura M, Tsuji O, Bregman BS, Toyama Y, Okano H (2011) Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the Differentiation Potential of Spinal Progenitors into Neuronal Cells. PLoS ONE 6(6): e20717. doi:10.1371/journal.pone.0020717 June 2011 \| Volume 6 \| Issue 6 \| e20717 Editor: Cesario V Borlongan, University of South Florida, United States of America Neutralization of endogenous CNTF inhibited the astrocytic differentiation of spinal progenitor cells To examine the role of endogenous CNTF in differentiation of spinal progenitor cells, we studied the effect of neutralization of endogenous CNTF on the fate of differentiating spinal progenitor cells. Different concentrations (0–500 mg/ml) of anti-CNTF antibody were applied to the differentiation medium at the time of plating. Neutralization of endogenous CNTF by polyclonal antibody (100 mg/ml and more) significantly decreased the number of astrocytes to approximately 20% of the total number of cells compared to 52% in control conditions (100 mg/ml normal rat IgG) (Fig. 4C, D). In contrast, neutralization of endogenous CNTF did not change the number of neurons or oligodendrocytes (Fig. 4A, B, E, F). There are two possible explanations for these findings. One is that neutralization of endogenous CNTF had an effect on the survival or proliferation of progenitor cells. Alternatively, neutralization of CNTF inhibited the differentiation of progenitor cells into astrocytes. Neurotrophic factor gene profile of spinal cord during normal development The development and maturation of progenitor cells depend not only on their genetic programming, but also on sequences and contributions of various environmental signals such as neuro- trophic factors that are appropriate to their developmental stages. How neurotrophic factors precisely control cell fate and orchestrate the generation of neurons, astrocytes and oligoden- drocytes in vivo is not clearly understood. To determine how neurotrophic factor gene expression is regulated in the spinal cord during normal development, we examined the neurotrophic factor gene expression profiles of developing spinal cord between E14 and P17 by RNase protection assay. A high level of NT-3 gene expression, a moderate level of CNTF and a low level of BDNF were observed in E14 spinal cord, but the bands for NGF, GDNF or NT-4 were undetectable (Fig. 1A). These neurotrophic factors showed distinct temporal patterns of their gene expression during embryonic and postnatal development. The high level of NT-3 gene expression at E14 decreased sharply by birth and subsequently there was almost no expression of NT-3 at P17 (Fig. 1D). BDNF gene expression, which was low at E14, increased between E19 and P10 (Fig. 1C). Following the increase of BDNF gene expression, NGF and GDNF gene expression increased (Fig. 1B, F). The temporal pattern of NGF and GDNF gene expression was similar to that of BDNF, but the magnitude of increase of NGF and GDNF gene expression was less than that of BDNF. Levels of NGF, BDNF and GDNF gene expression decreased simultaneously between P10 and P17. Consistent with this decrease of BDNF, GDNF and NGF gene expression, CNTF gene expression increased significantly at P17 (Fig. 1G). Neurotrophic factor gene profiles of spinal progenitor cells during differentiation Progenitor cells were isolated from E14 rat spinal cord and mechanically dissociated in culture medium. These cells expanded to form ‘‘neurospheres’’ in the presence of EGF/FGF2 and over 95% of these cells were nestin-immunoreactive (Fig. 2A, B). BrdU- labeling of these clusters confirmed that about 90% of the cells proliferated as determined by nuclear BrdU corporation (Fig. 2C). At 7 div, these neurospheres were centrifuged and dissociated mechanically. Single cells were subsequently re-seeded into EGF/ FGF2-containing medium. This procedure resulted in a second generation of neurospheres. After 2–4 passages, these cells were seeded into the PEI-coated flasks or coverslips with differentiation medium (EGF/FGF2-free and 1%FBS) following dissociation. At 1 div over 90% of these cells were still nestin immunoreactive (Fig. 2D, E) and there were also trkB and/or CNTFRa immunoreactive (Fig 2F, G). In the differentiation medium, these cells slowed or stopped dividing. At 7 div about 30% of these cells Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin In the present study, we used ribonuclease (RNase) protection assay to detect the expression of multiple neurotrophic factors genes in both developing spinal cord and embryonic spinal cord derived-progenitor cells. We used triple epitope immunocyto- chemistry to determine the phenotypic fate of progenitor cells in vitro under different treatments, including neutralization of endogenous CNTF and/or administration of exogenous BDNF or NT-3. The data indicate that endogenous CNTF and exogenous BDNF and NT-3 play crucial roles in the differentiation of E14 spinal cord derived- progenitor cells into astrocytes, neurons and oligodendrocytes, respectively. were labeled with BrdU during differentiation (Fig. 2H), and about 80% of these cells differentiated into neurons, astrocytes or oligodendrocytes (Fig. 2I, J, K ). Quantitative analysis revealed that majority of differentiated cells was astrocytes (52%), while fewer cells differentiated into oligodendrocytes (23%) and neurons (4%). This suggests that conditioning of the medium by the differenti- ating progenitor cells, especially endogenous neurotrophic factors may enhance glial cell differentiation. y g To determine how neurotrophic factor gene expression is regulated during the differentiation of E14 spinal cord derived progenitor cells, we examined the neurotrophic factor gene expression profiles of these cells by multi-probe RNase protection assay. BDNF and CNTF gene expression were observed in spinal progenitor cells before differentiation (0 div) (Fig. 3). During their differentiation BDNF gene expression declined and then became undetectable at 7 div. In contrast, CNTF gene expression increased significantly at 3 div and by 7 div reached the levels 3- fold greater than that of before differentiation (0 div). The expression of NGF, NT-3, NT-4 or GDNF mRNA was undetectable at any time point examined. Thus, E14 spinal cord derived progenitor cells showed a different pattern of neurotrophic factor gene expression during differentiation compared to the normal embryonic developing spinal cord (Fig. 1), including up- regulation of CNTF gene expression, down-regulation of BDNF gene expression and a lack of NT-3 gene expression. Interestingly, this pattern of neurotrophic factor gene expression is similar to that observed during postnatal development, and correlated temporally to active gliogenesis. Furthermore, spinal progenitor cells expressed CNTFR-immunoreactivity during differentiation. Taken together, these findings have led us to propose that CNTF acts as an autocrine and/or paracrine signal involved in the astrocytic differentiation from spinal progenitor cells. Introduction induces neuronal differentiation from embryonic striatum, subep- endymal zone and hippocampus derived progenitor cells [4,9,17,18]. Neurotrophin-3 (NT-3) induces neuronal differentia- tion from embryonic cortex and hippocampus derived progenitor cells [18,19]. Despite these extensive studies using the exogenous application of neurotrophic factors, little is known about the effect of endogenous neurotrophic factors on the differentiation of progenitor cells. We suggest that differentiation of neural progenitor cells is regulated not only by exogenous level of growth factors but rather, the endogenous expression of growth factors also play crucial roles in the differentiation of neural progenitor cells. In this study, we sought to determine the extent to which differentiation of spinal progenitor cells is altered by manipulation of endogenous as well as exogenous neurotrophic factors in vitro. We focused on the spinal progenitor cells because spinal progenitor cells may be distinct from cortical progenitor cells [20]. A better understanding of the control mechanism of spinal progenitor cell’s fate should enhance efforts to develop effective transplantation strategies aimed at restoring functional connectivity in the injured spinal cord. Neural stem/progenitor cells are an ideal source of tissue for neural transplantation, since these cells can be expanded in vitro, maintained in an undifferentiated state and retain the capacity to differentiate into neurons, astrocytes and oligodendrocytes [1,2,3]. Previous in vitro studies, however, showed that when neural progenitor cells were permitted to differentiate, they gave rise to mainly glial cells and only smaller fraction developed into neurons [4,5,6,7,8]. Although the culture systems used by various authors have differed with regard to the species, anatomic location, and developmental age of the harvested tissue, there is growing evidence to suggest that the choice of lineage is determined, at least in part, by environmental factors, among which neurotrophic factors play a potential modulating role in differentiation of progenitor cells [2,9,10]. For example, the exogenous addition of ciliary neurotrophic factor (CNTF) and subsequent gp130-signal activation promotes the differentiation of astrocytes from embry- onic hippocampus, cortex and spinal cord derived progenitor cells [11,12,13,14,15,16]. Brain-derived neurotrophic factor (BDNF) PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 June 2011 \| Volume 6 \| Issue 6 \| e20717 1 PLoS ONE \| www.plosone.org Neutralization of endogenous CNTF did not affect the survival or proliferation of spinal progenitor cells doi:10 1371/journal pone 0020717 g001 expression profile during their differentiation compared to that of the normal spinal cord during embryonic development: 1) there is a down-regulation of endogenous BDNF gene expression and 2) there is a lack of endogenous NT-3 gene expression in the spinal progenitor cells. In order to mimic the neurotrophic factor profile of spinal cord during embryonic development, we applied exogenous BDNF or NT-3 to culture cells in combination with anti-CNTF polyclonal antibody. numbers between control and neutralization groups at 7 div (Fig. 5A). Moreover, there were no significant differences in the percentages of BrdU-positive cells between control and neutral- ization groups at 7 div (Fig. 5B). In contrast, the percentage of nestin-positive cells of the neutralization group was significantly higher than that of control group at 7 div (p,0.05) (Fig. 5C). These findings suggest that neutralization of endogenous CNTF did not influence the survival or proliferation of the progenitor cells, but rather inhibited the astrocytic differentiation of these cells, resulting in the increase of the number of nestin-positive progenitor cells. The administration of exogenous BDNF alone increased the number of neurons, but did not change the number of either astrocytes or oligodendrocytes (Fig. 6A, D, G). The administration of exogenous NT-3 alone increased the number of oligodendro- cytes, but did not change the number of either neurons or astrocytes (Fig. 6A, D, G). However, the administration of exogenous BDNF or NT-3 did not have any apparent effect on the survival or proliferation of spinal progenitor cells (data not shown). In combination with anti-CNTF antibody, the adminis- tration of exogenous BDNF not only decreased the number of astrocytes (Fig. 6D, E) but also increased the number of neurons (Fig. 6A, B). There was, however, no significant change in the number of oligodendrocytes (Fig. 6G, H). In combination with anti-CNTF antibody, the administration of exogenous NT-3 not Neutralization of endogenous CNTF did not affect the survival or proliferation of spinal progenitor cells To determine the effect of neutralization of endogenous CNTF on survival and proliferation of spinal progenitor cells, we examined the number of total cells and the percentage of nestin- positive or BrdU-positive cell number to the total cell number. Since the application of 100 mg/ml anti-CNTF antibody signifi- cantly decreased the number of astrocytes as mentioned above, the subsequent experiments were performed for neutralization of endogenous CNTF using administration of 100 mg/ml anti-CNTF antibody. There was no significant difference in the total cell PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 2 Spinal Progenitors and Endogenous Neurotrophin Figure 1. Neurotrophic factor gene profile of spinal cord during development. Spinal cord tissues were harvested from embryos or neonates at each developmental age. Total RNA (10 mg) was loaded at each lane for RNase protection assay (A). There were three different temporal patterns of neurotrophic factor gene expression. NT-3 gene expression peaked at E14 and then declined sharply (D). BDNF gene expression (C), followed by NGF (B) and GDNF (F), increased between E19 and P10. Initial moderate expression of CNTF increased significantly at P10 and P17 (G). Each value represented mean 6 SEM of % GAPDH obtained from four independent experiments. GAPDH was used as an internal control to normalize for loading differences. Significant difference compared to the other developmental ages except (), # Significant difference compared to the other developmental ages including () (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g001 Figure 1. Neurotrophic factor gene profile of spinal cord during development. Spinal cord tissues were harvested from embryos or neonates at each developmental age. Total RNA (10 mg) was loaded at each lane for RNase protection assay (A). There were three different temporal patterns of neurotrophic factor gene expression. NT-3 gene expression peaked at E14 and then declined sharply (D). BDNF gene expression (C), followed by NGF (B) and GDNF (F), increased between E19 and P10. Initial moderate expression of CNTF increased significantly at P10 and P17 (G). Each value represented mean 6 SEM of % GAPDH obtained from four independent experiments. GAPDH was used as an internal control to normalize for loading differences. * Significant difference compared to the other developmental ages except (), # Significant difference compared to the other developmental ages including () (p,0.05, Tukey post hoc test). PLoS ONE \| www.plosone.org Neutralization of endogenous CNTF and administration of exogenous BDNF/NT-3 promote the differentiation into neurons/oligodendrocytes Neutralization of endogenous CNTF inhibited the differentia- tion of progenitor cells into astrocytes, but did not affect the differentiation into neurons or oligodendrocytes, resulting in the increase of the undifferentiated progenitor cells. These progenitor cells may require additional environmental cues for their differentiation into neurons or oligodendrocytes. Spinal progenitor cells show two major differences in their neurotrophic factor gene PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 June 2011 \| Volume 6 \| Issue 6 \| e20717 3 Spinal Progenitors and Endogenous Neurotrophin Figure 2. Characterization of E14 spinal cord-derived progenitor cells. Progenitor cells obtained from E14 fetal spinal cord formed sphere in the growth medium containing EGF/FGF2 (A: phase contrast image) and they were nestin (B: identical field to A) and BrdU immunoreactive (C). After dissociation, these cells were seeded on PEI-coated coverslips in no EGF/FGF2 and 1%FBS containing culture medium (D: phase contrast image). At 1div, over 90% of the dissociated progenitor cells were still nestin immunoreactive (E: identical field to D) and there were also trkB (F) and/or CNTFRa immunoreactive (G). These cells slowed or stopped dividing and during differentiation about 30% of the cells were labeled with BrdU at 7 div (H). At 7 div, these cells were triple-stained with anti-TuJ1 for neuron (I), GFAP for astrocyte (J) and O4 for oligodendrocyte (K). Approximately 50% of these cells differentiated into astrocytes. doi:10.1371/journal.pone.0020717.g002 Figure 2. Characterization of E14 spinal cord-derived progenitor cells. Progenitor cells obtained from E14 fetal spinal cord formed sphere in the growth medium containing EGF/FGF2 (A: phase contrast image) and they were nestin (B: identical field to A) and BrdU immunoreactive (C). After dissociation, these cells were seeded on PEI-coated coverslips in no EGF/FGF2 and 1%FBS containing culture medium (D: phase contrast image). At 1div, over 90% of the dissociated progenitor cells were still nestin immunoreactive (E: identical field to D) and there were also trkB (F) and/or CNTFRa immunoreactive (G). These cells slowed or stopped dividing and during differentiation about 30% of the cells were labeled with BrdU at 7 div (H). At 7 div, these cells were triple-stained with anti-TuJ1 for neuron (I), GFAP for astrocyte (J) and O4 for oligodendrocyte (K). Approximately 50% of these cells differentiated into astrocytes. doi:10.1371/journal.pone.0020717.g002 only decreased the number of astrocytes (Fig. 6D, F) but also increased the number of oligodendrocytes (Fig. 6G, I). Discussion Multipotent and self-renewing progenitor cells that generate neurons and macroglia are found in most areas of the developing nervous system [1]. Using a neurosphere culture system that allows us to follow the enriched expansion of spinal neural progenitor cells [5,21], we examined their fate when exposed to conditions that mimic the environment of late embryonic spinal cord in terms of neurotrophic factor profile: neutralization of endogenous CNTF and administration of exogenous BDNF or NT-3. Our results indicate that endogenous CNTF plays a role in differentiation of spinal progenitor cells into astrocytes. Furthermore, the additional administration of exogenous BDNF or NT-3 promotes their differentiation into neurons or oligodendrocytes, respectively. This pattern of neurotrophic factor gene expression of spinal progenitor cells in vitro is similar to that of the spinal cord during postnatal development correlated with active gliogenesis, but different from that of the spinal cord during embryonic development correlated with neurogenesis. In fact, we demonstrated the increase of NGF, BDNF and GDNF gene expression within the developmental spinal cord, which corresponds temporally with the period of differentiation and maturation of neurons [23–24], and also the simultaneous down-regulation of NGF, BDNF and GDNF gene expression and up-regulation of CNTF gene expression, which are coincident with postnatal gliogenesis [24,25]. Neutralization of endogenous CNTF and administration of exogenous BDNF/NT-3 promote the differentiation into neurons/oligodendrocytes This treatment did not affect the number of neurons (Fig. 6A, C). There was a significant increase in the number of oligodendrocytes in the cultures treated by NT-3 plus anti-CNTF antibody compared to NT-3 alone (Fig. 6G). endogenous neurotrophic factors may play a role in regulating the differentiation of spinal progenitor cells as well [9]. To the best of our knowledge, this is the first demonstration that spinal progenitor cells express a distinct profile of endogenous neurotrophic factor gene expression during their differentiation. We demon- strated that before differentiation, spinal progenitor cells expressed prominent CNTF and BDNF mRNAs in vitro. During their differentiation, endogenous BDNF gene expression significantly decreased, whereas CNTF gene expression significantly increased. Mimicking of the neurotrophic factor profile of spinal cord during embryonic development inhibits the differentiation of spinal progenitor cells into astrocytes, and promotes the differentiation into neurons and oligodendrocytes Mimicking of the neurotrophic factor profile of spinal cord during embryonic development inhibits the differentiation of spinal progenitor cells into astrocytes, and promotes the differentiation into neurons and oligodendrocytes Under baseline culture conditions approximately 50% of E14 spinal cord-derived progenitor cells differentiated into astrocytes. This differentiation of spinal progenitor cells towards an astrocytic phenotype may reflect autocrine and/or paracrine interactions between these cells. Such an autocrine mechanism of neurotrophic factor (NT-3 and BDNF) appears to regulate neurogenesis in progenitor cells isolated from the E14 mouse cortex [19,22]. Thus, Spinal progenitor cells were prepared from E14 embryos, a time when active neurogenesis occurs [23,24]. Neurotrophic factor gene expression of spinal progenitor cells in vitro differed from that of the embryonic spinal cord during normal development in vivo. The spinal progenitor cells had higher levels of CNTF, and lower PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 4 Spinal Progenitors and Endogenous Neurotrophin Figure 3. Neurotrophic factor gene profile of E14 spinal cord derived-progenitor cells during differentiation in vitro. After 2–4 passages, cells were seeded on the PEI-coated culture flasks at a density of 26105 cells/ml in the differentiation medium (no EGF/FGF2, 1% FBS). Culture cells were harvested at 1, 3 and 7 days in vitro (div). As a control cells were harvested immediately after dissociation (0 div). Total RNA (5 mg) was loaded at each lane for RNase protection assay. Before differentiation (0 div), there were two prominent gene expression of BDNF and CNTF. While BDNF gene expression decreased significantly at 3 and 7 div, CNTF gene expression increased significantly at 3 and 7 div. Each value represented mean 6 SEM of % GAPDH obtained from three independent experiments. GAPDH was used as an internal control to normalize for loading differences. * Significant difference compared to 0 div (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g003 Figure 3. Neurotrophic factor gene profile of E14 spinal cord derived-progenitor cells during differentiation in vitro. After 2–4 passages, cells were seeded on the PEI-coated culture flasks at a density of 26105 cells/ml in the differentiation medium (no EGF/FGF2, 1% FBS). Culture cells were harvested at 1, 3 and 7 days in vitro (div). As a control cells were harvested immediately after dissociation (0 div). Total RNA (5 mg) was loaded at each lane for RNase protection assay. Mimicking of the neurotrophic factor profile of spinal cord during embryonic development inhibits the differentiation of spinal progenitor cells into astrocytes, and promotes the differentiation into neurons and oligodendrocytes Before differentiation (0 div), there were two prominent gene expression of BDNF and CNTF. While BDNF gene expression decreased significantly at 3 and 7 div, CNTF gene expression increased significantly at 3 and 7 div. Each value represented mean 6 SEM of % GAPDH obtained from three independent experiments. GAPDH was used as an internal control to normalize for loading differences. * Significant difference compared to 0 div (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g003 in vivo, it is difficult to determine the function of endogenous CNTF by these knockout mice studies. Our in vitro data indicate that endogenous CNTF plays a role in inducing the differentiation of spinal progenitor cells into astrocytes, but does not effect their survival or proliferation. A possible explanation of this discrepancy between previous knockout mice studies and our in vitro study is that the redundancy of neurotrophic factors in vivo was not apparent in our cultures because these progenitor cells became more homogenous, at least morphologically, after 2-4 passages. levels of BDNF gene expression and failed to express NT-3. We suggest that these differences in the balance of neurotrophic factors environment may promote the differentiation of spinal progenitor cells in vitro into astrocytes. We hypothesize that by altering the neurotrophic factor profile that neural progenitor cells are exposed to in vitro to mimic that of the embryonic spinal cord in situ would inhibit astrocytic differentiation and promote neural differentiation of the spinal progenitor cells. First, we showed that neutralization of endogenous CNTF inhibited the differentiation of spinal progenitor cells into astrocytes, resulting in the increase of the number of undifferen- tiated progenitor cells that were nestin-positive. Neutralization of endogenous CNTF did not affect the survival or proliferation of spinal progenitor cells. Consistently, previous studies have demonstrated that administration of exogenous CNTF stimulates the two signal-transducing subunits of the CNTFRa-leukemia inhibitory factor receptor b (LIFRb) and gp130 and induces the differentiation of progenitor cells into astrocytes [11,15], and that there was a significant reduction in the numbers of neurons and astrocytes within the central nervous system of CNTFRa [26], LIFRb [27] or gp130 [14] knockout mice. In contrast to mice lacking CNTFRa, LIFRb or gp130, mice lacking CNTF developed in a normal manner and displayed only mild motor neuron problems later in adulthood without any major neurolog- ical abnormalities [28]. Mimicking of the neurotrophic factor profile of spinal cord during embryonic development inhibits the differentiation of spinal progenitor cells into astrocytes, and promotes the differentiation into neurons and oligodendrocytes Because there is a redundancy of CNTF related neurotrophic factors such as LIF, IL-6 and Oncostatin M Although CNTF was previously shown to promote survival of several kinds of neurons such as motor neurons [29,30], hippocampal neurons [31] and sensory neurons [32] and also survival and maturation of oligodendrocytes [33,34], neutraliza- tion of endogenous CNTF did not affect the number of neurons or oligodendrocytes in our cultures. Nakashima et al. demonstrated that in gp130 knockout mice there was a significant neuronal loss at E18.5, but not at E14.5[14]. Their findings suggest that signaling through gp130 is essential for survival of subgroups of differentiated neurons and that this effect depends on the developmental stage of neurons. In our short-term culture system, at least, neutralization of endogenous CNTF did not have apparent effects either on the differentiation of spinal progenitor cells into neurons or oligodendrocytes or on their survival. Further studies using long-term culture system will be needed to determine whether there are any effects of neutralization of endogenous CNTF on their long-term survival and maturation. PLoS ONE \| www.plosone June 2011 \| Volume 6 \| Issue 6 \| e20717 June 2011 \| Volume 6 \| Issue 6 \| e20717 5 PLoS ONE \| www.plosone.org Spinal Progenitors and Endogenous Neurotrophin Figure 4. Neutralization of endogenous CNTF inhibited the differentiation of spinal progenitor cells into astrocytes. After 2–4 passages, cells were seeded on PEI-coated coverslips at a density of 26105 cells/ml in the differentiation medium with different concentrations of anti-CNTF antibody (0–500 mg/ml). At 7div the culture cells were triple-stained with TuJ1 (A), GFAP (C) and O4 (E). Neutralization of endogenous CNTF by polyclonal antibody (100 mg/ml and more) significantly decreased the number of astrocytes (D), but did not affect the numbers of neurons (B) or oligodendrocytes (F). * Significant difference compared to the control (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g004 Figure 4. Neutralization of endogenous CNTF inhibited the differentiation of spinal progenitor cells into astrocytes. After 2–4 passages, cells were seeded on PEI-coated coverslips at a density of 26105 cells/ml in the differentiation medium with different concentrations of anti-CNTF antibody (0–500 mg/ml). At 7div the culture cells were triple-stained with TuJ1 (A), GFAP (C) and O4 (E). Neutralization of endogenous CNTF by polyclonal antibody (100 mg/ml and more) significantly decreased the number of astrocytes (D), but did not affect the numbers of neurons (B) or oligodendrocytes (F). Mimicking of the neurotrophic factor profile of spinal cord during embryonic development inhibits the differentiation of spinal progenitor cells into astrocytes, and promotes the differentiation into neurons and oligodendrocytes * Significant difference compared to the control (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g004 Interestingly, while neutralization of endogenous CNTF inhibited the differentiation of spinal progenitor cells into astrocytes, this led to an increase in the number of undifferentiated progenitor cells, rather than differentiation into the other cellular phenotypes. This suggests that these cells need additional environmental cues to promote the differentiation of spinal progenitor cells into neurons or oligodendrocytes. Second, we showed that administration of exogenous BDNF or NT-3 combined with neutralization of endogenous CNTF not only decreased the number of astrocytes, but also increased the number of neurons or oligodendrocytes, respectively. Previously BDNF and NT-3 were shown to promote neuronal differentiation of progenitor cells isolated from E14 striata [4], E14 cortex [19] and E16 hippocampus [18], and CNTF were shown to promote the astrocytic differentiation from NSCs in collaboration with Notch signals [35]. We also demonstrated that treatment of BDNF alone promoted the neuronal differentiation of spinal progenitor cells, but did not affect the glial differentiation. Treatments of BDNF plus anti- CNTF antibody promoted neuronal differentiation and also inhibited the astrocytic differentiation. There was no significant difference in the number of neurons between the cultures treated by BDNF plus anti-CNTF antibody and BDNF alone. Treatment of NT-3 alone promoted the differentiation of spinal progenitor cells into oligodendrocytes, but not into neurons. Treatment of NT-3 plus anti-CNTF antibody promoted the differentiation into oligodendrocytes and inhibited the astrocytic differentiation. Furthermore, there was a significant difference in the number of oligodendrocytes between the cultures treated by NT-3 plus anti- CNTF antibody and NT-3 alone. Rao and colleagues demon- strated that there are heterogeneous populations of E13.5 spinal cord derived progenitor cells including multipotent, neuronal- and glial- restricted progenitor cells [16,36]. Jean and colleagues reported that in the chemical demyelination model of rat CNS, treatment of NT-3 increased mature oligodendrocyte population and influenced directly on the oligodendrocyte lineage cells to enhance remyelination [37]. A possible explanation of our findings is that neutralization of endogenous CNTF inhibits the differen- tiation of multipotent and/or glial- restricted progenitor cells into astrocytes, and that they differentiate into oligodendrocytes in the presence of NT-3 and into neurons in the presence of BDNF. June 2011 \| Volume 6 \| Issue 6 \| e20717 Developmental study Thoracic spinal cord tissues were harvested from Sprague- Dawley rats (Zivic Laboratories, Zelienople, PA) at Embryonic Days 14 (E14) and E19 and Postnatal days 3 (P3), P7 and P17. Following perfusion with ice-cold saline, tissue was frozen on dry- ice quickly and stored at 280 uC until ribonuclease protection assay. Spinal Progenitor Cell Culture and Passage p g g Progenitor cells were cultured using the procedures of Weiss et al (1996b). Thoracic spinal cord tissues were removed from E14 embryos and mechanically dissociated with a fire-polished pipette in culture medium composed of a 1:1 mixture of DMEM and F-12 nutrient plus 50 U/ml penicillin and 50 mg/ml streptomycin (Life Technologies, Rockville, MD). After dissociation, cells were seeded in culture flasks (Nunclon) at a concentration of 26105 cells/ml with growth medium. The growth medium contained DMEM and F-12 nutrient (1:1), 50 U/ml penicillin, 50 mg/ml streptomycin, 0.6% glucose, 2 mM glutamine, 3 mM sodium bicarbonate, 5 mM HEPES buffer, 25 mg/ml insulin, 100 mg/ml transferrin (Life Technologies), 20 nM progesterone, 60 mM putrescine, 30 nM selenium chloride (Sigma, St Louis, MO), and 20 ng/ml EGF and FGF2 (Pepro Tech, Rocky Hill, NJ). After 7 days in vitro (div), cells formed well-developed floating clusters (spheres). To passage spheres, we pelleted 7 div spheres after centrifugation at 4006g for 5 min, resuspended them in fresh medium, and mechanically dissociated them with a fire-polished Pasteur pipette. Single cells were seeded into growth medium in culture flasks at a concentration of 26105 cells/ml. This procedure resulted in a second generation of spheres. Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin [41]. These endogenous stem/ progenitor cells may contribute the glial scar formation after spinal cord injury. CNTF gene expression increased significantly, but BDNF and NT-3 gene expression does not increase after spinal cord injury in the adult [42,43]. This notion is strongly supported by the previous study [44] showing that transplantation of fibroblasts producing BDNF or NT-3 into contused spinal cord promoted the differentiation of endogenous progenitor cells into oligodendrocytes. It was noteworthy that the blocking of CNTF at the beginning of SCI provides a more favorable environment for the differentiation of transplanted NSC and the regeneration of host axons, thereby promoting the functional recovery after SCI [45]. This strategy could be applied to cell transplantation of ES- and iPS-cell derived NSC for SCI. Figure 5. Effect of neutralization of endogenous CNTF on the survival and proliferation of spinal progenitor cells. Compari- sons of total cell number (A) and the percentage of BrdU (B) and nestin (C) positive cells to total cell number between control and neutraliza- tion groups (anti-CNTF antibody, 100 mg/ml). Neutralization of endog- enous CNTF did not affect the survival or proliferation of spinal progenitor cells, but increased the number of undifferentiated progenitor cells expressing nestin (C). * Significant difference between two groups (p,0.05, Mann-Whitney U-test). doi:10.1371/journal.pone.0020717.g005 Mimicking of the neurotrophic factor profile of spinal cord during embryonic development inhibits the differentiation of spinal progenitor cells into astrocytes, and promotes the differentiation into neurons and oligodendrocytes Previous studies demonstrated that there were also endogenous neural stem/progenitor cells in adult spinal cord [21,38,39], but after injury these endogenous stem/progenitor cell proliferated [40], migrated to the lesion site and differentiated into astrocytes PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 6 Treatments of Cultures After 2–4 passages, neurospheres were centrifuged and dissociated in EGF/FGF2-free medium. Cells were plated on the polyethyleneimine (PEI)-coated flasks at a density of 26105 cells/ml in EGF/FGF2-free and 1% fetal bovine serum (FBS) culture medium (differentiation medium). The culture flasks were coated by 0.1% PEI (sodium tetraborate decahyrate 0.15 M, pH 8.5) overnight at room temperature. At 1, 3 and 7 div, these cells were harvested for RNase protection assays of neurotrophic factor gene expression profiles. As a control, cells were also harvested before differentiation following dissociation (0 div). After 2–4 passages, neurospheres were centrifuged and dissociated in EGF/FGF2-free medium. Cells were plated on the polyethyleneimine (PEI)-coated flasks at a density of 26105 cells/ml in EGF/FGF2-free and 1% fetal bovine serum (FBS) culture medium (differentiation medium). The culture flasks were coated by 0.1% PEI (sodium tetraborate decahyrate 0.15 M, pH 8.5) overnight at room temperature. At 1, 3 and 7 div, these cells were harvested for RNase protection assays of neurotrophic factor gene expression profiles. As a control, cells were also harvested before differentiation following dissociation (0 div). For immunocytochemistry, after 2–4 passages neurospheres were centrifuged and dissociated in EGF/FGF2-free medium. Cells were plated onto PEI-coated 12 mm coverslips (Fisher Scientific, Pittsburgh, PA) in 24-well plates at a density of 26105 cells/ml with 1 ml differentiation medium. The role of Figure 5. Effect of neutralization of endogenous CNTF on the survival and proliferation of spinal progenitor cells. Compari- sons of total cell number (A) and the percentage of BrdU (B) and nestin (C) positive cells to total cell number between control and neutraliza- tion groups (anti-CNTF antibody, 100 mg/ml). Neutralization of endog- enous CNTF did not affect the survival or proliferation of spinal progenitor cells, but increased the number of undifferentiated progenitor cells expressing nestin (C). * Significant difference between two groups (p,0.05, Mann-Whitney U-test). doi:10.1371/journal.pone.0020717.g005 For immunocytochemistry, after 2–4 passages neurospheres were centrifuged and dissociated in EGF/FGF2-free medium. Cells were plated onto PEI-coated 12 mm coverslips (Fisher Scientific, Pittsburgh, PA) in 24-well plates at a density of 26105 cells/ml with 1 ml differentiation medium. The role of June 2011 \| Volume 6 \| Issue 6 \| e20717 June 2011 \| Volume 6 \| Issue 6 \| e20717 7 PLoS ONE \| www.plosone.org Spinal Progenitors and Endogenous Neurotrophin Figure 6. Effects of additional application of exogenous neurotrophic factors on the differentiation of spinal progenitor cells. Treatments of Cultures Treatments of cultures were as follows in each bar graph: (1) control (normal rat IgG 100 mg/ml); (2) BDNF (20 ng/ml); (3) NT-3 (20 ng/ml); (4) anti- CNTF (100 mg/ml); (5) anti-CNTF +BDNF; (6) anti-CNTF + NT-3. At 7 div after each treatment, cells were triple-stained with TuJ1 for neuron, GFAP for astrocyte and O4 for oligodendrocyte. Combined treatments of anti-CNTF plus exogenous BDNF (B, E, H) or NT-3 (C, F, I) not only inhibited the astrocytic differentiation (lanes 5 and 6 in D, E, F), but also promoted the differentiation of spinal progenitor cells into neurons (lane 5 in A, B) or oligodendrocytes (lane 6 in G, I), respectively. The application of exogenous BDNF alone (lane 2 in A) or NT-3 alone (lane 3 in G) also increased the numbers of neuron or oligodendrocytes, respectively. However, the differentiation of progenitor cells into astrocytes was not inhibited by these treatments (lanes 2 and 3 in D). Compared to the cultures treated by NT-3 alone (lane 3 in G), there was a significant increase in the number of oligodendrocytes in the cultures treated by anti-CNTF plus NT-3 (lane 6 in G). * Significant difference compared to the control (lane 1 in A, D, G) (p,0.05, Tukey post hoc test). # Significant difference compared to NT-3 alone (lane 3 in G) (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g006 Figure 6. Effects of additional application of exogenous neurotrophic factors on the differentiation of spinal progenitor cells. Treatments of cultures were as follows in each bar graph: (1) control (normal rat IgG 100 mg/ml); (2) BDNF (20 ng/ml); (3) NT-3 (20 ng/ml); (4) anti- CNTF (100 mg/ml); (5) anti-CNTF +BDNF; (6) anti-CNTF + NT-3. At 7 div after each treatment, cells were triple-stained with TuJ1 for neuron, GFAP for astrocyte and O4 for oligodendrocyte. Combined treatments of anti-CNTF plus exogenous BDNF (B, E, H) or NT-3 (C, F, I) not only inhibited the astrocytic differentiation (lanes 5 and 6 in D, E, F), but also promoted the differentiation of spinal progenitor cells into neurons (lane 5 in A, B) or oligodendrocytes (lane 6 in G, I), respectively. The application of exogenous BDNF alone (lane 2 in A) or NT-3 alone (lane 3 in G) also increased the numbers of neuron or oligodendrocytes, respectively. However, the differentiation of progenitor cells into astrocytes was not inhibited by these treatments (lanes 2 and 3 in D). Treatments of Cultures Rat anti-CNTF antibody (Sigma) was applied to the differentiation medium at various concentrations (0– 500 mg/ml) at the time of plating. In the control group, we added normal rat IgG (100 mg/ml, Sigma) to the differentiation medium. For analysis of the potential effects of exogenous BDNF or NT-3 on the differentiation of spinal progenitor cells, BDNF or NT-3 (20 ng/ml each, gift from Regeneron Pharmaceuticals, Inc.) was added to the medium at the time of plating and added every 2–3 days. At 7 div, these cultured cells were processed for immunocytochemistry. Treatments of Cultures Compared to the cultures treated by NT-3 alone (lane 3 in G), there was a significant increase in the number of oligodendrocytes in the cultures treated by anti-CNTF plus NT-3 (lane 6 in G). * Significant difference compared to the control (lane 1 in A, D, G) (p,0.05, Tukey post hoc test). # Significant difference compared to NT-3 alone (lane 3 in G) (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g006 gen, San Diego, CA). Briefly, a rat neurotrophin template set (45028P, PharMingen) was labeled with (a-32P) uridine triphos- phate. 4.66105 cpm of labeled probe and 5 mg total RNA were used for hybridization. After RNase treatments, the protected probes were resolved on a 5% urea-polyacrylamide gel. Autora- diographs were produced by exposing the labeled gels to Biomax film (Kodak, Rochester, NY) with intensifying screens at 280uC. The gels were exposed to a phosphor screen and analyzed using a phosphoimaging system for quantification (Molecular Dynamics, Sunnyvale, CA). GAPDH was used as an internal control to normalize for loading differences. gen, San Diego, CA). Briefly, a rat neurotrophin template set (45028P, PharMingen) was labeled with (a-32P) uridine triphos- phate. 4.66105 cpm of labeled probe and 5 mg total RNA were used for hybridization. After RNase treatments, the protected probes were resolved on a 5% urea-polyacrylamide gel. Autora- diographs were produced by exposing the labeled gels to Biomax film (Kodak, Rochester, NY) with intensifying screens at 280uC. The gels were exposed to a phosphor screen and analyzed using a phosphoimaging system for quantification (Molecular Dynamics, Sunnyvale, CA). GAPDH was used as an internal control to normalize for loading differences. CNTF in differentiation of spinal progenitor cells was investigated by neutralizing the endogenous CNTF with polyclonal antibody against CNTF IgG. Rat anti-CNTF antibody (Sigma) was applied to the differentiation medium at various concentrations (0– 500 mg/ml) at the time of plating. In the control group, we added normal rat IgG (100 mg/ml, Sigma) to the differentiation medium. For analysis of the potential effects of exogenous BDNF or NT-3 on the differentiation of spinal progenitor cells, BDNF or NT-3 (20 ng/ml each, gift from Regeneron Pharmaceuticals, Inc.) was added to the medium at the time of plating and added every 2–3 days. At 7 div, these cultured cells were processed for immunocytochemistry. CNTF in differentiation of spinal progenitor cells was investigated by neutralizing the endogenous CNTF with polyclonal antibody against CNTF IgG. PLoS ONE \| www.plosone.org References Svendsen C, Fawcett J, Bentlage C, Dunnett S (1995) Increased survival of rat EGF-generated CNS precursor cells using B27 supplemented medium. Exp Brain Res 102: 407–414. 21. Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, et al. (1996) Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16: 7599–7609. 8. Von VJ, Yeon D, Oh T, Markelonis G (1994) Differentiation and maturation of astrocytes derived from neuroepithelial progenitor cells in culture. Exp Neurol 128: 34–40. 22. Barnabe-Heider F, Miller FD (2003) Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J Neurosci 23: 5149–5160. 23. Nornes H, Das G (1974) Temporal pattern of neurogenesis in spinal cord of rat. I. An autoradiographic study--time and sites of origin and migration and settling patterns of neuroblasts. Brain Res 73: 121–138. 9. Islam O, Loo TX, Heese K (2009) Brain-derived neurotrophic factor (BDNF) has proliferative effects on neural stem cells through the truncated TRK-B receptor, MAP kinase, AKT, and STAT-3 signaling pathways. Curr Neurovasc Res 6: 42–53. p 24. Frederiksen K, McKay R (1988) Proliferation and differentiation of rat neuroepithelial precursor cells in vivo. J Neurosci 8: 1144–1151. 10. Lillien L (1998) Neural progenitors and stem cells: mechanisms of progenitor heterogeneity. Curr Opin Neurobiol 8: 37–44. 25. Ling E (1976) Study in the changes of the proportions and numbers of the various glial cell types in the spinal cord of neonatal and young adult rats. Acta Anat 96: 188–195. 11. Bonni A, Sun Y, Nadal-Vicens M, Bhatt A, Frank D, et al. (1997) Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway. Science 278: 477–483. 26. 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Kalyani A, Hobson K, Rao M (1997) Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterization, and clonal analysis. Dev Biol 186: 202–223. 7. Statistics Each value of RNase protection assays represented mean 6 SEM of percentage of each neurotrophic factor mRNA expression to GAPDH obtained from three independent experiments. Statistical comparisons were made using one-way ANOVA with a Tukey post hoc test on each neurotrophic factor among different time points. Each value of quantitative analysis of the immuno- cytochemistry represented mean 6 SEM of percentage of immunopositive cell number to total cell number obtained from three independent experiments. Statistical comparisons were made using one-way ANOVA with a Tukey post hoc test or Mann- Whitney U-test among different treatment groups. Statistical significance was set at p,0.05. Immunocytochemistry Immunocytochemical procedures were performed on spinal progenitor cells at 7 div using a slight modification of the procedures of Weiss et al (1996b). In brief, these cells were fixed with 4% paraformaldehyde for 20 min and washed three times successively with phosphate buffered saline (PBS, pH 7.4). For triple labeling of TuJ1, GFAP and O4, cells were incubated with the primary antibodies for TuJ1 and GFAP diluted in PBS containing 5% normal goat serum and 0.01% Triton-X100 at the same time at 4uC overnight. After washing, cells were incubated with the secondary antibodies of TRITC-conjugated anti-mouse IgG and Cy5-conjugated anti- rabbit IgG at 37uC for 30 min. After washing, cells were incubated with the primary antibody for O4 diluted in PBS containing 5% normal goat serum and 0.01% Triton-X100 at 37uC for 2 hr. After washing, cells were incubated with the secondary antibody of FITC-conjugated anti-mouse IgM at 37uC for 30 min. After washing, the cells were mounted with Fluorsave (Calbiochem, La Jolla, CA). The numbers of immuno-reactive cells and total cells per each field were counted over 206 fields (four coverslips, six random fields per each coverslip) after capturing each image by a confocal laser-scanning microscope (Fluoview, Olympus, Melville, NY). Each immunoreactive cell was counted only when the nucleus was detectable on the 206 field investigated. Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin CA). Appropriate secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). cells was performed as described previously by Ahmed et al. (1995). BrdU (1 mM; Sigma) was added to each of the wells containing progenitor cells (26105 cells/ml) in 1 ml differentiation medium with or without anti-CNTF antibody (100 mg/ml) at the time of plating for 7 div. The presence of BrdU did not modify total cell number. Cells were stained with ZYMED BrdU staining kit (Zymed, South San Francisco, CA) and counter stained with hematoxylin according to the supplier’s instructions. The percent- ages of BrdU positive cells to total cells of the examined field (four coverslips, six random fields per each coverslip) were calculated. Proliferation Assay To determine the effect of neutralization of endogenous CNTF on the proliferation of progenitor cells, BrdU labeling of dividing Author Contributions Conducted the cell cultures and most of the in vitro experiments: MN. Performed immunocytochemical analysis: OT. Provided instruction and supervised all the experiments: BSB YT. Supervised the whole project: HO. Wrote the manuscript HO MN. RNase protection assay Primary antibodies for indirect immunocytochemistry included a mouse monoclonal antibody to TuJ1 (BAbCO, West Grove, PA), rabbit antiserum to glial fibrillary acidic protein (GFAP, Dako, Carpinteria, CA), mouse-monoclonal antibody to O4 (Boeringer Mannheim, Indianapolis, IL), mouse anti-nestin monoclonal antibody (Chemicon, Temecula, CA), antibodies for trkB and CNTF receptor a (CNTFRa, Santa Cruz, Santa Cruz, Total RNA from each sample was extracted using TRIzol Reagent (Life Technologies) according to the supplier’s instruc- tions. RNase protection assays for relative quantification of neurotrophic factor mRNAs [nerve growth factor (NGF), BDNF, NT-3, NT-4, glial-derived neurotrophic factor (GDNF), CNTF] were performed according to the supplier’s instructions (PharMin- PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 8 Spinal Progenitors and Endogenous Neurotrophin comparison with other neurotrophic factors and cytokines. J Neurosci 10: 3507–3515. 38. Horner P, Power A, Kempermann G, Kuhn H, Palmer T, et al. (2000) Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord. J Neurosci 20: 2218–2228. 30. Martinou J, Martinou I, Kato A (1992) Cholinergic differentiation factor (CDF/ LIF) promotes survival of isolated rat embryonic motoneurons in vitro. Neuron 8: 737–744. p J 39. Johansson C, Momma S, Clarke D, Risling M, Lendahl U, et al. (1999) Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96: 25–34. 31. Ip N, Li Y, van dSI, Panayotatos N, Alderson R, et al. (1991) Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures. J Neurosci 11: 3124–3134. 40. Namiki J, Tator C (1999) Cell proliferation and nestin expression in the ependyma of the adult rat spinal cord after injury. J Neuropathol Exp Neurol 58: 489–498. 32. Thaler C, Suhr L, Ip N, Katz D (1994) Leukemia inhibitory factor and neurotrophins support overlapping populations of rat nodose sensory neurons in culture. Dev Biol 161: 338–344. 41. Frisen J, Johansson C, Torok C, Risling M, Lendahl U (1995) Rapid, widespread, and longlasting induction of nestin contributes to the generation of glial scar tissue after CNS injury. J Cell Biol 131: 453–464. 33. Louis J, Magal E, Takayama S, Varon S (1993) CNTF protection of oligodendrocytes against natural and tumor necrosis factor-induced death. Science 259: 689–692. 42. Nakamura M, Bregman BS (2001) Differences in neurotrophic factor gene expression profiles between neonate and adult rat spinal cord after injury. Exp Neurol 169: 407–415. 34. Mayer M, Bhakoo K, Noble M (1994) Ciliary neurotrophic factor and leukemia inhibitory factor promote the generation, maturation and survival of oligodendrocytes in vitro. Development 120: 143–153. 43. Oyesiku N, Wilcox J, Wigston D (1997) Changes in expression of ciliary neurotrophic factor (CNTF) and CNTF-receptor alpha after spinal cord injury. J Neurobiol 32: 251–261. 35. Nagao M, Sugimori M, Nakafuku M (2007) Cross talk between notch and growth factor/cytokine signaling pathways in neural stem cells. Mol Cell Biol 27: 3982–3994. J 44. McTigue D, Horner P, Stokes B, Gage F (1998) Neurotrophin-3 and brain- derived neurotrophic factor induce oligodendrocyte proliferation and myelina- tion of regenerating axons in the contused adult rat spinal cord. J Neurosci 18: 5354–5365. 36. References (1997) Embryonic precursor cells that express Trk receptors: induction of different cell fates by NGF, BDNF, NT-3, and CNTF. Exp Neurol 144: 350–360. 28. Masu Y, Wolf E, Holtmann B, Sendtner M, Brem G, et al. (1993) Disruption of the CNTF gene results in motor neuron degeneration. Nature 365: 27–32. 14. Nakashima K, Wiese S, Yanagisawa M, Arakawa H, Kimura N, et al. (1999) Developmental requirement of gp130 signaling in neuronal survival and astrocyte differentiation. J Neurosci 19: 5429–5434. 29. Arakawa Y, Sendtner M, Thoenen H (1990) Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 9 June 2011 \| Volume 6 \| Issue 6 \| e20717 Spinal Progenitors and Endogenous Neurotrophin Mayer-Proschel M, Kalyani A, Mujtaba T, Rao M (1997) Isolation of lineage- restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron 19: 773–785. 45. Ishii K, Nakamura M, Dai H, Finn TP, Okano H, et al. (2006) Neutralization of ciliary neurotrophic factor reduces astrocyte production from transplanted neural stem cells and promotes regeneration of corticospinal tract fibers in spinal cord injury. J Neurosci Res 84: 1669–1681. 37. Jean I, Lavialle C, Barthelaix-Pouplard A, Fressinaud C (2003) Neurotrophin-3 specifically increases mature oligodendrocyte population and enhances remye- lination after chemical demyelination of adult rat CNS. Brain Res 972: 110–118. PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e20717 10
https://openalex.org/W2086662001	https://europepmc.org/articles/pmc2515348?pdf=render	English	null	Evolutionary and Transmission Dynamics of Reassortant H5N1 Influenza Virus in Indonesia	PLOS pathogens	2,008	cc-by	13,248	Introduction transmission among family members were identified in 2005, raising concerns of possible human-to-human transmission of the virus [11,12]. As of April 8, 2008, Indonesia had 132 confirmed human cases with 107 deaths [13], the largest number of deaths among all affected countries. The H5N1 highly pathogenic avian influenza (HPAI) virus was originally isolated from a farmed goose in Guangdong province of China in 1996 [1], and soon spread to live-poultry markets in Hong Kong [2], resulting in 18 cases of human infection in 1997, 6 of which were fatal [3,4]. The first wave of H5N1 infection ceased after the depopulation of all poultry in Hong Kong, although the H5N1 virus was later found to circulate continuously in Southern China without causing apparent disease symptoms among infected poultry [5]. H5N1 outbreaks recurred in 2003, persistently affecting poultry farms in many Southeast Asia countries, such as China, Thailand, Vietnam, Indonesia and Cambodia. The viruses also spread outside Asia, including to some European countries. More importantly, occasional zoonotic transmissions to humans occurred in most of the affected Asian countries and the virus continued to pose a serious threat to global public health [6]. Previous studies have shown that several H5N1 genotypes have emerged in Asia through reassortment between H5N1 viruses and other subtypes [14,15]. One of these genotypes, Z, predominated the H5N1 outbreaks throughout 2003–2007, causing most H5N1 outbreaks in Asian countries, including Indonesia [16]. Moreover, a variety of antigenically distinct sublineages of Z genotype virus have been established [16]. Unlike Vietnam and Thailand, Indonesia was invaded by only a single sublineage of genotype Z virus. Previous phylogenetic analyses suggested that Hunan province of China may be the source of the initial H5N1 outbreak in Indonesia [17], and classified the Indonesian H5N1 HPAI viruses into three groups [10]; however, further statistical analysis is necessary to characterize and compare different aspects of their evolutionary histories. In this study, we examined molecular phylogeny of the most recent Indonesian H5N1 viruses isolated from avian and mammal hosts. A group of putative reassortant viruses was discovered and their genetic parents were identified. In addition, we investigated the evolutionary behaviors (including spatial migration, growth of genetic diversity, and evolutionary H5N1 outbreaks in Indonesia were initially detected in poultry farms in December 2003 [7]. It was suggested that the H5N1 virus was first introduced to Java and subsequently spread to other parts of the country [8]. Abstract H5N1 highly pathogenic avian influenza (HPAI) viruses have seriously affected the Asian poultry industry since their recurrence in 2003. The viruses pose a threat of emergence of a global pandemic influenza through point mutation or reassortment leading to a strain that can effectively transmit among humans. In this study, we present phylogenetic evidences for the interlineage reassortment among H5N1 HPAI viruses isolated from humans, cats, and birds in Indonesia, and identify the potential genetic parents of the reassorted genome segments. Parsimony analyses of viral phylogeography suggest that the reassortant viruses may have originated from greater Jakarta and surroundings, and subsequently spread to other regions in the West Java province. In addition, Bayesian methods were used to elucidate the genetic diversity dynamics of the reassortant strain and one of its genetic parents, which revealed a more rapid initial growth of genetic diversity in the reassortant viruses relative to their genetic parent. These results demonstrate that interlineage exchange of genetic information may play a pivotal role in determining viral genetic diversity in a focal population. Moreover, our study also revealed significantly stronger diversifying selection on the M1 and PB2 genes in the lineages preceding and subsequent to the emergence of the reassortant viruses, respectively. We discuss how the corresponding mutations might drive the adaptation and onward transmission of the newly formed reassortant viruses. Editor: Edward C. Holmes, The Pennsylvania State University, United States of America Received February 25, 2008; Accepted July 18, 2008; Published August 22, 2008 Copyright: 2008 Lam et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was partly funded and supported by research grant RFCID 06060672 from the Hong Kong Government and The University of Hong Kong Faculty of Science Research Development Fund to FCCL. OGP is supported by the Royal Society. This research was supported in part by the National Institutes of Health (AI43638, AI47745, and AI57167), the University of California Universitywide AIDS Research Program (grant number IS02-SD-701), and by a University of California-San Diego Center for AIDS Research/NIAID Developmental Award to SLKP (AI36214). Competing Interests: The authors have declared that no competing interests exist. * E-mail: fcleung@hkucc.hku.hk Tommy Tsan-Yuk Lam1, Chung-Chau Hon1, Oliver G. Pybus2, Sergei L. Kosakovsky Pond3, Raymond Tze- Yeung Wong1, Chi-Wai Yip1, Fanya Zeng1, Frederick Chi-Ching Leung1* Yeung Wong1, Chi-Wai Yip1, Fanya Zeng1, Frederick Chi-Ching Leung1* 1 School of Biological Sciences, The University of Hong Kong, Hong Kong Special Administrative Region, China, 2 Department of Zoology, University of Oxford, Oxford, United Kingdom, 3 Department of Pathology, University of California San Diego, La Jolla, California, United States of America 1 School of Biological Sciences, The University of Hong Kong, Hong Kong Special Administrative Region, China, 2 Department of Zoology, University of Oxford, Oxford, United Kingdom, 3 Department of Pathology, University of California San Diego, La Jolla, California, United States of America PLoS Pathogens \| www.plospathogens.org Evolutionary and Transmission Dynamics of Reassortant H5N1 Influenza Virus in Indonesia Tommy Tsan-Yuk Lam1, Chung-Chau Hon1, Oliver G. Pybus2, Sergei L. Kosakovsky Pond3, Raymond Tze- Yeung Wong1, Chi-Wai Yip1, Fanya Zeng1, Frederick Chi-Ching Leung1* Phylogenetic relationships among Indonesian H5N1 viruses Phylogenetic trees of Indonesian H5N1 viruses were reconstruct- ed from 12 separate gene datasets (Table S6), using a maximum likelihood (ML) approach with bootstrapping analyses to assess clade robustness (Figures 1, S1–S3; computer files of dendrogram are available as Dataset S1). In all the phylogenies, viruses sampled from avian species during earlier years of outbreaks (predominantly 2003– 2004) tended to cluster near the root as expected, but with a poorly resolved branching structure that is likely due to relatively low sequence divergence. In contrast, viruses sampled from recent infections (2005–2007) from avian and mammalian hosts formed three well-supported lineages with bootstrap support (or posterior probabilities) over 90 (or 0.9) under neighbor-joining (NJ), ML and Bayesian Markov Chain Monte Carlo (BMCMC) methods. We denote these lineages as groups 1, 2, and 3 in the hemagglutinin (HA) and neuraminidase (NA) phylogenies (Figures 1A and S2C). This structure was preserved in the phylogenies of other genes for which sufficient sequence data were available (viruses from group 3 were missing sequence data for the NP, NS, NS1, NS2, and PB2 genes). The group 3 lineage in the MP, M1, M2, and PB1 phylogenies was only represented by the A/Indonesia/6/05 strain. Author Summary H5N1 highly pathogenic avian influenza (HPAI) virus emerged in China in 1996, and has spread beyond Asia since 2003. Following the first outbreak reported in Indonesian poultry farms in December 2003, the virus spilled over to 27 Indonesian provinces by June 2006, and became endemic in the country. In the following years, repeated sporadic human infections in Indonesia had been attributed to H5N1 HPAI viruses. Nonetheless, the viral evolution and transmission have not been fully under- stood. Here, we report phylogenetic evidence of a group of interlineage reassortant viruses isolated from human and cats in Java. Our comparative study of the reassortant viruses and one group of genetic parents found that although their rates of evolution were similar and both of their phylogenies were not geographically structured within mainland Java, the growths of genetic diversity were different. We also detected significant positive selection on the viral matrix and polymerase genes preceding and subsequent to the emergence of the reassortant viruses, which might correspond to viral adaptation. Based on our findings, we discuss the possibility of host switching in facilitating the emergence of the reassortant strain, and call for more extensive viral surveillances in the non-avian population in Indonesia. Identification of reassortant viruses We found previously unrecognized phylogenetic discordance between gene trees involving human and cat isolates (n = 25, denoted in red in Figures 1, S1–S3)—the main focus of our study—suggesting that they are reassortant viruses descending from group 2 and 3 lineages. In addition, the placement of two avian viruses isolates from Java (Ck/IDN/Semerang1631-62/07 and Ck/IDN/Magelang1631-57/07, shown in blue in Figures 1A and S2C) differed between HA and NA phylogenies, suggesting another reassortment event. To further investigate the putative reassortant human and cat viruses, a selected dataset (n = 24) of manually concatenated full genomes (Figure 2A; see Methods) of Indonesian H5N1 HPAI viruses were analyzed using more sophisticated analysis methods, including similarity plots, bootscan analyses and GARD analyses (genetic algorithm for recombination detection). In the similarity and bootscan plots (Figure 2B and 2C), the putative reassortants (represented by a consensus sequence) showed a high degree of sequence similarity and phylogenetic clustering with the group 3 strain A/Indonesia/6/05 in the MP and PB1 segments, but not in other genomic regions, where they were more similar to the consensus sequence of group 2 viruses (Figure 2B and 2C). Moreover, GARD detected two well-supported breakpoints near the boundaries of MP and PB1 segments in the concatenated genomes (Figure 2D), suggesting that the phylogenetic incongru- ence was significant between the three regions. In summary, all three analyses agreed that the newly reassortant strains had arisen from acquiring PB1 and MP genome segments from the group 3 lineage and the remaining segments from the group 2 lineage. drift and selection) of the reassortant viruses and compare with those of the parental strain, thereby providing insights into the nature and impact of this emerging reassortant strain. PLoS Pathogens \| www.plospathogens.org Introduction The virus rapidly became endemic in Indonesia [9,10], and continued to cause sporadic zoonotic transmissions to humans beginning in July 2005 [9]. Three clusters of H5N1 1 August 2008 \| Volume 4 \| Issue 8 \| e1000130 Indonesian Reassortant H5N1 Influenza Virus group B taxa are earlier viruses. Groups 1 and 2 in this study correspond to groups C and A defined by Smith et al. respectively, plus some more recent viruses. Smith did not report group 3, because the sequences were unavailable at that time. Author Summary Genetic, temporal, and geographical origin of reassortant viruses Based on the HA phylogeny (Figure 1A), we further classified the reassortant viruses into three subgroups (R1, R2, and R3) with bootstrap support of 80% or better, as shown in the phylogenies containing only reassortant viruses (Figure S4). Similar groupings were observed in the NA phylogeny (Figures S2C and S4B), although here subgroup R3 clustered with subgroup R1, and two reassortant viruses isolated in 2007 (IDN/CDC1046/07, IDN/ CDC1047/07) moved to a different subgroup. These inferred clustering patterns can be explained by multiple reassortment events, or by a single reassortment followed by divergence due to mutation and selection in different populations. We note that some group 2 viruses also cluster inside the reassortant subgroups (Figures 1A and S2C) and may indicate more reassortment events; however, most of them formed polytomies close to the most recent common ancestor (MRCA) of the reassortant subgroups and had poor bootstrap support for their exact placement. As the divergence between the reassortant subgroups and other intercalating group 2 viruses are low, the three subgroups may actually be linked uninterruptedly, implying a single origin. Therefore, the times and number of reassortment events that generated the putative mosaic reassortant viruses remains elusive. We examine both the single and multiple origin hypotheses in subsequent analyses, excluding the intercalating group 2 viruses from the reassortant group. It is important to recognize that our phylogenetic groupings (groups 1, 2, and 3) of Indonesian H5N1 viruses (Figure 1 and Table S6) are slightly different to those by Smith and coworkers [10] who did not require the same level of clustering support for each group, leading to the inclusion of earlier viruses (predomi- nantly 2003–2004). We chose to be conservative, and did not include poorly supported branches (e.g., earlier viruses) in our viral group definition. Therefore, we did not define a group corresponding to the group B of Smith et al., because most of To estimate the times of the reassortment events that generated the putative reassortant viruses, the times of the MRCA (tMRCA) of the three reassortant subgroups were estimated using BMCMC methods [18,19]. Genetic, temporal, and geographical origin of reassortant viruses Phylogenetic trees of Indonesian H5N1 influenza viruses. ML phylogenies reconstructed from (A) HA gene; (B) M1 gene; (C) M2 gene. Topological supports (.90) summarized from 1,000 ML bootstrap replications are shown. For major lineages, NJ bootstrap support (1,000 replications) and posterior probability from BMCMC analyses (5,000 tree samples) are also shown inside parentheses (ML/NJ/BMCMC). Putative human and cat reassortant viruses are in red. Reassortant subgroups (R1, R2, and R3) are further indicated with dashed lines in (A). Putative avian reassortant viruses are in blue. The pre-emergence lineage (refer to main text) is highlighted in gray in M1 phylogeny (B). Arrows indicate the roots. The distance unit is substitutions/site. doi:10.1371/journal.ppat.1000130.g001 The results of tMRCA estimation are summarized in Figure 3C and 3D. In addition, sequence isolation dates were plotted against their genetic distance (units of substitutions/site) to their MRCA, to graphically show the accumulation of mutations through time (Figure 3A and 3B). The tMRCA of all reassortant viruses (All- among strict and relaxed clock models of evolution [21]. The uncorrelated exponentially-distributed clock model (UCED) significantly outperformed the other models (lnBF.3) for most datasets, except for the NA gene of the reassortant viruses, for which the strict clock model was not rejected (lnBF,1; Table S4). Genetic, temporal, and geographical origin of reassortant viruses Bayes Factors (BF) [20] were used to select August 2008 \| Volume 4 \| Issue 8 \| e1000130 August 2008 \| Volume 4 \| Issue 8 \| e1000130 2 Indonesian Reassortant H5N1 Influenza Virus R2 R3 Group 3 Group 1 R1 Group 2 A (HA) IDN/CDC940/06 IDN/CDC759/06 IDN/CDC669/06 IDN/CDC836/06 IDN/CDC739/06 IDN/CDC835/06 IDN/CDC610/06 Ck/IDN/Bandung1631 49/06 Muscovy Dk/Jakarta/HABWIN/06 Ck/IDN/Garut1631 51/06 Qa/Jakarta/JU1/06 Ck/West Java/TASIKSOL/06 IDN/CDC644/06 IDN/CDC1046/07 IDN/CDC1047/07 IDN/CDC1032/07 IDN/CDC938/06 IDN/CDC887/06 IDN/CDC1031/07 IDN/CDC523/06 IDN/CDC699/06 IDN/CDC634/06 IDN/CDC582/06 Ck/IDN/Lampung1631 23/06 Ck/IDN/Semerang1631 62/07 Sn/IDN/Malang1631 61/07 IDN/CDC370/06 IDN/CDC390/06 Ck/West Java/SMI ENDRI1/06 Ck/West Java/SMI ENDRI2/06 IDN/298H/06 Pg/IDN/Rokhit1631 6/06 Ck/IDN/Padang1631 1/06 Ck/IDN/Siak1631 2/06 Ck/IDN/Agam1631 3/06 Ck/IDN/Pekenbaru1631 11/06 IDN/CDC624/06 Ck/IDN/Gunung Kidul1631 33/06 IDN/CDC623/06 MDk/IDN/Kedri1631 24/06 Ck/West Java/SMI CSLK EB/06 Ck/West Java/SMI CSLK EC/06 Ck/West Java/SMI PAT/06 Ck/West Java/PWT WIJ/06 IDN/CDC292T/05 IDN/CDC742/06 IDN/CDC287E/05 Ck/IDN/Belitung Timor1631 18/06 IDN/CDC357/06 Ck/West Java/TASIKSOB/06 IDN/195H/05 Ck/IDN/Rejang Lebong1631 22/06 IDN/5/05 Ck/Pakun Baru/BPPV II/05 Ck/Murao Jambi/BBPV II/05 Dk/Indramayu/BBPW109/06 IDN/CDC194P/05 IDN/CDC184/05 IDN/CDC7/05 Ck/IDN/Wates1/05 Ck/Magetan/BBVW/05 Ck/IDN/Wates126/05 Ck/Gunung Kidal/BBVW/05 Ck/IDN/Wates130/05 Ck/Papua/TB1/06 Ck/Papua/TB15/06 Ck/Papua/TA5/06 Dk/Parepare/BBVM/05 Ck/Wajo/BBVM/05 Ck/IDN/CDC25/05 Ck/IDN/CDC24/05 Ck/Way Kanan/BBPVIII/06 Ck/Bandar Lampung/BBPVIII/06 Ck/Sembawa/BPPV III/05 Ck/Palembang/BPPV III/05 Ck/IDN/Bangka Seletan1631 21/06 Ck/IDN/Bangka Seletan1631 20/06 Ck/Purworejo/BBVW/05 Ck/Yogjakarta/BBVet IX/04 Qa/Yogjakarta/BBVet IX/04 Qa/Boyolali/BPPV4/04 Ck/IDN/Wates80/05 Ck/Kupang 1 NTT/BPPV6/04 Ck/Kulon Progo/BBVet XII 1/04 Ck/Kulon Progo/BBVet XII 2/04 Ck/IDN/Soppeng1631 71/07 Ck/West Java/GARUT MAY/06 Qa/Central Java/SMRG/06 Ck/IDN/Magelang1631 57/07 Ck/IDN/Kulon1631 47/06 Ck/Gunung Kidul/BBVW/06 IDN/6/05 Ck/Madiun/BBVW1420/05 Tk/Kedaton/BPPV3/04 Ck/Pangkalpinang/BPPV3/04 Ck/IDN/Wates83/05 Ck/IDN/Wates77/05 Ck/Salatiga/BBVet I/05 Ck/Padang/BBPVII/06 Ck/Salam/BBPV II/05 Ck/Siak/BPPV II/05 Ck/Rokan Hilli/BPPV II/05 Ck/Duma/BBPV II/05 Ck/Agam/BBPVI/05 Ck/Pulau Rampang/BBPVII/06 Ck/Simalanggang/BPPVI/05 Dk/Madiun/BBVW1358/05 IDN/CDC595/06 IDN/CDC625/06 IDN/CDC594/06 IDN/CDC597/06 IDN/CDC596/06 IDN/CDC599/06 Tk/Langkat/BBPVI/05 Ck/Langkat/BBPV1 576/05 Ck/Dairi/BPPVI/05 Ck/Taput/BBPV1 576/05 Ck/Tebing Tinggi/BPPVI/05 Ck/Medan/BBPV1 576/05 Ck/Medan/BPPV1 498/05 Ck/Tarutung/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Deli Derdang/BBPVI/05 Ck/Karo/BBPVII/06 Ck/Pidie/BPPV1/05 Ck/Medan/BBPV1 571/05 Ck/Medan/BPPV1 534/05 Ck/Deli Serdang/BPPV1/05 Ck/Wonosobo/BPPV4/03 Ck/Pekalongan/BPPV4/03 Ck/West Java/HAMD/06 Ck/IDN/11/03 Ck/IDN/4/04 Ck/IDN/PA/03 Ck/Malang/BBVet IV/04 Ck/Purwakarta/BBVet IV/04 Qa/Tasikmalaya/BPPV4/04 Ck/IDN/7/03 Ck/Sragen/BPPV4/03 Ck/IDN/BL/03 Ck/Ngawi/BPPV4/04 Dk/Tabanan/BPPV1/05 Dk/Bufeleng/BPPV1/05 Dk/Pali/BBVW1358/05 Dk/IDN/MS/04 Ck/IDN/2A/03 Ck/IDN/5/04 Ck/Bantul/BBVet I/05 Ck/Kupang 3 NTT/BPPV6/04 Ck/Kupang 2 NTT/BPPV6/04 Ck/Mangarai NTT/BPPV6/04 Ck/Jembrana/BPPV6/04 Ck/Bangli Bali/BPPV6 2/04 Ck/Bangli Bali/BBPV6 1/04 Ck/IDN/R60/05 100 IDN/CDC326/06 IDN/CDC329/06 Ct/IDN/CDC1/06 Ck/West Java/TASIK1/06 Ck/West Java/TASIK2/06 99 99 98 90 100 94 95 100 91 90 98 96 94 98 99 99 99 99 100 98 90 (97/97/1.00) 100 97 100 99 99 (100/100/1.00) 100 100 98 98 (100/100/1.00) 95 96 100 0.005 B (M1) Group 3 Group 1 Group 2 IDN/6/05 IDN/195H/05 IDN/5/05 IDN/298H/06 IDN/CDC194P/05 IDN/CDC370/06 IDN/CDC390/06 IDN/CDC624/06 IDN/CDC623/06 Dk/Parepare/BBVM/05 Ck/Wajo/BBVM/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/CDC184/05 IDN/CDC7/05 Ck/IDN/CDC25/05 Ck/IDN/CDC24/05 Ck/Yogjakarta/BBVet IX/04 Qa/Yogjakarta/BBVet IX/04 Ck/Purworejo/BBVW/05 Ck/Malang/BBVet IV/04 Ck/Purwakarta/BBVet IV/04 Ck/Kupang 1 NTT/BPPV6/04 Ck/Salatiga/BBVet I/05 Ck/Tebing Tinggi/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Simalanggang/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Dairi/BPPVI/05 IDN/CDC625/06 IDN/CDC599/06 IDN/CDC596/06 IDN/CDC597/06 IDN/CDC594/06 IDN/CDC595/06 Ck/Bantul/BBVet I/05 Ck/Kupang 2 NTT/BPPV6/04 Qa/Boyolali/BPPV4/04 Qa/Tasikmalaya/BPPV4/04 Ck/IDN/7/03 Ck/Kulon Progo/BBVet XII 1/04 Ck/Kulon Progo/BBVet XII 2/04 IDN/CDC1047/07 IDN/CDC1046/07 IDN/CDC644/06 IDN/CDC938/06 IDN/CDC887/06 IDN/CDC1032/07 IDN/CDC1031/07 IDN/CDC523/06 Ct/IDN/CDC1/06 IDN/CDC326/06 IDN/CDC329/06 IDN/CDC292T/05 IDN/CDC357/06 IDN/CDC582/06 IDN/CDC634/06 IDN/CDC699/06 IDN/CDC669/06 IDN/CDC835/06 IDN/CDC759/06 IDN/CDC836/06 IDN/CDC739/06 IDN/CDC940/06 IDN/CDC610/06 IDN/CDC287E/05 Ck/IDN/11/03 Ck/Bangli Bali/BBPV6 1/04 Ck/IDN/PA/03 Ck/Mangarai NTT/BPPV6/04 Ck/Pekalongan/BPPV4/03 Ck/IDN/BL/03 Ck/Bangli Bali/BPPV6 2/04 Ck/Kulon Progo/BBVW/05 Dk/IDN/MS/04 Ck/IDN/2A/03 Ck/Jembrana/BPPV6/04 Ck/Sragen/BPPV4/03 Tk/Kedaton/BPPV3/04 Ck/Pangkalpinang/BPPV3/04 Ck/Ngawi/BPPV4/04 Ck/Kupang 3 NTT/BPPV6/04 Ck/Wonosobo/BPPV4/03 Ck/IDN/5/04 Ck/IDN/4/04 90 (91/87/1.00) 95 97 96 98 0.002 (70/64/0.73) (68/68/1.00) (91/87/1.00) dN/dS = 1.514 Group 3 Group 1 Group 2 C (M2) 97 Ck/Simalanggang/BPPVI/05 Ck/Salatiga/BBVet I/05 Ck/Tarutung/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Tebing Tinggi/BPPVI/05 IDN/CDC599/06 IDN/CDC625/06 IDN/CDC596/06 IDN/CDC595/06 IDN/CDC594/06 IDN/CDC597/06 IDN/CDC940/06 IDN/CDC835/06 IDN/CDC836/06 IDN/CDC759/06 IDN/CDC329/06 IDN/CDC326/06 Ct/IDN/CDC1/06 IDN/CDC739/06 IDN/CDC610/06 IDN/CDC669/06 IDN/CDC938/06 IDN/CDC1031/07 IDN/CDC1032/07 IDN/CDC887/06 IDN/CDC582/06 IDN/CDC523/06 IDN/CDC292T/05 IDN/CDC287E/05 IDN/CDC644/06 IDN/CDC699/06 IDN/CDC634/06 96 (76/74/1.00) (31/27/0.47) (40/34/0.97) IDN/CDC357/06 IDN/CDC1047/07 IDN/CDC1046/07 IDN/6/05 Ck/Pangkalpinang/BPPV3/04 Tk/Kedaton/BPPV3/04 Ck/IDN/7/03 Ck/IDN/2A/03 Ck/Malang/BBVet IV/04 Ck/Purwakarta/BBVet IV/04 Ck/Kupang 1 NTT/BPPV6/04 Ck/Kulon Progo/BBVW/05 IDN/298H/06 IDN/CDC184/05 Ck/Magetan/BBVW/05 IDN/CDC7/05 IDN/5/05 Dk/Parepare/BBVM/05 Ck/Wajo/BBVM/05 Ck/Gunung Kidal/BBVW/05 Ck/IDN/CDC25/05 Ck/IDN/CDC24/05 IDN/CDC194P/05 IDN/CDC624/06 IDN/CDC623/06 IDN/CDC390/06 IDN/CDC370/06 Ck/Bantul/BBVet I/05 Qa/Yogjakarta/BBVet IX/04 Ck/Kupang 2 NTT/BPPV6/04 Ck/Mangarai NTT/BPPV6/04 Ck/Purworejo/BBVW/05 Dk/IDN/MS/04 Ck/Jembrana/BPPV6/04 Ck/IDN/11/03 Ck/IDN/BL/03 Ck/Sragen/BPPV4/03 Ck/Kupang 3 NTT/BPPV6/04 Qa/Boyolali/BPPV4/04 Ck/Ngawi/BPPV4/04 Ck/Wonosobo/BPPV4/03 Ck/Yogjakarta/BBVet IX/04 Ck/Bangli Bali/BBPV6 1/04 Qa/Tasikmalaya/BPPV4/04 Ck/IDN/4/04 Ck/Pekalongan/BPPV4/03 Ck/IDN/PA/03 Ck/Bangli Bali/BPPV6 2/04 Ck/IDN/5/04 Ck/Kulon Progo/BBVet XII 1/04 Ck/Kulon Progo/BBVet XII 2/04 0.01 R2 R3 R1 p A (HA) IDN/CDC940/06 IDN/CDC759/06 IDN/CDC669/06 IDN/CDC836/06 IDN/CDC739/06 IDN/CDC835/06 IDN/CDC610/06 Ck/IDN/Bandung1631 49/06 Muscovy Dk/Jakarta/HABWIN/06 Ck/IDN/Garut1631 51/06 Qa/Jakarta/JU1/06 Ck/West Java/TASIKSOL/06 IDN/CDC644/06 IDN/CDC1046/07 IDN/CDC1047/07 IDN/CDC1032/07 IDN/CDC938/06 IDN/CDC887/06 IDN/CDC1031/07 IDN/CDC523/06 IDN/CDC699/06 IDN/CDC634/06 IDN/CDC582/06 Ck/IDN/Lampung1631 23/06 Ck/IDN/Semerang1631 62/07 Sn/IDN/Malang1631 61/07 IDN/CDC370/06 IDN/CDC390/06 Ck/West Java/SMI ENDRI1/06 Ck/West Java/SMI ENDRI2/06 IDN/298H/06 Pg/IDN/Rokhit1631 6/06 Ck/IDN/Padang1631 1/06 Ck/IDN/Siak1631 2/06 Ck/IDN/Agam1631 3/06 Ck/IDN/Pekenbaru1631 11/06 IDN/CDC624/06 Ck/IDN/Gunung Kidul1631 33/06 IDN/CDC623/06 MDk/IDN/Kedri1631 24/06 Ck/West Java/SMI CSLK EB/06 Ck/West Java/SMI CSLK EC/06 Ck/West Java/SMI PAT/06 Ck/West Java/PWT WIJ/06 IDN/CDC292T/05 IDN/CDC742/06 IDN/CDC287E/05 Ck/IDN/Belitung Timor1631 18/06 IDN/CDC357/06 Ck/West Java/TASIKSOB/06 IDN/195H/05 Ck/IDN/Rejang Lebong1631 22/06 IDN/5/05 Ck/Pakun Baru/BPPV II/05 Ck/Murao Jambi/BBPV II/05 Dk/Indramayu/BBPW109/06 IDN/CDC194P/05 IDN/CDC184/05 IDN/CDC7/05 Ck/IDN/Wates1/05 Ck/Magetan/BBVW/05 Ck/IDN/Wates126/05 Ck/Gunung Kidal/BBVW/05 Ck/IDN/Wates130/05 Ck/Papua/TB1/06 Ck/Papua/TB15/06 Ck/Papua/TA5/06 Dk/Parepare/BBVM/05 Ck/Wajo/BBVM/05 Ck/IDN/CDC25/05 Ck/IDN/CDC24/05 Ck/Way Kanan/BBPVIII/06 Ck/Bandar Lampung/BBPVIII/06 Ck/Sembawa/BPPV III/05 Ck/Palembang/BPPV III/05 Ck/IDN/Bangka Seletan1631 21/06 Ck/IDN/Bangka Seletan1631 20/06 Ck/Purworejo/BBVW/05 Ck/Yogjakarta/BBVet IX/04 Qa/Yogjakarta/BBVet IX/04 Qa/Boyolali/BPPV4/04 100 IDN/CDC326/06 IDN/CDC329/06 Ct/IDN/CDC1/06 Ck/West Java/TASIK1/06 Ck/West Java/TASIK2/06 99 99 98 90 100 94 95 100 91 90 98 96 94 98 99 99 99 99 100 98 90 (97/97/1.00) 100 Group 2 B (M1) Group 3 Group 1 Group 2 IDN/6/05 IDN/195H/05 IDN/5/05 IDN/298H/06 IDN/CDC194P/05 IDN/CDC370/06 IDN/CDC390/06 IDN/CDC624/06 IDN/CDC623/06 Dk/Parepare/BBVM/05 Ck/Wajo/BBVM/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/CDC184/05 IDN/CDC7/05 Ck/IDN/CDC25/05 Ck/IDN/CDC24/05 Ck/Yogjakarta/BBVet IX/04 Qa/Yogjakarta/BBVet IX/04 Ck/Purworejo/BBVW/05 Ck/Malang/BBVet IV/04 Ck/Purwakarta/BBVet IV/04 Ck/Kupang 1 NTT/BPPV6/04 Ck/Salatiga/BBVet I/05 Ck/Tebing Tinggi/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Simalanggang/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Dairi/BPPVI/05 IDN/CDC625/06 IDN/CDC599/06 IDN/CDC596/06 IDN/CDC597/06 IDN/CDC594/06 IDN/CDC595/06 Ck/Bantul/BBVet I/05 Ck/Kupang 2 NTT/BPPV6/04 Qa/Boyolali/BPPV4/04 Qa/Tasikmalaya/BPPV4/04 Ck/IDN/7/03 Ck/Kulon Progo/BBVet XII 1/04 Ck/Kulon Progo/BBVet XII 2/04 IDN/CDC1047/07 IDN/CDC1046/07 IDN/CDC644/06 IDN/CDC938/06 IDN/CDC887/06 IDN/CDC1032/07 IDN/CDC1031/07 IDN/CDC523/06 Ct/IDN/CDC1/06 IDN/CDC326/06 IDN/CDC329/06 IDN/CDC292T/05 IDN/CDC357/06 IDN/CDC582/06 IDN/CDC634/06 IDN/CDC699/06 IDN/CDC669/06 IDN/CDC835/06 IDN/CDC759/06 IDN/CDC836/06 IDN/CDC739/06 IDN/CDC940/06 IDN/CDC610/06 IDN/CDC287E/05 Ck/IDN/11/03 Ck/Bangli Bali/BBPV6 1/04 Ck/IDN/PA/03 Ck/Mangarai NTT/BPPV6/04 Ck/Pekalongan/BPPV4/03 Ck/IDN/BL/03 Ck/Bangli Bali/BPPV6 2/04 Ck/Kulon Progo/BBVW/05 Dk/IDN/MS/04 Ck/IDN/2A/03 Ck/Jembrana/BPPV6/04 Ck/Sragen/BPPV4/03 Tk/Kedaton/BPPV3/04 Ck/Pangkalpinang/BPPV3/04 Ck/Ngawi/BPPV4/04 Ck/Kupang 3 NTT/BPPV6/04 Ck/Wonosobo/BPPV4/03 Ck/IDN/5/04 Ck/IDN/4/04 90 (91/87/1.00) 95 97 96 98 0.002 (70/64/0.73) (68/68/1.00) (91/87/1.00) dN/dS = 1.514 A A (HA) B B (M1) A (HA) Group 2 0.002 Group 3 Group 1 Group 2 C (M2) 97 Ck/Simalanggang/BPPVI/05 Ck/Salatiga/BBVet I/05 Ck/Tarutung/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Tebing Tinggi/BPPVI/05 IDN/CDC599/06 IDN/CDC625/06 IDN/CDC596/06 IDN/CDC595/06 IDN/CDC594/06 IDN/CDC597/06 IDN/CDC940/06 IDN/CDC835/06 IDN/CDC836/06 IDN/CDC759/06 IDN/CDC329/06 IDN/CDC326/06 Ct/IDN/CDC1/06 IDN/CDC739/06 IDN/CDC610/06 IDN/CDC669/06 IDN/CDC938/06 IDN/CDC1031/07 IDN/CDC1032/07 IDN/CDC887/06 IDN/CDC582/06 IDN/CDC523/06 IDN/CDC292T/05 IDN/CDC287E/05 IDN/CDC644/06 IDN/CDC699/06 IDN/CDC634/06 96 (76/74/1.00) (31/27/0.47) (40/34/0.97) IDN/CDC357/06 IDN/CDC1047/07 IDN/CDC1046/07 IDN/6/05 Ck/Pangkalpinang/BPPV3/04 Tk/Kedaton/BPPV3/04 Ck/IDN/7/03 Ck/IDN/2A/03 Ck/Malang/BBVet IV/04 Ck/Purwakarta/BBVet IV/04 Ck/Kupang 1 NTT/BPPV6/04 Ck/Kulon Progo/BBVW/05 IDN/298H/06 IDN/CDC184/05 Ck/Magetan/BBVW/05 IDN/CDC7/05 IDN/5/05 Dk/Parepare/BBVM/05 Ck/Wajo/BBVM/05 Ck/Gunung Kidal/BBVW/05 Ck/IDN/CDC25/05 Ck/IDN/CDC24/05 IDN/CDC194P/05 IDN/CDC624/06 IDN/CDC623/06 IDN/CDC390/06 IDN/CDC370/06 Ck/Bantul/BBVet I/05 Qa/Yogjakarta/BBVet IX/04 Ck/Kupang 2 NTT/BPPV6/04 Ck/Mangarai NTT/BPPV6/04 Ck/Purworejo/BBVW/05 Dk/IDN/MS/04 Ck/Jembrana/BPPV6/04 Ck/IDN/11/03 Ck/IDN/BL/03 Ck/Sragen/BPPV4/03 Ck/Kupang 3 NTT/BPPV6/04 Qa/Boyolali/BPPV4/04 Ck/Ngawi/BPPV4/04 Ck/Wonosobo/BPPV4/03 Ck/Yogjakarta/BBVet IX/04 Ck/Bangli Bali/BBPV6 1/04 Qa/Tasikmalaya/BPPV4/04 Ck/IDN/4/04 Ck/Pekalongan/BPPV4/03 Ck/IDN/PA/03 Ck/Bangli Bali/BPPV6 2/04 Ck/IDN/5/04 Ck/Kulon Progo/BBVet XII 1/04 Ck/Kulon Progo/BBVet XII 2/04 0.01 C Group 1 PLoS Pathogens \| www.plospathogens.org August 2008 \| Volume 4 \| Issue 8 \| e1000130 August 2008 \| Volume 4 \| Issue 8 \| e1000130 3 Indonesian Reassortant H5N1 Influenza Virus Indonesian Reassortant H5N1 Influenza Virus Figure 1. Genetic, temporal, and geographical origin of reassortant viruses doi:10.1371/journal.ppat.1000130.g002 PLoS Pathogens \| www.plospathogens.org 4 August 2008 \| Volume 4 \| Issue 8 \| e100 Early variants without significant grouping Group 1 Group 2 Group 3 IDN/CDC739/06 IDN/CDC940/06 IDN/CDC759/06 IDN/CDC836/06 IDN/CDC669/06 IDN/CDC835/06 IDN/CDC699/06 IDN/CDC523/06 IDN/CDC887/06 IDN/CDC938/06 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/6/05 Ck/Simalanggang/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 IDN/CDC594/06 IDN/CDC595/06 IDN/CDC597/06 IDN/CDC596/06 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC887/06 IDN/CDC938/06 IDN/CDC523/06 IDN/CDC699/06 IDN/CDC835/06 IDN/CDC669/06 IDN/CDC739/06 IDN/CDC836/06 IDN/CDC759/06 IDN/CDC940/06 IDN/6/05 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Simalanggang/BPPVI/05 IDN/CDC597/06 IDN/CDC594/06 IDN/CDC596/06 IDN/CDC595/06 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC836/06 IDN/CDC940/06 IDN/CDC759/06 IDN/CDC669/06 IDN/CDC739/06 IDN/CDC835/06 IDN/CDC699/06 IDN/CDC523/06 IDN/CDC887/06 IDN/CDC938/06 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/6/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Simalanggang/BPPVI/05 Ck/Dairi/BPPVI/05 IDN/CDC595/06 IDN/CDC594/06 IDN/CDC597/06 IDN/CDC596/06 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC523/06 IDN/CDC887/06 IDN/CDC938/06 IDN/CDC836/06 IDN/CDC940/06 IDN/CDC739/06 IDN/CDC759/06 IDN/CDC669/06 IDN/CDC835/06 IDN/6/05 IDN/CDC699/06 Ck/Dairi/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Simalanggang/BPPVI/05 IDN/CDC594/06 IDN/CDC595/06 IDN/CDC597/06 IDN/CDC596/06 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/5/05 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC836/06 IDN/CDC940/06 IDN/CDC835/06 IDN/CDC739/06 IDN/CDC669/06 IDN/CDC759/06 IDN/CDC938 IDN/CDC523/06 IDN/CDC699/06 IDN/CDC887/06 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBV Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Simalanggang/BPPVI/0 IDN/CDC594/06 IDN/CDC595/06 IDN/CDC597/06 IDN/CDC596/06 IDN/6/05 Dk/IDN/MS/04 Ck/IDN/PA/03 2256bp PB2 PB1 PA HA NP NA MP NS 4524bp 6636bp 8319bp 9778bp 11113bp 12045bp 1285 Early variants Group 1 Group 3 Group 2 Similarity 1.0 0.99 0.98 0.97 % of Permuted Trees 100 90 80 70 60 50 40 30 20 10 Bootscan analysis Concatenated influenza genome Breakpoint placement support using c-AIC in GARD Phylogenies inferred from non-recombinant fragments Similarity plot A B C D E 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2kbp 4kbp 6kbp 8kbp 10kbp 12kbp 12023bp 11080bp 4524bp 2197bp Model average support Figure 2. Recombination analyses on concatenated influenza virus genomes. (A) Schematic diagram of concatenated influenza genomes. (B) Similarity plot. (C) Bootscan analysis. (D) GARD analysis. (E) Individual phylogenies reconstructed from non-recombinant frag identified by GARD. Consensus sequences representing viral groups, window size of 600 bp and step size of 10 bp, were used for the similarit and bootscan analysis. The distance bar for the trees in (D) is 0.004 substitutions/site. Taxa of putative reassortant viruses are in red. Genetic, temporal, and geographical origin of reassortant viruses Early variants without significant grouping Group 1 Group 2 Group 3 IDN/CDC739/06 IDN/CDC940/06 IDN/CDC759/06 IDN/CDC836/06 IDN/CDC669/06 IDN/CDC835/06 IDN/CDC699/06 IDN/CDC523/06 IDN/CDC887/06 IDN/CDC938/06 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/6/05 Ck/Simalanggang/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 IDN/CDC594/06 IDN/CDC595/06 IDN/CDC597/06 IDN/CDC596/06 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC887/06 IDN/CDC938/06 IDN/CDC523/06 IDN/CDC699/06 IDN/CDC835/06 IDN/CDC669/06 IDN/CDC739/06 IDN/CDC836/06 IDN/CDC759/06 IDN/CDC940/06 IDN/6/05 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Simalanggang/BPPVI/05 IDN/CDC597/06 IDN/CDC594/06 IDN/CDC596/06 IDN/CDC595/06 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC836/06 IDN/CDC940/06 IDN/CDC759/06 IDN/CDC669/06 IDN/CDC739/06 IDN/CDC835/06 IDN/CDC699/06 IDN/CDC523/06 IDN/CDC887/06 IDN/CDC938/06 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/6/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Simalanggang/BPPVI/05 Ck/Dairi/BPPVI/05 IDN/CDC595/06 IDN/CDC594/06 IDN/CDC597/06 IDN/CDC596/06 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC523/06 IDN/CDC887/06 IDN/CDC938/06 IDN/CDC836/06 IDN/CDC940/06 IDN/CDC739/06 IDN/CDC759/06 IDN/CDC669/06 IDN/CDC835/06 IDN/6/05 IDN/CDC699/06 Ck/Dairi/BPPVI/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Simalanggang/BPPVI/05 IDN/CDC594/06 IDN/CDC595/06 IDN/CDC597/06 IDN/CDC596/06 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 IDN/5/05 Dk/IDN/MS/04 Ck/IDN/PA/03 IDN/CDC836/06 IDN/CDC940/06 IDN/CDC835/06 IDN/CDC739/06 IDN/CDC669/06 IDN/CDC759/06 IDN/CDC938/0 IDN/CDC523/06 IDN/CDC699/06 IDN/CDC887/06 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Simalanggang/BPPVI/05 IDN/CDC594/06 IDN/CDC595/06 IDN/CDC597/06 IDN/CDC596/06 IDN/6/05 Dk/IDN/MS/04 Ck/IDN/PA/03 2256bp PB2 PB1 PA HA NP NA MP NS 4524bp 6636bp 8319bp 9778bp 11113bp 12045bp 12855 Early variants Group 1 Group 3 Group 2 Similarity 1.0 0.99 0.98 0.97 % of Permuted Trees 100 90 80 70 60 50 40 30 20 10 Bootscan analysis Concatenated influenza genome Breakpoint placement support using c-AIC in GARD Phylogenies inferred from non-recombinant fragments Similarity plot A B C D E 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2kbp 4kbp 6kbp 8kbp 10kbp 12kbp 12023bp 11080bp 4524bp 2197bp Model average support Figure 2. Recombination analyses on concatenated influenza virus genomes. (A) Schematic diagram of concatenated influenza genomes. (B) Similarity plot. (C) Bootscan analysis. (D) GARD analysis. (E) Individual phylogenies reconstructed from non-recombinant fragm identified by GARD. Consensus sequences representing viral groups, window size of 600 bp and step size of 10 bp, were used for the similarity and bootscan analysis. The distance bar for the trees in (D) is 0.004 substitutions/site. Taxa of putative reassortant viruses are in red. Genetic, temporal, and geographical origin of reassortant viruses doi:10.1371/journal.ppat.1000130.g002 PLoS Pathogens \| www.plospathogens.org 4 August 2008 \| Volume 4 \| Issue 8 \| e10 2256bp PB2 PB1 PA HA NP NA MP NS 4524bp 6636bp 8319bp 9778bp 11113bp 12045bp 12855bp Early variants Group 1 Group 3 Group 2 Similarity 1.0 0.99 0.98 0.97 s 100 Concatenated influenza genome Similarity plot A B C A Co Similarity plot B B C D Group 3 IDN/CDC836/06 IDN/CDC940/06 IDN/CDC835/06 IDN/CDC739/06 IDN/CDC669/06 IDN/CDC759/06 IDN/CDC938/06 IDN/CDC523/06 IDN/CDC699/06 IDN/CDC887/06 IDN/5/05 Ck/Magetan/BBVW/05 Ck/Gunung Kidal/BBVW/05 Ck/Deli Serdang/BPPVI/05 Ck/Tarutung/BPPVI/05 Ck/Dairi/BPPVI/05 Ck/Simalanggang/BPPVI/05 IDN/CDC594/06 IDN/CDC595/06 IDN/CDC597/06 IDN/CDC596/06 IDN/6/05 Dk/IDN/MS/04 Ck/IDN/PA/03 12kbp E Group 2 Group 3 Figure 2. Recombination analyses on concatenated influenza virus genomes. (A) Schematic diagram of concatenated influenza virus genomes. (B) Similarity plot. (C) Bootscan analysis. (D) GARD analysis. (E) Individual phylogenies reconstructed from non-recombinant fragments identified by GARD. Consensus sequences representing viral groups, window size of 600 bp and step size of 10 bp, were used for the similarity plot and bootscan analysis. The distance bar for the trees in (D) is 0.004 substitutions/site. Taxa of putative reassortant viruses are in red. doi:10.1371/journal.ppat.1000130.g002 PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org August 2008 \| Volume 4 \| Issue 8 \| e1000130 4 Indonesian Reassortant H5N1 Influenza Virus Time (year) 10 1 10 2 10 -1 10 1 10 2 10 -1 2007 2006.5 2006 2005.5 2005 10 1 10 -1 10 -2 0 5 10 15 2007 2006.5 2006 2005.5 2005 R3-tMRCA R2-tMRCA R1-tMRCA All-tMRCA 2007 2006.5 2006 2005.5 2005 2004.5 Time (year) 10 1 10 -1 10 -2 0 5 10 Genetic distance ( subs/site) Relative genetic diversity (NeT) Relative genetic diversity (NeT) 10 -3 Linear regression BMCMC tMRCA Reassortant BSP Parent (group 2) BSP A C E G B D F H Regression mean Regression CI tMRCA median tMRCA HPD BSP median BSP HPD BSP median BSP HPD Slope = 0.0035 0.0009 Slope = 0.0079 0.0011 + + Slope = 0.0047 0.0008 + R3-tMRCA R2-tMRCA R1-tMRCA All-tMRCA 2007 2006.5 2006 2005.5 2005 2004.5 Figure 3. Estimation of tMRCA and relative genetic diversity for reassortant viruses and its genetic parent (group 2). (A) and (B) show the plots of the genetic distance from MRCA to each reassortant taxa, and the linear regression line depicting the tMRCA for HA and NA genes, respectively. 95% confidence intervals are shown by dashed lines. Genetic, temporal, and geographical origin of reassortant viruses Red dots and regression line indicate the removal of two sequences from the NA dataset. (C) and (D) show the tMRCAs estimated respectively from HA and NA genes using the BMCMC method. 95% higher probability density (HPD) is shown by the error bar. (E) and (F) show the Bayesian Skyline plots (BSP) illustrating the change of relative genetic diversity of reassortant viruses through time estimated from the HA and NA gene datasets, respectively. (G) and (H) show the BSP for group 2 viruses estimated from HA and NA gene datasets, respectively. doi:10.1371/journal.ppat.1000130.g003 A E Reassortant BSP G Parent (group 2) BSP Figure 3. Estimation of tMRCA and relative genetic diversity for reassortant viruses and its genetic parent (group 2). (A) and (B) show the plots of the genetic distance from MRCA to each reassortant taxa, and the linear regression line depicting the tMRCA for HA and NA genes, respectively. 95% confidence intervals are shown by dashed lines. Red dots and regression line indicate the removal of two sequences from the NA dataset. (C) and (D) show the tMRCAs estimated respectively from HA and NA genes using the BMCMC method. 95% higher probability density (HPD) is shown by the error bar. (E) and (F) show the Bayesian Skyline plots (BSP) illustrating the change of relative genetic diversity of reassortant viruses through time estimated from the HA and NA gene datasets, respectively. (G) and (H) show the BSP for group 2 viruses estimated from HA and NA gene datasets, respectively. d i 10 1371/j l t 1000130 003 tMRCA) was dated to July 2005 (highest probability density, HPD confidence interval: April–October), which is consistent with the linear regression estimate in Figure 3A and 3B. However, the regression estimate of All-tMRCA for the NA gene (April 2005) is slightly older than BMCMC estimate (July 2005). If strains IDN/ CDC1046/07 and IDN/CDC1047/07 are excluded from regres- sion analyses then the All-tMRCA date (July 2005; as indicated by the red regression line in Figure 3B) becomes consistent with the BMCMC estimate (July 2005). This suggests that the regression tMRCA) was dated to July 2005 (highest probability density, HPD confidence interval: April–October), which is consistent with the linear regression estimate in Figure 3A and 3B. However, the regression estimate of All-tMRCA for the NA gene (April 2005) is slightly older than BMCMC estimate (July 2005). PLoS Pathogens \| www.plospathogens.org Genetic, temporal, and geographical origin of reassortant viruses If strains IDN/ CDC1046/07 and IDN/CDC1047/07 are excluded from regres- sion analyses then the All-tMRCA date (July 2005; as indicated by the red regression line in Figure 3B) becomes consistent with the BMCMC estimate (July 2005). This suggests that the regression method was sensitive to rate variation caused by the two potential recent reassortants. The tMRCA estimates (R1-tMRCA, R2- tMRCA, and R3-tMRCA) for the individual reassortant sub- groups range from May 2005 to April 2006 (Figure 3C and 3D). The majority (17/25) of reassortant viruses were isolated from Greater Jakarta and surrounding areas such as Bekasi and Banten (Table 1 and Figure 4). The remaining eight samples were isolated from more distant locations in West Java province, such as Indramayu and Karawang. The two earliest reassortant viruses PLoS Pathogens \| www.plospathogens.org August 2008 \| Volume 4 \| Issue 8 \| e1000130 5 Indonesian Reassortant H5N1 Influenza Virus Table 1. Cases of human infections caused by Indonesian reassortant H5N1 HPAI viruses. Genetic, temporal, and geographical origin of reassortant viruses migrations between mainland Java, Sumatera and Sulawesi Selatan including Papua) are insignificantly (p.0.2) and significantly (p,0.0002) lower than the corresponding null values respectively, suggesting that the phylogeny of group 2 viruses is not geographically structured within Java, but is subdivided by island-to-island migrations. However, we could not address whether the viral migrations inside Sumatera and Sulawesi Selatan including Papua are panmictic or structured due to limited operative localities in our dataset to distinguish between different regions inside these islands. We also found that the migration of group 2 viruses from Greater Jakarta and surroundings to Sumatera and Sulawesi Selatan including Papua was more frequent than expected under the null hypothesis, and there is relatively little viral migration from the rest of Java to Sumatera and Sulawesi Selatan including Papua (Table S3). This observation suggests Greater Jakarta played a more salient role in dispersing group 2 viruses to other Indonesian islands than other parts of Java did. were isolated from central and east Jakarta (Table 1). Parsimony reconstruction (see Methods) of binary ancestral geographical states (either Greater Jakarta or West Java) upon the HA and NA ML phylogenies suggested that the MRCA of all reassortants (and the MRCAs of each reassortant subgroup) likely originated from Greater Jakarta and surroundings (Table S2; result robust to random resolution of polytomies; see Methods). Genetic, temporal, and geographical origin of reassortant viruses Sub- group Strain name Case index Fatal Sex Age Family cluster Placef Onset date Death date Sampling date R1 IDN/CDC292/05 15 Y M 8 Central Jakarta (1) 8-12-05 15-12-05 15-12-05 R1 IDN/CDC742/06 56 Y F 17 Jakarta (1) 28-7-06 8-8-06 7-8-06 R1 IDN/CDC287/05 16 Y M 39 East Jakarta (2) 9-12-05 12-12-05 13-12-05 R1 IDN/CDC357/06 21 Y M 15 Padalarang, Bandung, West Java (12) n/a 1-2-06 30-1-06 R1 IDN/CDC326/06 19 Y M 4 Ec, brother Indramayu, West Java (8) 8-1-06 17-1-06 15-1-06 R1 IDN/CDC329/06 18 Y F 13 Ec, sister Indramayu, West Java (8) 6-1-06 14-1-06 14-1-06 R3 IDN/CDC940/06 74 Y M 30 m Karawang, West Java (9) 5-11-06 13-11-06 12-11-06 R3 IDN/CDC759/06 59 Y F 35 Cikelet, West Java (10) 8-8-06 17-8-06 17-8-06 R3 IDN/CDC669/06 51 Y M 13 South Jakarta (3) 9-6-06 14-6-06 13-6-06 R3 IDN/CDC836/06 68 Y M 20 Bandung, West Java (7) 17-9-06 28-9-06 24-9-06 R3 IDN/CDC739/06 55 Y M 16 Bekasi, West Java (4) 26-7-06 7-8-06 5-8-06 R3 IDN/CDC835/06 67 Y M 9 South Jakarta (3) 13-9-06 22-9-06 22-9-06 R3 IDN/CDC610/06 41 Y M 12 Bekasi, West Java (4)a 7-5-06 13-5-06 11-5-06 R2 IDN/CDC644/06 49 Y M 15 Tasikmalaya, West Java (11) 24-5-06 30-5-06 30-5-06 R2 IDN/CDC1046/07 77 Y F 22 Tangerang, Banten (5) 3-1-07 12-1-07 11-1-07 R2 IDN/CDC1047/07 78 Y F 27 South Jakarta (3) 6-1-07 12-1-07 12-1-07 R2 IDN/CDC1032/07 76 Y F 37 Md, mother Tangerang, Banten (5) 1-1-07 11-1-07 6-1-07 R2 IDN/CDC938/06 73 Y F 35 Tangerang, Banten (5) 7-11-06 28-11-06 10-11-06 R2 IDN/CDC887/06 71 Y M 11 South Jakarta (3) 2-10-06 14-10-06 14-10-06 R2 IDN/CDC1031/07 75 Y M 14 West Jakarta (6) 31-12-06 10-1-07 5-1-07 R2 IDN/CDC523/06 30 Y F 10 m Kapuk, West Jakarta (6) 17-3-06 23-3-06 23-3-06 R2 IDN/CDC699/06 53 Y F 3 suburb of Jakarta (5)b 23-6-06 6-7-06 6-7-06 R2 IDN/CDC634/06 44 Y F 10 He, sister Bandung, West Java (7) 16-5-06 23-5-06 23-5-06 R2 IDN/CDC582/06 33 Y M 30 Jakarta (1) 17-4-06 26-4-06 26-4-06 aSome information sources refer to this as East Jakarta. b bSome information sources refer to this as Tangerang. than the null value. However, the observed value of GSC within Java (i.e., migrations between Greater Jakarta and the rest of Java) and between the three islands (i.e. There are two other family members (father and sister) suspected with H5N1 infections (non-fatal). dThere is another family member (son) who was confirmed with H5N1 infection (index #79, non-fatal); however, virus sequences are not available. eThere is another family member (brother) who was confirmed with H5N1 infection (index #45, fatal); however, virus sequences are not available. fNumbers in parentheses are the unique references to the localities shown in Figure 4. Numbers 1–6 were assigned to Greater Jakarta and surroundings; numbers 7–12 were assigned to West Java. n/a = Information not available. doi:10.1371/journal.ppat.1000130.t001 PLoS Pathogens \| www.plospathogens.org bSome information sources refer to this as Tangerang. cTh t th f il b (f th d i t ) t d ith H5N1 i f ti ( f t l) August 2008 \| Volume 4 \| Issue 8 \| e1000130 Population dynamics of reassortant and its parental strain the MRCA of the group 3 viruses and preceding the emergence of the reassortant viruses (highlighted in Figure 1B). The dN/dS values for the M1 gene in this lineage (which we call the pre- emergence lineage) was estimated to be 1.514 (95% CI: 0.447– 3.814; see Table S5), significantly higher (LRT p,0.002) than the mean estimates for other lineages (dN/dS = 0.077) in the Indonesian clade and for lineages in other H5N1 HPAI clades (e.g., Fujian, Qinghai, Thailand and Vietnam clades which have dN/dS ranging from 0.05 to 0.09). This lineage-specific elevation of dN/dS was not significant (LRT p.0.1) for other genes (i.e. HA, NA, M2, PB1; see Table S5). Four amino acid changes in M1 occurred along the pre-emergence lineage, including threonine to alanine at reside 37, arginine to lysine at reside 95, threonine to alanine at reside 137, glutamine to histidine at reside 249. Three (residue 37, 95, and 137) of them are located close to the electrostatic positive surface of the N-terminal domain of the M1 protein molecule (Figure S7), and one (residue 249) is located in the remaining C-terminal fragment. We used the Bayesian skyline plot (BSP) [23] to estimate the change of relative genetic diversity of the reassortant viruses and of the group 2 parental strain over time, as shown in Figure 3E–3H. For both the HA and NA datasets, the group 2 viruses consistently show a slow growth in relative genetic diversity over time which appears to follow a constant size or exponential growth model, whereas the reassortant viruses initially exhibited an abrupt rise in relative genetic diversity followed by stabilization, which visually resembles a logistic growth curve with two phases [24,25] (Figure 3E and 3F). When the BSPs are superimposed upon the demographic results obtained under parametric growth models (i.e., constant, exponential and logistic growth; Figure S8), then a similar observation can also be made. However, BF tests (Table S4) indicate there is insufficient statistical power to discriminate between the three parametric growth models (lnBF,2.99), suggesting a lack of strong demographic signal in these data. When the parametric demographic models were fitted to the data, the median estimates of growth rates for the reassortant datasets are generally higher than those estimated for the datasets of group 2 viruses (Table S1). However, the confidence intervals of some growth rate estimates are fairly large and overlapped among the reassortant and group 2 viral datasets. Discussion This study classified H5N1 HPAI viruses in Indonesia into three distinct viral lineages (groups 1, 2, and 3) and discovered a group of naturally occurring reassortant viruses that represent a newly emergent H5N1 HPAI strain in Java in 2006. Several phylogenetic methods concurred that two (MP and PB1) of the reassortant viruses’ genome segments descended from the group 3 ancestral viruses, and the remaining six (PB2, PA, HA, NP, NA, NS) segments descended from the group 2 ancestral viruses. Although the majority of reassortant viruses (24/25) are human isolates, few of the associated human infections are epidemiologically linked (Table 1), suggesting multiple sporadic zoonotic transmissions from birds. The phylogeographic results indicate that the parental viruses of the reassortants have been co-circulating in Java since 2005. Despite the identification of parental lineages, the exact number of reassortment events remains difficult to assess. Although the three fairly consistent phylogenetic subgroups (subgroups R1, R2, and R3 in Figure S4) formed by the reassortant viruses suggest three independent reassortments, the Spatial migration of reassortant and its parental strain The mean numbers of observed geographical state changes (GSC) of the reassortant and of the group 2 parental strains were estimated independently and compared with the null distribution of GSC values under the null hypothesis of completely unrestricted migration (i.e. panmixis; Figure S5) [22]. For the reassortant strain, the observed GSC value was not significantly lower than the GSC value expected under panmixis (Slatkin-Maddison test: p.0.2). Therefore the observed geographic structure is not significantly different to that expected by chance alone. For group 2 viruses, the observed GSC value for all geographical state pairs is significantly (p,0.0002) lower August 2008 \| Volume 4 \| Issue 8 \| e1000130 August 2008 \| Volume 4 \| Issue 8 \| e1000130 6 Indonesian Reassortant H5N1 Influenza Virus WEST JAVA G. JAKARTA G. JAKARTA CENTRAL JAVA D.I YOGYAKARTA EAST JAVA Group 2 (2006) Group 2 (2005) Reassortant Group 2 (2007) Group 3 (2006) Group 3 (2005) Group 3 (2007) Legend: G. Jakarta and surroundings 5 5 1 3 1 3 6 6 4 4 8 7 2 2 9 12 10 11 25 km 100 km Figure 4. Map of mainland Java in Indonesia. Locations for the putative reassortant viruses focused in this study are indicated with green triangles. Numbers inside the triangles refer to the locations of the reassortant viruses described in Table 1. Locations of group 2 and 3 viruses are indicated with orange circles and squares, respectively. Red circles and squares denote the locations of putative avian reassortant viruses in 2007. Only those parental strains genetically close to the reassortant viruses are shown in the map. A zoom-in of Greater Jakarta (G. Jakarta) and surroundings is illustrated separately in a dashed-line bounded box in the centre of the figure. doi:10.1371/journal.ppat.1000130.g004 D.I YOGYAKARTA EAST JAVA EAST JAVA Figure 4. Map of mainland Java in Indonesia. Locations for the putative reassortant viruses focused in this study are indicated with green triangles. Numbers inside the triangles refer to the locations of the reassortant viruses described in Table 1. Locations of group 2 and 3 viruses are indicated with orange circles and squares, respectively. Red circles and squares denote the locations of putative avian reassortant viruses in 2007. Only those parental strains genetically close to the reassortant viruses are shown in the map. A zoom-in of Greater Jakarta (G. Spatial migration of reassortant and its parental strain Jakarta) and surroundings is illustrated separately in a dashed-line bounded box in the centre of the figure. doi:10.1371/journal.ppat.1000130.g004 Figure 4. Map of mainland Java in Indonesia. Locations for the putative reassortant viruses focused in this study are indicated with green triangles. Numbers inside the triangles refer to the locations of the reassortant viruses described in Table 1. Locations of group 2 and 3 viruses are indicated with orange circles and squares, respectively. Red circles and squares denote the locations of putative avian reassortant viruses in 2007. Only those parental strains genetically close to the reassortant viruses are shown in the map. A zoom-in of Greater Jakarta (G. Jakarta) and surroundings is illustrated separately in a dashed-line bounded box in the centre of the figure. doi:10.1371/journal.ppat.1000130.g004 PLoS Pathogens \| www.plospathogens.org Indonesian Reassortant H5N1 Influenza Virus Future studies on economic and social geography (e.g., addressing the modes of inter-provincial poultry transport) in Indonesia might help to further elucidate the effect on the viral dispersal by human, agricultural and industrial activities. In this study, we opted for a lower geographical resolution (i.e., four widely ranged geographical states instead of distinct geographical coordinate for each viral isolate) in our phylogeographic analyses because of the varying precision of the geographical data we have. Therefore, more complex hypotheses of viral origin and migration trajectory cannot be investigated here, but can be explored when more high-quality geographical data of Indonesian H5N1 viral samples is available. Mechanisms of viral transmissions are sometimes correlated with genetic diversity dynamics. For example, hepatitis C viruses transmitted by drug injection or blood products have a faster rate of spread than endemic strains circulating in Asia and Africa [32]. It has also been suggested that mosquito susceptibility may affect the growth of dengue viruses [33]. Therefore, it is possible that a change of host species could generate the difference in the viral dynamics we observe. In our study, the majority of the reassortant viruses (24/25) were isolated from humans, whereas only a minority of the group 2 viruses were isolated from humans (10/57 and 10/41 in the HA and NA datasets, respectively). It has been previously shown that the receptor binding specificity of hemagglutinin [34] and mutations in the viral polymerase (e.g., lysine at residue 627 of PB2) [35–37] can determine viral transmissibility and replication in different host species. None of the aforementioned HA mutations which confer recognition to human-type host cell receptors [34,38] were found in the Indonesian reassortant viruses; however, our detection of positively selected sites in the PB2 gene of the reassortant viruses could potentially reflect adaptation to mammalian hosts, and requires further investigation. In particular, amino acid changes on two positively selected sites (threonine to methionine at reside 76, glutamic acid to glycine and alanine at reside 677) were found on the internal branches of the reassortant lineage, corresponding to molecular changes during sustainable transmissions. However, some of these positively selected changes may also result from the compensatory evolution as the mix of genome segments from different strains might alter their epistatic physiochemistry [39]. Although most of the human isolates in our datasets were epidemiologically unlinked, such linkage is theoretically possible if many asymptotic or mildly manifested human infections are not reported. Indonesian Reassortant H5N1 Influenza Virus We expect future development of analysis methods will help to shed more light on the interaction between viral migration and genetic diversity. Analysis of clinical records (Table 1) found that the mean duration from onset to death in those fatal human cases caused by Indonesian reassortant H5N1 viruses is 9.1 days (standard deviation [SD] = 3.9; n = 23) and those caused by other Indone- sian H5N1 viruses is 7.7 days (SD = 2.7; n = 10), and their means are not significantly different (student t-test, p.0.25, two-tails). Therefore, based on the clinical records, the reassortant viruses did not kill human faster than other Indonesian H5N1 viruses did. However, we would recommend more experimental studies addressing the virulence, pathogenicity and immunogenicity of the reassortant viruses and the parent strains to verify this claim in the future. According to our analyses, the common ancestor of the reassortant viruses is dated to July 2005 (HPD: April–October), approximately 5 months prior to the first case of human infection caused by the reassortant virus (index case #15 defined by WHO; see Table 1). Our analysis of virus phylogeography suggests the ancestors of these reassortant viruses first arose in Greater Jakarta and surroundings, which agrees with the observation that the first two cases of human infection by the reassortant viruses occurred in Central and East Jakarta (index cases #15 and #16). The molecular dating and phylogenetic analyses suggest that nascent reassortant viruses might take several months to spread and expand their diversity in the local bird population, eventually leading to the exposure of human population. The subsequent spread of the reassortant strain seems to become more rapid and extensive, as human cases were reported outside Greater Jakarta one month later, and the reassortant virus spread to as far as the south and east of West Java in the following six months (Table 1 and Figure 4). Commercial poultry transportation, as well as carriage by migratory birds, may facilitate the viral migration, but their tangible contributions need further studies. Our results suggest that the circulations of reassortant viruses and their genetic parent (group 2) were not restricted by geography within Java. The viral migration back to Greater Jakarta could be driven by the inter-province transfer of infected poultry, in particular the importation of live poultry or fresh poultry products to the densely human populated Jakarta from the remote provinces engaged in poultry-farming. Indonesian Reassortant H5N1 Influenza Virus underlying uncertainty in our estimated phylogenies means that we cannot rule out the possibility of a single origin. The BSP analyses (Figure 3E–3H) indicate that the reassortant viruses follow a logistic-like growth curve, which is typical for virus invasion and maintenance, especially in a structured population [24,25]. In contrast, the group 2 viruses followed a more continuous and relatively slow growth in diversity. There was insufficient data in our samples to definitively discriminate between alternative population growth models and provide narrow confidence intervals for parameter estimates, but our results are suggestive and future sequencing will add to the needed statistical power. The hypothesis of three reassortments implies that the viruses have acquired exactly the same genome segments from the same group of parental viruses, which seems unlikely to occur by chance (probability = 0.0089, assuming panmixis and that exactly two genomic segments are swapped out). This probability might be increased if reassortments confer a selective advantage. We did detect a significantly stronger selection pressure on the M1 protein in the pre-emergence lineage of group 3 parental strain that led to the reassortant viruses (Table S5). Previous reports suggested a few amino acid changes in M1 of influenza A and B viruses can confer a growth advantage in mouse lungs [27–29]. Although the M1 mutations identified in this pre-emergence lineage have not been functional characterized elsewhere in the authors’ knowledge, one (residue 137, TRA) of them is close to a previously characterized mutation (residue 139, TRA) which controls the virulence in mouse model [27,29]. Three of the inferred residue changes are located close to the electropositive surface of N-terminal domain of M1 protein (Figure S7) that acts to bind viral RNA [30,31]. The M1 matrix protein mediates encapsulation of viral RNA- nucleoproteins into membrane envelope during packaging [31], and has close contact with other viral proteins inside the viral particle. It seems possible that some of these changes may be involved in the adaptation of reassortant viruses, through promotion of structural interactions among viral proteins. What factors have contributed to the apparent difference in the growth of genetic diversity? Rates of molecular evolution between the two groups were similar (Figure S6) and therefore are not likely to be the cause. Since our analyses could not resolve the temporal dynamics of population subdivision by geography, we cannot directly investigate how viral genetic variation is affected by the population structure. PLoS Pathogens \| www.plospathogens.org Diversifying selection in the PB2 and M1 genes Diversifying selection in the PB2 and M1 genes Using the Random Effects Likelihood (REL) method [26] we found sites under positive selection in the PB2 gene (codons 76, 534, 627, 677 and 740) and the PA gene (codon 409) of the reassortant viruses. The Fixed Effects Likelihood (FEL) method [26] was more conservative and only identified PB2 codon 534 as being positively selected. For the group 2 viruses, HA codon 129 (starting from HA1) and M1 gene codon 205 were the only selected sites identified by the FEL and REL methods, respectively. Using a lineage-specific selection model (see Meth- ods), we identified elevated rates of diversifying selection, measured by the ratio of non-synonymous to synonymous substitutions (dN/dS), on the M1 gene in the lineage leading to PLoS Pathogens \| www.plospathogens.org August 2008 \| Volume 4 \| Issue 8 \| e1000130 7 Indonesian Reassortant H5N1 Influenza Virus Recently, some evidence of subclinical or asymptotic H5N1 infection in humans has been put forward [40,41]; PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org August 2008 \| Volume 4 \| Issue 8 \| e1000130 8 Indonesian Reassortant H5N1 Influenza Virus however, the ability of the viruses to transmit from these infected individuals to other susceptible individuals remains unknown. version 4beta10 [58], respectively. A general-time-reversal (GTR) substitution model with gamma distributed rate heterogeneity of 4 rate categories (C4) and a proportion of invariable sites were used in all tree reconstruction methods. Phylogenies were rooted with the H5N1 HPAI strain A/Ck/HK/YU324/2003, which is genetically close to the newly reported Hunan strains [17], and shares comparable genetic proximity to Indonesian clade. The possible role of other animal host species in the transmissions of reassortant viruses in Indonesia should not be neglected. In particular, one of the reassortant viruses was isolated from a dead cat in Jakarta, where H5N1 outbreaks in poultry and sporadic human infections have been reported [42]. Moreover, unusual high mortality of cats in the vicinity of H5N1 HPAI outbreaks has been reported [43]. An unofficial report also detected H5N1 HPAI sero-positivity in around 20% of 500 blood samples taken from stray cats near poultry markets in Java and Sumatera [44]. In addition to small cats in Germany [45], Iran [46], and Indonesia [42], dogs and zoo tigers were also found infected with H5N1 HPAI viruses in Thailand [47,48]. Further- more, previous experimental studies have demonstrated that cats can be infected with H5N1 HPAI virus [49,50], and that cat-to-cat transmission is possible [49,51]. Could cats, or other non-avian species, have played a role in spreading the reassortant viruses in Java? Similarly, could cats act as amplifying hosts facilitating viral expansion and cross-species transmission, as civets did in the SARS outbreaks [52]? Future experimental studies on these reassortant viruses, that assess viral transmissibility between species, together with epidemiological studies, such as viral monitoring within Indonesian animal populations using serological tests and PCR detection, would give more clues to these questions. Recombination and reassortment detection Homologous recombination within each gene segment among Indonesian H5N1 isolates was extensively searched using Recom- bination Detection Program version 2 (RDP2) [59], and the datasets are found to be free of homologous recombination. Putative reassortant viruses were preliminarily identified by their topological incongruity across the phylogenies of different gene segments. This was further investigated using a smaller set of Indonesian H5N1 virus isolates with full genome sequences, which included sequences of early viruses (n = 2), group 1–3 lineages (n = 12) and putative reassortant viruses (n = 10). The eight gene segment alignments were manually concatenated in the order of their length to generate a single alignment of complete genome sequences, and was further analyzed using 1) similarity plots and 2) bootscan analyses [60] implemented in SIMPLOT version 3.5.1 [61], and 3) GARD [62] available via the Datamonkey website [63]. The hypothesis of reassortment was supported if the recombinant breakpoints were detected near the junctions where the genome segments were manually concatenated. H5N1 HPAI viruses have been endemic and evolved into different genetic lineages that have spread across Indonesia. Areas where more than one lineage of virus is co-circulating, such as Jakarta, are most likely to generate novel viruses by inter-lineage reassortment. These reassortant viruses have distinctive evolution- ary and transmission dynamics, as shown in this study. We suggest that more intensive and timely field surveillance and analysis of influenza viruses, including H5N1 HPAI and human H3N2, H1N1, and H1N2 epidemic strains, should be employed, so that bio-security can be undertaken promptly and appropriate strains can be selected for vaccine production whenever a novel reassortant strain emerges. The reassortant viruses reported in this study should be also added to the watch list for the future epidemiological surveillance. Phylogeography and migration analyses The geographic locations of virus isolation were either obtained from the sequence databases, or obtained through personal communication with Catherine Smith (from Disease Control and Prevention, Atlanta, USA), or inferred from their strain names (Tables 1 and S6). The locations of isolates were indicated on the map of main island of Java in Indonesia (Figure 4). Due to the limit of our geographical data, the localities of the isolates shown in the map (Figure 4) should be regarded as arbitrary within the province which is the highest precision level shared by all viral samples. Each of the reassortant viruses was assigned with a state of either Greater Jakarta (surroundings included) or West Java depending on its place of origin (Table 1). The migratory history of the reassortant viruses (n = 25) between these geographical states were inferred based on the refined ML phylogeny of HA and NA (Figure S4) independently using two parsimony optimization methods, called ACCTRAN (accelerated transformation) and DELTRAN (delayed transformation) implemented in PAUP* software. The geographical states of all ancestral nodes in the tree were estimated to achieve minimum state changes in overall, and therefore the number of state changes and state of the MRCA of the reassortant was obtained. Polytomies were randomly resolved 1,000 times, and state changes were estimated separately for each resolution. The mean number of state changes was then calculated. To test against the null hypothesis of completely unrestricted migration between geographical states (panmixis), the mean number of observed state changes was compared with the frequency distribution of the mean number of expected state changes under the null hypotheses. The null distribution and critical values were generated by randomly shuffling the states of isolates 5,000 times (the Slatkin-Maddison test [22,24,64]). The migratory history of group 2 viruses was also studied using the HA gene in a similar manner, while each group 2 virus was assigned to either of four widely ranged geographical states: Greater Jakarta and surround- ings, the rest of Java, Sumatra, and Sulawesi Selatan, including Sequence data collection and alignment Sequence data collection and alignment H5N1 influenza viruses isolated from avian and mammalian hosts in Indonesia during 2003–2007 were studied. Their genomic sequences (n = 807) were extracted from the Influenza Virus Resource [53] and the Influenza Sequence Database [54] in September 2007, and aligned using MUSCLE version 3.6 [55]. Columns with gaps were removed from the alignments, and sequences from the same virus strain (duplicated submission in the two databases) were filtered such that one copy was retained. Eight genome segment alignment datasets (PB2, PB1, PA, HA, NP, NA, MP, and NS), as well as four coding sequences (M1, M2, NS1, and NS2), were generated. Full details of our datasets can be found in Table S6 and S7. PLoS Pathogens \| www.plospathogens.org Indonesian Reassortant H5N1 Influenza Virus Indonesian Reassortant H5N1 Influenza Virus Papua. This assignment scheme is comparable to that of reassortant viruses, as West Java is part of Java. and cat reassortant viruses are in red. Putative avian reassortant viruses are in blue. Arrows indicate the roots. Distance unit is substitutions/site. and cat reassortant viruses are in red. Putative avian reassortant viruses are in blue. Arrows indicate the roots. Distance unit is substitutions/site. Identifying mutations fixed along the lineage The ancestral nucleotide sequences of all internal nodes were reconstructed using joint ML method [68] implemented in HYPHY. Amino acid changes along the pre-emergence lineage were determined, and were then mapped onto the three-dimensional structure of the N-terminal domain of M1 matrix protein molecule [30] available (PDB-ID: 1EA3) in RCSB Protein Data Bank. Found at: doi:10.1371/journal.ppat.1000130.s005 (1.24 MB EPS) Figure S6 Substitution rates of HA and NA genes from reassortant and group 2 parental strains. 95% higher probability densities (HPDs) are indicated by the error bars. 1st, 2nd, 3rd, and C denote the rate for the 1st codon position, 2nd codon position, 3rd codon position, and whole sequence (non-partitioned), respectively. Substitution rate units for codon partitioned and non-partitioned sequences are substitution/codon/year and sub- stitution/site/year, respectively. Detecting positively selected sites and lineages g y g Positively selected sites were detected using random effect likelihood (REL) and fixed effect likelihood (FEL) methods [26] via the Datamonkey website [63]. Bayes factors larger than 50 and p- values smaller than 0.1 were used as thresholds for strong evidence of selection in REL and FEL, respectively. To test lineage-specific positive selection, the two-ratio branch model was used, which pre-specifies a single rate of synonymous substitution (dS) for the whole phylogeny and two rates of non-synonymous substitution (dN1 and dN2). The dN1 was specified for the pre-emergence lineage (indicated as the ancestral branch connecting the group 3 MRCA; see Figure 1B) for the group 3 viruses (including the reassortant viruses for M1, M2, and PB1 genes). The dN2 was specified for other lineages across the phylogenies. The ML estimates of these rate parameters were performed in HYPHY version 0.99 [67]. The resulting likelihood score of the two-ratio model was then compared with that of the one-ratio model, which assumes the same dN and dS across the phylogeny, using the likelihood ratio test (LRT, with degree of freedom = 1). The substitution model MG94XGTR+C4 was used. Figure S4 Refined phylogenies of reassortant viruses inferred from their HA and NA genes. ML phylogenies reconstructed from (A) HA gene and (B) NA gene. Topological supports summarized from 1,000 ML bootstrap replications are shown. For major lineages, NJ bootstrap support (1,000 replications) and posterior probability from BMCMC analyses (5,000 tree samples) are also shown inside parentheses (ML/NJ/BMCMC). Subgroups R1, R2, and R3 reassortant viruses are in red, blue, and green, respectively. Arrows indicate the rooting using strain IDN/5/05 as the outgroup. Distance unit is substitutions/site. Found at: doi:10.1371/journal.ppat.1000130.s004 (1.18 MB EPS) Figure S5 Mean number of observed geographical state changes and the frequency distribution of mean number of expected geographical state changes under null hypothesis of panmixis. (A– D) show the frequency distribution of reassortant viruses from the HA ([A–B]) and NA ([C–D]) datasets using delayed transforma- tion (DEL; [A,C]) and accelerated transformation (ACC; [B,D]) methods. (E) and (F) show the frequency distribution of group 2 viruses from the HA dataset using DEL and ACC methods, respectively. Arrowheads indicate the mean number of observed geographical state changes (GSCs) and the corresponding p-value tested against the null distribution. PLoS Pathogens \| www.plospathogens.org Estimating the rate of evolution and genetic diversity dynamics Found at: doi:10.1371/journal.ppat.1000130.s001 (2.81 MB EPS) Found at: doi:10.1371/journal.ppat.1000130.s001 (2.81 MB EPS) Figure S2 Phylogenies of PA, NP, and NA genes of Indonesian H5N1 HPAI viruses. ML phylogenies reconstructed from (A) PA gene, (B) NP gene, and (C) NA gene. Topological supports (.90) summarized from 1,000 ML bootstrap replications are shown. For major lineages, NJ bootstrap support (1,000 replications) and posterior probability from BMCMC analyses (5,000 tree samples) are also shown inside parentheses (ML/NJ/BMCMC). Putative human and cat reassortant viruses are in red. Putative avian reassortant viruses are in blue. Arrows indicate the roots. Distance unit is substitutions/site. y Parameters of codon-partitioned substitution rates, demograph- ic functions, tMRCA and tree topologies were co-estimated from HA and NA gene datasets of reassortant and group 2 viruses separately in a BMCMC framework [18] using BEAST version 1.4.6 [65]. Substitution model HKY+C4 with invariable site portion was used. Isolation dates were used to calibrate the molecular clock. Three clock models including strict clock, UCEN and UCLN relaxed clocks [21] were attempted independently, and the best-fit clock model was selected by comparing the BF calculated from their posterior distributions [20]. The Bayesian skyline plot [23] was used to estimate population dynamics, in terms of relative genetic diversity. Less complex parametric demographic models (constant size, exponential growth and logistic growth) were applied independently, and the best-fit models selected by BF tests were used to quantitatively estimate the growth rate and other demographic parameters. The BMCMC analyses contained 26108 states, with sampling every 1,000 states, and the first 10% of each chain was discarded as burn-in. Convergences and effective sample sizes of the estimates were checked using Tracer v1.4 [66]. Found at: doi:10.1371/journal.ppat.1000130.s002 (3.06 MB EPS) Figure S3 Phylogenies of MP and NS segments, NS1 and NS2 genes of Indonesian H5N1 HPAI viruses. ML phylogenies reconstructed from (A) MP segment, (B) NS segment, (C) NS1 gene, and (D) NS2 gene. Topological supports (.90) summarized from 1,000 ML bootstrap replications are shown. For major lineages, NJ bootstrap support (1,000 replications) and posterior probability from BMCMC analyses (5,000 tree samples) are also shown inside parentheses (ML/NJ/BMCMC). Putative human and cat reassortant viruses are in red. Arrows indicate the roots. Distance unit is substitutions/site. Found at: doi:10.1371/journal.ppat.1000130.s003 (3.19 MB EPS) Phylogenetic analyses Phylogenetic trees of 12 alignment datasets were reconstructed using the ML approach implemented in PhyML 3.412 [56]. The robustness of the ML tree topology was assessed by comparing the ML topology with the topologies sampled in the BMCMC analysis performed in MrBayes version 3.1.2 [57], and with bootstrapping analyses of 1,000 pseudo-replicate datasets. ML and NJ trees were estimated from the bootstrap datasets using PhyML [56] and PAUP* PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org August 2008 \| Volume 4 \| Issue 8 \| e1000130 9 Supporting Information Empty entries indicate the unavailability (e.g., no sequence found, too short, too many ambiguous codes, and too many gaps) of the sequence. Table S6 Information and phylogenetic groupings of sequences used in this study. 1, 2, 3, and X denotes groups 1, 2, 3, and unclassified (early viruses; see main text for explanation). Empty entries indicate the unavailability (e.g., no sequence found, too short, too many ambiguous codes, and too many gaps) of the sequence. Dataset S1 Computer files of phylogenetic trees of Indonesian H5N1 influenza viruses. The zip file includes dendrogram (in NEWICK format) of ML phylogenies reconstructed from PB2, PB1, PA, HA, NP, NA, MP, M1, M2, NS, NS1, and NS2 gene datasets (described in main text). Topological supports (percent- ages) shown in the internal nodes were summarized from 1,000 ML bootstrap replications. NJ bootstrap replications and BMCMC sampled trees are available upon request. Found at: doi:10.1371/journal.ppat.1000130.s009 (0.02 MB ZIP) Found at: doi:10.1371/journal.ppat.1000130.s015 (0.35 MB DOC) Table S7 Accession numbers of the sequences used in this study. Found at: doi:10.1371/journal.ppat.1000130.s016 (0.93 MB DOC) p p q Found at: doi:10.1371/journal.ppat.1000130.s009 (0.02 MB ZIP) Table S1 Parameter estimates of best-fit parametric demograph- ic models in HA and NA gene datasets of reassortant and its parental strain. 95% highest probability densities (HPDs) of the estimates are shown in the parentheses. Found at: doi:10.1371/journal.ppat.1000130.s010 (0.04 MB DOC) Table S1 Parameter estimates of best-fit parametric demograph- ic models in HA and NA gene datasets of reassortant and its parental strain. 95% highest probability densities (HPDs) of the estimates are shown in the parentheses. Acknowledgements p Found at: doi:10.1371/journal.ppat.1000130.s010 (0.04 MB DOC) We gratefully acknowledge the sharing of virus samples by Ministry of Health, Indonesia, and thank the scientists in H5N1 reference laboratories for genome sequences. We thank Catherine Smith for her help in refining the geographical data used in this study. We also thank A.M. Vandamme, Philippe Lemey, Andrew Rambaut, Alexei Drummond and the anony- mous reviewers for their helpful advices. We gratefully acknowledge the support of BIOSUPPORT and HPCPOWER projects for providing bioinformatic and computational services from Computer Centre in The University of Hong Kong. We also thank W. K. Kwan and Frankie Cheung for their computational assistance. Table S2 Proportion of geographical state of the MRCAs of reassortant viruses inferred from 1,000 polytomy-resolved trees by parsimony methods. Ambiguous states estimated are ignored. Two parsimony optimizations, including delayed transformation (DEL) and accelerated transformation (ACC), were used. Found at: doi:10.1371/journal.ppat.1000130.s011 (0.04 MB DOC) Table S3 Difference between mean observed and expected number of geographical state changes in the parental strain viruses (group 2). Author Contributions Conceived and designed the experiments: TTYL FCCL. Analyzed the data: SLKP RTYW. Contributed reagents/materials/analysis tools: RTYW. Wrote the paper: SLKP. Collected and organized the sequence data: TTYL RTYW CWY FZ. Performed the research experiments and analyses: TTYL. Analyzed the data and wrote the manuscript: TTYL CCH OGP SLKP FCCL. Conceived and designed the experiments: TTYL FCCL. Analyzed the data: SLKP RTYW. Contributed reagents/materials/analysis tools: RTYW. Wrote the paper: SLKP. Collected and organized the sequence data: TTYL RTYW CWY FZ. Performed the research experiments and analyses: TTYL. Analyzed the data and wrote the manuscript: TTYL CCH OGP SLKP FCCL. Found at: doi:10.1371/journal.ppat.1000130.s012 (0.05 MB DOC) Table S4 Bayes factor testing of different molecular clock and demographic models in BMCMC analyses. Underlined are the References 9. Sedyaningsih ER, Isfandari S, Setiawaty V, Rifati L, Harun S, et al. (2007) Epidemiology of cases of H5N1 virus infection in Indonesia, July 2005–June 2006. J Infect Dis 196: 522–527. 1. Xu X, Subbarao, Cox NJ, Guo Y (1999) Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261: 15–19. 10. Smith GJ, Naipospos TS, Nguyen TD, de Jong MD, Vijaykrishna D, et al. 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Supporting Information Figure S1 Phylogenies of PB2 and PB1 genes of Indonesian H5N1 HPAI viruses. ML phylogenies reconstructed from (A) PB2 gene and (B) PB1 gene. Topological supports (.90) summarized from 1,000 ML bootstrap replications are shown. For major lineages, NJ bootstrap support (1,000 replications) and posterior probability from BMCMC analyses (5,000 tree samples) are also shown inside parentheses (ML/NJ/BMCMC). Putative human Found at: doi:10.1371/journal.ppat.1000130.s006 (0.88 MB EPS) Found at: doi:10.1371/journal.ppat.1000130.s006 (0.88 MB EPS) Figure S7 Molecular structure of the dimer formed by two N- terminal domains of the M1 matrix protein. There are 8 snapshots of the M1 dimer structure (PDB-ID: 1EA3) in which each is August 2008 \| Volume 4 \| Issue 8 \| e1000130 10 Indonesian Reassortant H5N1 Influenza Virus selected best-fit models that could not be rejected by the alternative models. Found at: doi:10.1371/journal.ppat.1000130.s013 (0.05 MB DOC) selected best-fit models that could not be rejected by the alternative models. rotated 45 degrees counter-clockwise (on x-plane) from its left snapshot. Blue and red indicate the positively and negatively charged surfaces, respectively. The residues in which the substitutions occurred along the pre-emergence lineage are highlighted with green (residues 37, 95, and 137 are indicated by white, orange, and yellow arrows, respectively). The structure is visualized by DeepView software. rotated 45 degrees counter-clockwise (on x-plane) from its left snapshot. Blue and red indicate the positively and negatively charged surfaces, respectively. The residues in which the substitutions occurred along the pre-emergence lineage are highlighted with green (residues 37, 95, and 137 are indicated by white, orange, and yellow arrows, respectively). The structure is visualized by DeepView software. Table S5 Estimations of dN/dS using 1-ratio and 2-ratio lineage-specific selection models. These estimations were per- formed in HYPHY software. Gene datasets other than PB1, HA, NA, M1, and M2 were not analyzed because group 3 is represented by the single virus IDN/6/05. Found at: doi:10.1371/journal.ppat.1000130.s014 (0.03 MB DOC) y p Found at: doi:10.1371/journal.ppat.1000130.s007 (3.54 MB TIF) y p Found at: doi:10.1371/journal.ppat.1000130.s007 (3.54 MB TIF) Figure S8 Growth curves of genetic diversity estimated by simple parametric models are superimposed on Bayesian skyline plots. at: doi:10.1371/journal.ppat.1000130.s008 (2.82 MB E Table S6 Information and phylogenetic groupings of sequences used in this study. 1, 2, 3, and X denotes groups 1, 2, 3, and unclassified (early viruses; see main text for explanation). Indonesian Reassortant H5N1 Influenza Virus Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. 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https://openalex.org/W2108074319	https://scholarshare.temple.edu/bitstream/20.500.12613/5284/2/Musashi%20proteins%20are%20post-transcriptional%20regulators%20of%20the%20epithelial-luminal%20cell%20state.pdf	English	null	Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state	eLife	2,014	cc-by	18,264	elifesciences.org elifesciences.org RESEARCH ARTICLE elifesciences.org elifesciences.org Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state Yarden Katz1,2,4†, Feifei Li3†, Nicole J Lambert4, Ethan S Sokol2,4, Wai-Leong Tam2, Albert W Cheng2,4, Edoardo M Airoldi5,6, Christopher J Lengner7,8, Piyush B Gupta2,4, Zhengquan Yu3, Rudolf Jaenisch2,4, Christopher B Burge4* 1Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, United States; 2Whitehead Institute for Biomedical Research, Cambridge, United States; 3State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China; 4Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; 5Department of Statistics, Harvard University, Cambridge, United States; 6The Broad Institute, Cambridge, United States; 7Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States; 8Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, United States Abstract The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types. DOI: 10 7554/eLife 03915 001 *For correspondence: zyu@cau. edu.cn (ZY); jaenisch@wi.mit.edu (RJ); cburge@mit.edu (CBB) †These authors contributed equally to this work Competing interests: See page 24 Competing interests: See page 24 Funding: See page 24 Received: 06 July 2014 Accepted: 05 November 2014 Published: 07 November 2014 Introduction During both normal development and cancer progression, cells undergo state transitions marked by distinct gene expression profiles and changes in morphology, motility, and other properties. The Epithelial-to-Mesenchymal Transition (EMT) is one such transition, which is essential in development and is thought to be co-opted by tumor cells undergoing metastasis (Polyak and Weinberg, 2009). Much work on cell state transitions in both the stem cell and cancer biology fields has focused on the roles that transcription factors play in driving these transitions (Polyak and Weinberg, 2009; Lee and Young, 2013), such as the induction of EMT by ectopic expression of the transcription factors Snail, Slug, or Twist (Mani et al., 2008). Reviewing editor: Benjamin J Blencowe, University of Toronto, Canada Canada now find that the Musashi proteins are also often overexpressed in human breast, lung, and prostate tumors. In addition, Musashi proteins are much less abundant in cells that have completed an epithelial-to-mesenchymal transition. When Katz, Li et al. artificially reduced the amounts of Musashi proteins in breast cancer cells, the cells migrated and dispersed, as if becoming mesenchymal cells. Furthermore, many of the genes normally used in epithelial cells were switched off. In comparison, artificially increasing the levels of Musashi proteins halted the movement of mesenchymal cells and led to increased levels of genes used in epithelial cells, as if they were reverting to epithelial cells. Therefore, it appears that the Musashi proteins prevent epithelial cells from developing mesenchymal properties. When Katz, Li et al. artificially reduced the amounts of Musashi proteins in breast cancer cells, the cells migrated and dispersed, as if becoming mesenchymal cells. Furthermore, many of the genes normally used in epithelial cells were switched off. In comparison, artificially increasing the levels of Musashi proteins halted the movement of mesenchymal cells and led to increased levels of genes used in epithelial cells, as if they were reverting to epithelial cells. Therefore, it appears that the Musashi proteins prevent epithelial cells from developing mesenchymal properties. Katz, Li et al. investigated how Musashi proteins work at the molecular level by studying neural and mammary cells in mice. This revealed that Musashi proteins control the steps that lead to the epithelial-to-mesenchymal transition by binding to the tail end of the RNA molecules that include the instructions to make certain proteins. This affects how often these proteins can be made from the RNA molecules. Katz, Li et al. suggest that Musashi proteins may similarly control the behavior of progenitor and stem cells in many other tissues as well; however, further study is needed to confirm this. DOI: 10 7554/eLife 03915 002 reprogramming of somatic cells to induced pluripotent stem cells (iPSCs), which have the essential characteristics of embryonic stem cells (ESCs). For example, overexpression of the translational regu lator and microRNA processing factor Lin28 along with three transcription factors is sufficient to repro gram somatic cells (Yu et al., 2007). The Muscleblind-like (Mbnl) family of RBPs promote differentiation by repressing an ESC-specific alternative splicing program, and inhibition of Mbnls promotes cellular reprogramming (Han et al., 2013). Canada Copyright Katz et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Recent work has shown that RNA-binding proteins (RBPs) also play important roles in cell state transitions, by driving post-transcriptional gene expression programs specific to a particular cell state. The epithelial specific regulatory protein (ESRP) family of RBPs are RNA splicing factors with epithelial tissue-specific expression whose ectopic expression can partially reverse EMT (Warzecha et al., 2009; Shapiro et al., 2011). RBPs have also been implicated in other cell state transitions, such as Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 1 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine eLife digest All living things start life as a single cell, but many organisms develop into a collection of different, specialized cells. Most of the cells in an organism can only divide to make more of the same type of cell; however, stem cells are different because they can ‘differentiate’ and develop into several different cell types. eLife digest All living things start life as a single cell, but many organisms develop into a collection of different, specialized cells. Most of the cells in an organism can only divide to make more of the same type of cell; however, stem cells are different because they can ‘differentiate’ and develop into several different cell types. A key step in the development of an embryo is called the epithelial-to-mesenchymal transition, in which an epithelial cell—a cell type that normally lines body surfaces and cavities—begins to crawl away from the tissue it is in and starts to differentiate. This transition also allows cancer cells to leave tumors and spread around the body, in a process known as metastasis. In mammals, two proteins called Musashi1 and Musashi2 are abundant in stem cells and brain cancers, but are rarely found in specialized tissues and cells. Katz, Li et al. now find that the Musashi proteins are also often overexpressed in human breast, lung, and prostate tumors. In addition, Musashi proteins are much less abundant in cells that have completed an epithelial-to-mesenchymal transition. In mammals, two proteins called Musashi1 and Musashi2 are abundant in stem cells and brain cancers, but are rarely found in specialized tissues and cells. Katz, Li et al. Msi genes are frequently overexpressed in multiple human cancers Msi genes are frequently overexpressed in multiple human cancers To obtain a broad view of the role Msis might play in human cancer, we surveyed the expression and mutation profiles of Msi genes in primary tumors using genomic and RNA sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) (Cancer Genome Atlas Network., 2012). To determine whether Msi genes are generally upregulated in human cancers, we analyzed RNA-Seq data from five cancer types for which matched tumor-control pairs were available. In these matched designs, a pair of RNA samples was obtained in parallel from a single patient's tumor and healthy tissue-matched biopsy, thus minimizing the contribution of individual genetic variation to expression differences. We observed that Msi1 was upregulated in at least 40% of breast, lung, and prostate tumors, while Msi2 was upregulated in at least 50% of breast and prostate tumors (Figure 1A, top). Overall, Msi1 or Msi2 were significantly upregulated in matched tumor-control pairs for 3 of the 5 cancer types, compared to control pairs. Kidney tumors showed the opposite expression pattern, with Msi1 and Msi2 downregulated in a majority of tumors and rarely upregulated, and in thyroid cancer neither Msi1 nor Msi2 showed a strong bias towards up- or down-regulation (Figure 1A, top). In breast tumors, a bimodal distribution of Msi1 expression was observed, with a roughly even split between up- and down-regulation of Msi1, consistent with the idea that Msi1 upregulation might be specific to a subtype of breast tumors. The bimodality of Msi1 expression was not seen when comparing control pairs, so is not explained by general variability in Msi1 levels (Figure 1A, bottom, solid vs dotted lines). Examining genome sequencing data from matched tumor-control pairs across nine diverse cancer types, we found that Msi1 and Msi2 were not significantly mutated in most of these cancers (Figure 1B). One notable exception was kidney cancer (KIRC), where non-silent mutations in Msi1 were significantly overrepresented, detectable in 9% of tumors (ranked in the 99th percentile of mutations per gene in this cancer) (Figure 1—figure supplement 1A). This observation, together with the lower Msi mRNA levels observed in matched kidney tumors (Figure 1A), is consistent with a model in which loss of Msi function is selected for in kidney tumor cells, either as a result of downregulation or mutation. Canada For ESRP, Lin28, and Mbnl proteins, the developmental or cell- type-specific expression pattern of the protein provided clues to their functions in the maintenance of epithelial, stem cell, or differentiated cell state. The Musashi (Msi) family comprises some of the most highly conserved and tissue-specific RBPs, with Drosophila Msi expressed exclusively in the nervous system (Nakamura et al., 1994; Busch and Hertel, 2011). In mammals, the two family members Msi1 and Msi2 are highly expressed in stem cell compartments but are mostly absent from differentiated tissues. Msi1 is a marker of neural stem cells (NSCs) (Sakakibara et al., 1996) and is also expressed in stem cells in the gut (Kayahara et al., 2003) and epithelial cells in the mammary gland (Colitti and Farinacci, 2009), while Msi2 is expressed in hematopoietic stem cells (HSCs) (Kharas et al., 2010). This expression pattern led to the proposal that Msi proteins generally mark the epithelial stem cell state across distinct tissues (Okano et al., 2005), with HSCs being an exception. Msi1 is not expressed in the normal adult brain outside a minority of adult NSCs but is induced in glioblastoma (Muto et al., 2012). Msi proteins affect cell proliferation in several cancer types. In glioma and medulloblastoma cell lines, knockdown of Msi1 reduced the colony-forming capacity of these cells and reduced their tumor igenic growth in a xenograft assay in mice (Muto et al., 2012). Msi expression correlates with HER2 expression in breast cancer cell lines, and knockdown of Msi proteins resulted in decreased prolifera tion (Wang et al., 2010). These observations, together with the cell-type specific expression of Msi proteins in normal development, suggested that Msi proteins might function as regulators of cell state, with potential relevance to cancer. Msi proteins have been proposed to act as translational repressors of mRNAs—and sometimes activators (MacNicol et al., 2011)—when bound to mRNA 3′ UTRs, and were speculated to aff Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 2 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine pre-mRNA processing in Drosophila (Nakamura et al., 1994; Okano et al., 2002). However, no conclusive genome-wide evidence for either role has been reported for the mammalian Msi family. Here, we aimed to investigate the roles of these proteins in human cancers and to gain a better under standing of their genome-wide effects on the transcriptome using mouse models. Msi genes are frequently overexpressed in multiple human cancers The observation that Msi1/Msi2 was not significantly mutated in most tumors but are overexpressed in several tumor types (including glioblastoma) makes their profile more similar to oncogenes like FOS or HER2, than to tumor suppressors like PTEN and TP53, which tend to have the opposite pattern (Verhaak et al., 2010; Cancer Genome Atlas Network., 2012) (Figure 1B). Msi expression marks an epithelial-luminal state and is downregulated upon EMT upon EMT To determine whether Msi overexpression is specific to a particular cancer cell state, we focused on breast cancer, where tumors with distinct properties can be robustly classified by gene expression (Parker et al., 2009; Cancer Genome Atlas Network., 2012). Unsupervised hierarchical clustering of matched tumor and control samples produced a nearly perfect separation of tumors from control samples, rather than clustering by patient/genome of origin (Figure 1—figure supplement 1B). We overlaid on top of our clustering a classification of samples into Normal, HER2+, Luminal A, Luminal B, and Basal states using RNA-Seq data to measure expression of the PAM50 gene set (Parker et al., 2009). Our clustering using all genes corresponded well to the PAM50 classification (Cancer Genome Atlas Network., 2012), separating most Luminal A from Luminal B tumors and showing a general grouping of HER2+ tumors (Figure 1—figure supplement 1B). Using this classification, we found that Msi2 was highly expressed in Luminal tumors (Figure 2A). Msi1 was more variable across tumor subtypes, often showing a bimodal profile, split between up- and down-regulation (Figure 1A and Figure 2—figure supplement 1B). Msi2 expression was highest in Luminal B tumors, which are known to be more aggressive and highly proliferating (Ki67-high) than Luminal A types and are thought to share properties with epithelial mammary progenitor cells (Das et al., 2013). These observations prompted the hypothesis that Msi proteins might be localized to epithelial cells in breast cancer tumors. The splicing factors Rbfox2 and Mbnl1 were previously identified as regulators of EMT and are Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 3 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Figure 1. Msi genes are frequently overexpressed in breast, lung, and prostate cancer but downregulated in kidney cancer. (A) Top: percentage of matched tumor–control pairs with upregulated (black-fill bars) or downregulated (grey-fill bars) Msi1 or Msi2 in five cancer types with matched RNA-Seq data. Upregulated/downregulated defined as at least two-fold change in expression in tumor relative to matched control. Asterisks indicate one-tailed statistical significance levels relative to control pairs. Bottom: distribution of fold changes for Msi1 and Msi2 in matched tumor–control pairs (solid red and green lines, respectively) and in an equal number of control pairs (dotted red and green lines, respectively.) Shaded gray density shows the fold change across all genes. Msi expression marks an epithelial-luminal state and is downregulated upon EMT (B) Percentage of tumors with non-silent mutations in Msi1/Msi2 and a select set of oncogenes and tumor suppressors across nine cancer types. Bold entries indicate genes whose mutation rate is at least two-fold above the cancer type average mutation rate. DOI: 10.7554/eLife.03915.003 The following figure supplement is available for figure 1: Figure supplement 1 Analysis of Msi1/Msi2 mutation and expression profiles in TCGA datasets Figure 1. Msi genes are frequently overexpressed in breast, lung, and prostate cancer but downregulated in kidney cancer. (A) Top: percentage of matched tumor–control pairs with upregulated (black-fill bars) or downregulated (grey-fill bars) Msi1 or Msi2 in five cancer types with matched RNA-Seq data. Upregulated/downregulated defined as at least two-fold change in expression in tumor relative to matched control. Asterisks indicate one-tailed statistical significance levels relative to control pairs. Bottom: distribution of fold changes for Msi1 and Msi2 in matched tumor–control pairs (solid red and green lines, respectively) and in an equal number of control pairs (dotted red and green lines, respectively.) Shaded gray density shows the fold change across all genes. (B) Percentage of tumors with non-silent mutations in Msi1/Msi2 and a select set of oncogenes and tumor suppressors across nine cancer types. Bold entries indicate genes whose mutation rate is at least two-fold above the cancer type average mutation rate. DOI: 10.7554/eLife.03915.003 The following figure supplement is available for figure 1: Figure supplement 1. Analysis of Msi1/Msi2 mutation and expression profiles in TCGA datasets. DOI: 10.7554/eLife.03915.004 4 of 27 4 of 27 Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 Research article Research article Research article Genomics and evolutionary biology \| Human biology and medicine y gy \| gy Figure 2. Msi is associated with the epithelial-luminal state in breast cancer. (A) mRNA expression of Msi2 across different breast tumor types RNA-Seq. (B) Immunofluorescence staining for Ecadherin (ECAD, red) and Msi1 (MSI1, green). Top: luminal human breast tumor with high num ECAD-positive cells. MSI1 shows primarily cytoplasmic localization (white arrowheads). Inset shows magnified version of ECAD and MSI sta Figure 2. Continued on next page Figure 2. Msi is associated with the epithelial-luminal state in breast cancer. (A) mRNA expression of Msi2 across different breast tumor types in TCGA RNA-Seq. (B) Immunofluorescence staining for Ecadherin (ECAD, red) and Msi1 (MSI1, green). Top: luminal human breast tumor with high number of ECAD-positive cells. MSI1 shows primarily cytoplasmic localization (white arrowheads). Figure 2. Continued Bottom: triple negative, basal-like tumor. ECAD-positive cells showed strong cytoplasmic MSI1 stain (blue arrowheads) while ECAD-negative cells were MSI1-negative (red). Single confocal stacks shown, 10 μm scale. (C) mRNA expression of Msi1, Msi2, Ecad, Fn1, Vim, and Jag1 in breast cancer cell lines by RNA-Seq (datasets are listed in Supplementary file 1). (D) Western blot for MSI1/2 (MSI1/2 cross react. antibody), MSI2, phosphorylated HER2 (p-HER2) and HER2 in panel of breast cell lines. ‘HMLE + pB’ indicates HMLE cells infected with pB empty vector, ‘HMLE + Twist’ indicates HMLE cells infected with Twist transcription factor to induce EMT. MDAMB231-derived metastatic lines (231-Brain, 231-Bone) and Sum159 are basal, HER2- negative cancer cell lines. BT474 and SKBR3 are HER2-positive, epithelial-luminal cancer cell lines. Epithelial-luminal (HER2-positive) lines show increased expression of Msi proteins compared with basal lines, and Twist-induced EMT reduces Msi expression. (E) mRNA expression of Msi1, Msi2, Ecad, Fn1, Vim, and Twist1 in GBM tumors classified as mesenchymal (n = 20) or epithelial (n = 20) using an EMT gene signature. DOI: 10.7554/eLife.03915.005 e following figure supplements are available for figure 2: Figure supplement 1. Expression of Msi1/Msi2 in subtypes of breast cancer cell lines and breast cancer tumors. DOI: 10.7554/eLife.03915.006 Figure supplement 2. Expression of Rbfox2 (Rbm9) and Mbnl1 in subtypes of breast cancer tumors from TCGA. DOI: 10.7554/eLife.03915.007 upregulated during this transition (Venables et al., 2013). Using TCGA expression analysis, we con firmed that Rbfox2 and Mbnl1 are more highly expressed in luminal tumors compared with mesenchy mal tumors, as predicted by their role in EMT (Figure 2—figure supplement 2). upregulated during this transition (Venables et al., 2013). Using TCGA expression analysis, we con firmed that Rbfox2 and Mbnl1 are more highly expressed in luminal tumors compared with mesenchy mal tumors, as predicted by their role in EMT (Figure 2—figure supplement 2). To examine the expression and distribution of Msi proteins in tumors, we stained a panel of human breast cancer tumors for MSI1 and the epithelial marker E-cadherin (ECAD). MSI1 expression was predominantly cytoplasmic (Figure 2B, top panel). Across luminal tumors, MSI1 was co-expressed with ECAD (as in Figure 2B, top panel). In triple negative/basal-like tumors, a minority of ECAD-positive cells showed strong MSI1 staining, whereas ECAD-negative cells showed little to no expression (Figure 2B, blue and red arrowheads, respectively), supporting an association between Msi and epithelial cell state in tumors. Figure 2. Continued Given the heterogeneity of human tumor samples, it is possible that the increased expression of Msi genes in luminal tumors (compared with basal) reflects the gener ally higher fraction of epithelial cells in these tumors. To explore whether Msi expression is associated with a luminal as opposed to basal state in a more homogenous system, we analyzed RNA-Seq data for luminal and basal breast cancer cell lines gener ated by multiple independent labs (RNA-Seq data sets used are listed in Supplementary file 1). Gene expression profiles from the same cell lines generated independently tended to cluster together in unsupervised clustering (supporting consistency of data across labs), and overall the basal cell lines were distinguishable from the luminal lines (Figure 2—figure supplement 1A). Matching the pattern observed in primary tumors, we observed higher Msi1 and Msi2 expression in luminal breast cancer lines than in basal lines (Figure 2C, left panel). Expression of Fibronectin (Fn1), Vimentin (Vim), and Jagged1 (Jag1), which are associated with the basal/mesenchymal state (Yamamoto et al., 2013), had the opposite pattern, showing strong enrichment in basal over luminal lines (Figure 2C, right panel). The enrichments of these four genes for either the luminal or basal state were unusual when compared to the background distribution of these enrichments across all expressed genes (Figure 2—figure supplement 1C), indicating that these genes are strong indicators of the two states. To further investigate the connection between Msi expression and EMT in breast cancer, we examined Msi expression in a panel of breast cancer-derived cell lines. Consistent with the RNA-Seq data from primary tumors, HER2+ epithelial cell lines expressed higher levels of Msi1 and Msi2 com pared with HER2– lines (Figure 2D, lane 6 and 7). A standard cell culture model of EMT is the immor talized inducible-Twist human mammary epithelial (HMLE-Twist) cell line, which undergoes EMT when induced to express the transcription factor Twist (Mani et al., 2008). We found that Msi1 was strongly downregulated in HMLE cells following Twist-induced EMT (Figure 2D), consistent with the epithelial-associated expression pattern of Msis in primary tumors (Figure 2A–C). Similarly, Msi protein expression was higher in luminal, HER2+ breast cancer lines (BT474, SKBR3 in Figure 2D) compared with basal HER2– breast cancer lines (brain and bone metastatic derivatives of MDAMB231, 231- Brain and 231-Bone, and SUM159 in Figure 2D). We next asked whether the epithelial expression signature of Msis is present in other primary tumors. Msi expression marks an epithelial-luminal state and is downregulated upon EMT Inset shows magnified version of ECAD and MSI staining. Figure 2. Continued on next page Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 5 of 27 Research article Research article Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Figure 2. Continued Figure 2. Continued Genetic system for inducible overexpression and depletion of Msi1/2 in NSCs The upregulation of Msi genes in glioblastoma motivated the choice of NSCs as a system to study the molecular roles of Msi proteins, a cell type where both proteins are highly expressed in normal devel opment, and where their target mRNAs are likely to be present. NSCs provide a well-characterized system for homogeneous cell culture (Kim et al., 2003), which is not always available for progenitor/ stem cell types cultured from other primary tissues like the mammary gland, making NSCs grown in culture amenable to analysis by genome-wide techniques. Furthermore, the conserved expression of Msi genes in the nervous system and their reactivation in human glioblastoma suggests that molecular insights obtained in this system could be informative about the roles of Msi proteins in glioblastoma cells. We cultured cortical NSCs from E12.5 embryos obtained from transgenic mice with a Dox-inducible Msi1 or Msi2 allele, and from double conditional knockout mice for Msi1/Msi2, whose deletion was driven by a Tamoxifen-inducible Cre (Figure 3A). These systems enabled robust overexpression or depletion of Msi proteins (Figure 3B) within 48–72 hr of induction. To study the effects of Msi deple tion and induction on mRNA processing, expression, and translation, we used ribosome footprint profiling (Ribo-Seq) (Ingolia et al., 2009) and high-throughput sequencing of polyA-selected RNA (RNA-Seq) (Mortazavi et al., 2008) (Figure 3A). Overexpression of Msi1 alters translation of targets without causing large changes in mRNA levels When Msi1 or Msi2 were overexpressed, few significant changes in mRNA expression were observed after 48 hr (Figure 3C). This observation suggests that these factors do not directly impact transcrip tion or mRNA stability/decay but leaves open possible effects on other steps in gene expression such as mRNA translation. To determine the genome-wide effects of Msi proteins on translation, we performed Ribo-Seq on Msi1-overexpressing cells and double knockout cells. Reads from these Ribo-Seq libraries showed the expected enrichment in coding exons relative to UTRs and introns, and yielded high scores in various quality control (QC) metrics (Figure 3—figure supplement 1). These QC metrics were highly consistent across libraries, supporting comparative analysis of the resulting data (Figure 3— figure supplement 1). To examine changes in translation, we computed ‘Translational Efficiency’ (TE) values for all protein-coding genes, a measure of ribosome occupancy along messages that is defined as the ratio of the ribosome footprint read density in the ORF to the RNA-seq read density. Examination of TEs across overexpression and knockout samples yielded a handful of genes with very large changes in ribosome occupancy (Figure 3D, ‘Materials and methods’). Figure 2. Continued Given the established role of Msi proteins as regulators of Glioblastoma (GBM) cell growth and as markers of primary tumors (Muto et al., 2012), we examined whether there is a similar sub type expression pattern in GBM tumors from TCGA (Verhaak et al., 2010). We used an EMT gene Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 6 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine signature to rank GBM tumors from more epithelial to more mesenchymal, based on the similarity of each tumor's gene expression profile to that of cells undergoing EMT in culture (Feng et al., 2014). Using this ranking, we found that the top 20 most epithelial tumors expressed higher levels of Msi and epithelial markers like ECAD (Figure 2E). By contrast, the top 20 most mesenchymal tumors expressed lower levels of Msi and higher levels of mesenchymal markers like Fibronectin and Vimentin (Figure 2E). Thus, Msi expression is enriched in epithelial tumors in GBM as well, consistent with the results obtained in breast cancer tumors and cell lines. signature to rank GBM tumors from more epithelial to more mesenchymal, based on the similarity of each tumor's gene expression profile to that of cells undergoing EMT in culture (Feng et al., 2014). Using this ranking, we found that the top 20 most epithelial tumors expressed higher levels of Msi and epithelial markers like ECAD (Figure 2E). By contrast, the top 20 most mesenchymal tumors expressed lower levels of Msi and higher levels of mesenchymal markers like Fibronectin and Vimentin (Figure 2E). Thus, Msi expression is enriched in epithelial tumors in GBM as well, consistent with the results obtained in breast cancer tumors and cell lines. Taken together, these results show that Msi genes are rarely mutated but frequently overexpressed across human cancers and are strong markers of the epithelial-luminal state. This pattern suggests that Msi proteins may play a role in the maintenance of an epithelial state and/or repression of EMT, in both breast and neural cell types. To better understand the molecular functions of Msi proteins, we turned to a controlled cell culture system. Msi1 represses translation of Notch ligand Jagged1 and regulates translation of RBPs Several genes exhibited substantial changes in their translation efficiency in response to overexpression of Msi1, including six genes with increased TE and three with reduced TE (Figure 3D). Genes with increased translation included the RNA processing factor Prpf3/Prp3p, a U4/U6 snRNP-associated factor, and genes involved in epithelial cell biology such as Kirrel3/NEPH2. Genes with repressed trans lation included: Rbm22/Cwc2, another splicing factor associated with U6 snRNP; Dhx37, an RNA helicase with possible role in alternative splicing (Hirata et al., 2013); and Jag1, a ligand of Notch receptors and an important regulator of Notch signaling. No change was detected in translation of previously Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 7 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and Research article Figure 3. Genetic system for studying effects of Msi loss/gain of function on gene expression. (A) Experimental setup and use of Msi1/2 overexpression and conditional double knockout mice for derivation of neural stem cells, which were then used for ribosome profiling (Ribo-Se mRNA sequencing (RNA-Seq). (B) Western blot analysis of Musashi overexpression and knockout in neural stem cells. Overexpression and con knockout cells were exposed to Dox and 4-OHT for 72 hr, respectively. (C) mRNA-Seq expression values (RPKM) scatters between Msi1 overexp cells and controls (left), Msi2 overexpressing cells and controls right (72 hr Dox). Msi1/2 each robustly overexpressed with similar magnitude fol Dox. (D) Comparison of translational efficiency (TE) values using Ribo-Seq on Msi1 overexpressing cells on Dox (72 hr) vs controls (left) and con knockout cells following 4-OHT for 48 hr (right). Colored points indicate select genes with large changes in TE. Figure 3. Continued on next page Figure 3. Genetic system for studying effects of Msi loss/gain of function on gene expression. (A) Experimental setup and use of Msi1/2 inducible overexpression and conditional double knockout mice for derivation of neural stem cells, which were then used for ribosome profiling (Ribo-Seq) and mRNA sequencing (RNA-Seq). (B) Western blot analysis of Musashi overexpression and knockout in neural stem cells. Overexpression and conditional knockout cells were exposed to Dox and 4-OHT for 72 hr, respectively. (C) mRNA-Seq expression values (RPKM) scatters between Msi1 overexpressing cells and controls (left), Msi2 overexpressing cells and controls right (72 hr Dox). Msi1/2 each robustly overexpressed with similar magnitude following Dox. MSI1 shows high affinity for specific RNA motifs containing one or more UAGs To determine sequence-specific RNA binding preferences of Msi proteins, we used ‘RNA Bind-n-Seq’ (RBNS) to obtain quantitative and unbiased measurement of the spectrum of RNA motifs bound by recombinant MSI1 protein in vitro (Lambert et al., 2014) (Figure 4A). For each 6mer, the ‘R value’ was defined as the occurrence frequency in libraries derived from MSI1-bound RNAs divided by the corre sponding frequency in the input RNA library, and 6mer ‘enrichment’ was defined as the maximum R value observed across all protein concentrations. The fold enrichment profiles obtained by RBNS for the top five most enriched 6mers and five randomly chosen 6mers are shown in Figure 4B. Enriched 6mers exhibited similar enrichment profiles across concentrations, peaking in fold enrichment at con centrations typically between 16–64 nM (Figure 4B). To summarize the binding preferences of MSI1 from RBNS, we aligned the most enriched 6mers to generate a motif, which emphasizes that MSI1 binds predominantly to UAG-containing sequences, preferentially flanked by Us (Figure 4C). The MSI1 binding site (G/A)UAGU from a previous SELEX study was ∼threefold enriched by RBNS, along with highly similar sequences, confirming binding under our assay conditions (Imai et al., 2001; Ray et al., 2013). Closer examination of the RBNS data revealed evidence for longer, higher-affinity motifs containing multiple UAGs with short intervening spacers (not shown). Previous studies suggested that MSI1 binds 3′ UTR regions of mRNAs to regulate translation (Okano et al., 2005). We calculated the density of RBNS-enriched 6mers in 3′ UTR regions genome-wide and ranked genes by the density of enriched 6mers in their 3′ UTR (‘Materials and methods’). We observed that the 3′ UTR of Jag1—which is translationally repressed by Msi (Figure 3D)—contains a moderately high density of RBNS-enriched 6mers, ranking in the 85th percentile of all 3′ UTRs (Figure 4D). To ask whether Msi proteins can directly bind the Jag1 mRNA and test the RBNS motif, we selected two regions of the Jag1 3′ UTR that contained the highest density of RBNS-enriched 6mers for in vitro analysis (Figure 4B, top). A gel-shift assay detected strong binding of RNAs representing both regions by recom binant Msi protein, with estimated Kd values of 15 nM and 9 nM for regions 1 and 2, respectively (repre sentative gel shifts are shown in Figure 4—figure supplement 1). Since both sequences contain UAGs (Figure 4—figure supplement 1), we hypothesized that the UAGs nucleate binding. Msi1 represses translation of Notch ligand Jagged1 and regulates translation of RBPs (D) Comparison of translational efficiency (TE) values using Ribo-Seq on Msi1 overexpressing cells on Dox (72 hr) vs controls (left) and conditional knockout cells following 4-OHT for 48 hr (right). Colored points indicate select genes with large changes in TE. Figure 3. Continued on next page Figure 3. Genetic system for studying effects of Msi loss/gain of function on gene expression. (A) Experimental setup and use of Msi1/2 inducible overexpression and conditional double knockout mice for derivation of neural stem cells, which were then used for ribosome profiling (Ribo-Seq) and mRNA sequencing (RNA-Seq). (B) Western blot analysis of Musashi overexpression and knockout in neural stem cells. Overexpression and conditional knockout cells were exposed to Dox and 4-OHT for 72 hr, respectively. (C) mRNA-Seq expression values (RPKM) scatters between Msi1 overexpressing cells and controls (left), Msi2 overexpressing cells and controls right (72 hr Dox). Msi1/2 each robustly overexpressed with similar magnitude following Dox. (D) Comparison of translational efficiency (TE) values using Ribo-Seq on Msi1 overexpressing cells on Dox (72 hr) vs controls (left) and conditional knockout cells following 4-OHT for 48 hr (right). Colored points indicate select genes with large changes in TE. Figure 3. Continued on next page Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 8 of 27 Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine DOI: 10.7554/eLife.03915.008 The following figure supplement is available for figure 3: Figure supplement 1. Quality control metrics for Ribo-Seq libraries. DOI: 10.7554/eLife.03915.009 Figure 3. Continued reported Msi target Numb (Okano et al., 2002), though Numb had low coverage of Ribo-Seq reads in NSCs, reducing our statistical power to detect regulation (‘Materials and methods’). To explore whether the observed changes are mediated by direct protein binding to RNA targets, we mapped the RNA binding specificity of Msis. MSI1 shows high affinity for specific RNA motifs containing one or more UAGs (E) Percent binding of MSI1 protein to region 1 and region 2 (red curves) and mutants where UAG sites are disrupted (blue curves), measured by gel-shift (see Figure 4—figure supplement 1). Kd estimates for region 1 and region 2 are shown (mean o 2 gel-shifts per sequence). (F) Western blot analysis of Jag1 regulation by Msi: top left panel, Jag1 expression in Msi1 overexpression cells and controls ll l f i (T l l C l i d N l f i ) J 1 i l i ll d i d i f M i1 d d d Research article Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Research article Figure 4. Profiling MSI1 binding preferences by RNA Bind-n-Seq. (A) Schemaic of Bind-n-Seq experiment for MSI1 protein. Increased concentrations of MSI1-SBP fusion protein incubated with random RNA pool, pulled by straptavidin pull-down, reverse-transcribed and sequenced. (B) Fold enrichment of top five enriched 6mers (red curves) and five randomly chosen 6mers (blue curves) across protein concentrations. (C) Binding motif for MSI1. Position- weight matrix generated by global alignment of top 20 enriched 6mers. (D) Two sites in Jag1 3' UTR, region 1 and region 2, containing a high density of enriched 6mers. Top: PhyloP conservation score for 3' UTR in 20 nt windows (based on UCSC vertebrates multiple alignment). Bottom: number of enriched 6mers from BNS in 20 nt windows of 3' UTR. (E) Percent binding of MSI1 protein to region 1 and region 2 (red curves) and mutants where UAG sites are disrupted (blue curves), measured by gel-shift (see Figure 4—figure supplement 1). Kd estimates for region 1 and region 2 are shown (mean of 2 gel-shifts per sequence). (F) Western blot analysis of Jag1 regulation by Msi: top left panel, Jag1 expression in Msi1 overexpression cells and controls in cellular fractions (T—total lysate, C—cytoplasmic and N—nuclear fractions). Jag1 is translationally repressed upon induction of Msi1 and detected Figure 4. Continued on next page Figure 4. Profiling MSI1 binding preferences by RNA Bind-n-Seq. (A) Schemaic of Bind-n-Seq experiment for MSI1 protein. Increased concentrations of MSI1-SBP fusion protein incubated with random RNA pool, pulled by straptavidin pull-down, reverse-transcribed and sequenced. (B) Fold enrichment of top five enriched 6mers (red curves) and five randomly chosen 6mers (blue curves) across protein concentrations. (C) Binding motif for MSI1. MSI1 shows high affinity for specific RNA motifs containing one or more UAGs Mutation of the UAG sites to UCC reduced binding to MSI1 protein by an order of magnitude or more in each case (Figure 4E), supporting a model where MSI1 binding occurs primarily at these sites. Following Msi overexpression, the Ribo-Seq density of the Jag1 coding region was reduced by ∼fivefold, while its mRNA level was little changed, suggesting a predominant effect at the translational level (Figure 4—figure supplement 2). In double knockout cells, Jag1 mRNA increased ∼1.5-fold by RNA-Seq (Figure 4—figure supplement 2), with a similar increase in Ribo-Seq density, suggesting effects on message stability either in the absence of or as a consequence of translational derepression. Western blot analysis confirmed repression of JAG1 protein by Msi1 overexpression (Figure 4F) and derepression in double knockout cells (Figure 4G). The high similarity between MSI1 and MSI2 pro teins (over 70% identity at the amino acid level, with highly similar RNA recognition motifs) suggests similarity in function, and we confirmed that Msi2 overexpression also repressed JAG1 protein expres sion by Western analysis (Figure 4H). To directly test the hypothesis that Msi proteins regulate Jag1 translation via UTR binding, we constructed luciferase reporters for the Jag1 3' UTR and transfected these into 293T cells. Knockdown of MSI1 or knockdown of both MSI1 and MSI2 increased luciferase expression in these cells, relative to mock knockdown treatments (Figure 4—figure supplement 3). This observation also indicates that Msi-dependent regulation of Jag1 translation is conserved from murine Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 9 of 27 Genomics and evolutionary biology \| Human biology and medicin Research article Figure 4. Profiling MSI1 binding preferences by RNA Bind-n-Seq. (A) Schemaic of Bind-n-Seq experiment for MSI1 protein. Increased concentrations of MSI1-SBP fusion protein incubated with random RNA pool, pulled by straptavidin pull-down, reverse-transcribed and sequenced. (B) Fold enrichment o top five enriched 6mers (red curves) and five randomly chosen 6mers (blue curves) across protein concentrations. (C) Binding motif for MSI1. Position weight matrix generated by global alignment of top 20 enriched 6mers. (D) Two sites in Jag1 3' UTR, region 1 and region 2, containing a high density of enriched 6mers. Top: PhyloP conservation score for 3' UTR in 20 nt windows (based on UCSC vertebrates multiple alignment). Bottom: number of enriched 6mers from BNS in 20 nt windows of 3' UTR. Msi proteins regulate alternative splicing Since some of the largest changes in translation observed by Ribo-Seq affected RBPs with functions in RNA splicing, we hypothesized that Msi overexpression might trigger changes in pre-mRNA splic ing. Changes in mRNA splicing following Msi overexpression or depletion were assessed by analysis of RNA-seq data using the MISO software (Katz et al., 2010). For example, exon 38 in the Myo18a gene, which is predominantly included under control conditions, was modestly repressed following Msi2 overexpression and strongly repressed following Msi1 overexpression (Figure 5A). In total, we observed several hundred alternatively spliced exons that were either repressed or enhanced by overexpression or knockout of Msis (Figure 5B). Msi proteins are predominantly localized in the cyto plasm (Figure 5—figure supplement 1), even when overexpressed (Figure 3F), suggesting that these changes in pre-mRNA splicing are indirect. For example, these splicing changes may result from changes in the levels of splicing factors whose mRNAs are translationally regulated by Msi proteins. To test whether Msi1 and Msi2 affect pre-mRNA splicing in similar ways, we compared the direction of splicing changes following Msi1 or Msi2 overexpression. Exons with increased inclusion following Msi1 overexpression tended to show increased inclusion following Msi2 overexpression as well, while Msi1 OE-induced splicing changes were uncorrelated with Dox-induced changes (Figure 5C). A similar pattern was observed for exons with decreased inclusion (Figure 5C). These observations suggested that Msi1 and Msi2 trigger similar effects on mRNA splicing. Splicing changes observed in the Msi1/ Msi2 double knockout cells exposed to 4-OHT were inversely correlated to those observed following Msi overexpression (Figure 5C). This observation further supports that Msi proteins affect splicing at physiological expression levels. No correlation in splicing was observed between Msi1-induced cells and exposure to 4-OHT of double floxed cells lacking the Cre driver (Figure 5C). to human cells. In sum, our results support a model where Msi proteins directly bind to the Jag1 3′ UTR to mediate post-transcriptional repression of protein levels. to human cells. In sum, our results support a model where Msi proteins directly bind to the Jag1 3′ UTR to mediate post-transcriptional repression of protein levels. Research article Research article Genomics and evolutionary biology \| Human biology and medicine Figure 4. Continued Figure 4. Continued only in total and cytoplasmic lysates. hnRNP A1, known to shuttle between the nucleus and the cytoplasm and alpha-Tubulin used as loading controls. (G) Increased JAG1 protein levels in double knockout cells. (H) Reduced JAG1 protein levels upon Msi2 overexpression. DOI: 10.7554/eLife.03915.010 e following figure supplements are available for figure 4: Figure supplement 1. Validation by gel-shift of MSI1 binding to Jag1 3' UTR sequences. DOI: 10.7554/eLife.03915.011 Figure supplement 2. Effect of Msi1 gain and loss of function on Jag1 mRNA levels and protein expression. DOI: 10.7554/eLife.03915.012 Figure supplement 3. Validation of Msi-dependent regulation of Jag1 protein levels using luciferase reporters containing Jag1 3' UTR. DOI: 10 7554/eLife 03915 013 Figure supplement 3. Validation of Msi-dependent regulation of Jag1 protein levels using luciferase reporters containing Jag1 3' UTR. DOI: 10.7554/eLife.03915.013 to human cells. In sum, our results support a model where Msi proteins directly bind to the Jag1 3′ UTR to mediate post-transcriptional repression of protein levels. MSI1 shows high affinity for specific RNA motifs containing one or more UAGs Position- weight matrix generated by global alignment of top 20 enriched 6mers. (D) Two sites in Jag1 3' UTR, region 1 and region 2, containing a high density of enriched 6mers. Top: PhyloP conservation score for 3' UTR in 20 nt windows (based on UCSC vertebrates multiple alignment). Bottom: number of enriched 6mers from BNS in 20 nt windows of 3' UTR. (E) Percent binding of MSI1 protein to region 1 and region 2 (red curves) and mutants where UAG sites are disrupted (blue curves), measured by gel-shift (see Figure 4—figure supplement 1). Kd estimates for region 1 and region 2 are shown (mean of 2 gel-shifts per sequence). (F) Western blot analysis of Jag1 regulation by Msi: top left panel, Jag1 expression in Msi1 overexpression cells and controls in cellular fractions (T—total lysate, C—cytoplasmic and N—nuclear fractions). Jag1 is translationally repressed upon induction of Msi1 and detected Figure 4. Continued on next page Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 10 of 27 Research article R Msi-associated splicing changes are observed in cancer lines and associated with luminal state We next considered whether the splicing changes associated with Msi mis-expression in NSCs might be related to splicing changes observed in human breast cancer cells or with a particular cell state. The natural variation in Msi levels across breast cancer cell lines (Figure 2C–E) enabled a com parison of splicing patterns between Msi-high (luminal) vs Msi-low (basal) cells. To compare mouse and human splicing patterns, we identified human alternative exon trios orthologous to mouse alternative and flanking exon trios using synteny in a multi-genome alignment (Figure 5D and Supp. ‘Materials and methods’). We first compared changes (ΔΨ) in the percent spliced in (PSI or Ψ) values of mouse exons between Msi1 overexpressing cells vs controls, to ΔΨ values of orthologous exons between luminal and basal breast cancer cell lines (Figure 5E). The splicing patterns were consistent: the human orthologs of exons up-regulated in Msi1-OE NSCs had higher inclusion in luminal (Msi-high) than in basal (Msi-low) cell lines, and similarly for down-regulated exons (Figure 5E). Such agreement was observed for several different luminal and basal pairs, but was strongest when comparing HER2+ luminal lines such as BT474 and SKBR3 to basal lines, consistent with the higher Msi levels observed in HER2+ cell lines (Figure 2D). These observations support the proposition that Msi contributes to a luminal splicing program in human breast cancers by triggering changes similar to those induced in mouse NSCs. Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 11 of 27 Research article Research article Research article Genomics and evolutionary biology \| Human biology and medicine Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 12 o Figure 5. Global impact of Msi proteins on alternative splicing. (A) Sashimi plot for Myo18a alternative exon 38 with Percent Spliced In (Ψ) estimates MISO (values with 95% confidence intervals, right panel.) Exon splicing is repressed by Msi1 overexpression and slightly increased in knockout Msi1/ cells. ‘+’ indicates samples treated with Dox/Tam for overexpression/knockout cells, respectively. E12.5 neural stem cells were used for all samples Figure 5. Continued on next page Figure 5. Global impact of Msi proteins on alternative splicing. (A) Sashimi plot for Myo18a alternative exon 38 with Percent Spliced In (Ψ) estimates MISO (values with 95% confidence intervals, right panel.) Exon splicing is repressed by Msi1 overexpression and slightly increased in knockout Msi1/ Figure 5. Global impact of Msi proteins on alternative splicing. Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Figure 5. Continued Figure 5. Continued except Msi1 overexpression for which an additional E13.5 NSC time point was sequenced. (B) Number of differential events (MISO Bayes factor ≥10, ΔΨ ≥ 0.12) in each alternative RNA processing category (SE—skipped exons, A5SS—alternative 5′ splice site, A3SS—alternative 3′ splice site, MXE—mutually exclusive exons, RI—retained introns) for Msi1 overexpression (‘Msi1 OE’), Msi2 overexpression (‘Msi2 OE’), double knockouts (‘Double KO’), and a Dox control pair (‘Control’). (C) Comparison of ΔΨ in Msi1 overexpression vs control binned by direction (‘Spliced in’ or ‘Spliced out’, x-axis) to ΔΨ in Msi2 overexpression cells and in double knockout cells (along with respective Tam and Dox controls, y-axis). (D) Computational strategy for identifying human orthologs of alternative exon trios regulated in mouse neural stem cells. Orthologous exon trios were identified by synteny using multiple genome alignments. (E) Comparison of ΔΨ mouse alternative exons by Msi1 (comparing overexpression to control, x-axis) and ΔΨ of their orthologous exon trios in human (comparing luminal and basal cell lines, y-axis). Two pairs of luminal and basal cells compared: BT474 vs MDAMB231 and SKBR3 vs MDAMB231. ΔΨ value distributions summarized by violin plots with a dot indicating the mean ΔΨ value. DOI: 10.7554/eLife.03915.014 The following figure supplements are available for figure 5: Figure supplement 1. Subcellular localization of MSI1 protein in murine NSCs. DOI: 10.7554/eLife.03915.015 Figure supplement 2. Analysis of two conserved Msi-induced splicing changes in breast cancer tumors. DOI: 10.7554/eLife.03915.016 Two of the most strongly affected alternative exons in murine NSCs, Myo18a exon 38 (Figure 5A) and Erbin exon 21 (Erbb2ip, a direct binding-partner of the breast cancer oncogene HER2/Erbb2) were conserved in the human genome and detected in the transcriptomes of all analyzed breast tumors and controls. In primary tumors, these exons showed a striking cancer-associated splicing pattern, with the ERBIN exon enhanced in tumors and the MYO18A exon repressed in tumors (Figure 5— figure supplement 2A). To test whether the regulation of these exons is responsive to Msi levels, we correlated the fold change in Msi expression for each matched tumor–control pair with the ΔΨ value of the ERBIN and MYO18A exons in that pair (Figure 5—figure supplement 2B). We observed high correlation between the extent of Msi overexpression and the change in splicing in luminal tumors, particularly for MSI2. Research article As in mouse NSCs, increased expression of Msis was associated with increased inclusion of the ERBIN exon and repression of MYO18A exon splicing, suggesting that Msi-dependent regulation of splicing may be conserved not only in breast cancer cell lines but also in primary tumors. Msi-associated splicing changes are observed in cancer lines and associated with luminal state (A) Sashimi plot for Myo18a alternative exon 38 with Percent Spliced In (Ψ) estimates by MISO (values with 95% confidence intervals, right panel.) Exon splicing is repressed by Msi1 overexpression and slightly increased in knockout Msi1/2 cells. ‘+’ indicates samples treated with Dox/Tam for overexpression/knockout cells, respectively. E12.5 neural stem cells were used for all samples Figure 5. Continued on next page Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 12 of 27 Research article Msi proteins are required to maintain epithelial-luminal state in breast cancer cells and regulate EMT processes To address whether Msi proteins are functionally required for the maintenance of the luminal state, we performed RNAi knockdown of Msi1 and Msi2 in two luminal breast cancer cell lines, BT474 and MCF7- Ras, where Msi proteins are highly expressed (Figure 2C and Figure 6—figure supplement 1A). In the HER2+ luminal cell line BT474, cells grow in tightly packed epithelial colonies (Figure 6A). We observed a striking morphological change upon knockdown of MSI1 or MSI2, where cells progressively separated and acquired a basal-like appearance 3–5 days after knockdown (Figure 6A), accompa nied by reduced proliferation (not shown). A similar phenotype was observed in MCF7-Ras cells upon knockdown of MSI1 or MSI2 (Figure 6—figure supplement 1B). These results argue that Msi expres sion is required for the maintenance of the epithelial-luminal state in breast cancer cell lines. The Notch pathway regulator Jag1, which we found was translationally repressed by Msi, is known to be required for EMT. Jag1-depleted keratinocytes undergoing TGFβ-induced EMT fail to express mesenchymal markers and retain epithelial morphology (Zavadil et al., 2004). Furthermore, knock down of Jag1 in keratinocytes strongly impairs wound healing (Chigurupati et al., 2007), a process that requires cells to acquire mesenchymal properties such as migration and protrusion. Our gene expression analysis also supported the mesenchymal-basal specific expression of Jag1, which is partic ularly pronounced in breast cancer (Figure 2). The epithelial-associated expression pattern of Msi genes and the antagonistic relation between Msi and Jag1 (Figure 2) prompted the hypothesis that Msi activation promotes an epithelial cell identity, effectively blocking EMT. To test the hypothesis that Msi activation may hinder EMT processes by promoting the epithelial state, we assessed the effect of Msi knockdown and overexpression on EMT marker expression. Knockdown of MSI1 or MSI2 in the luminal cell line BT474 generally resulted in a decrease in epithelial marker expression and an increase in mesenchymal marker expression, consistent with Msi loss pro moting EMT (Figure 6B). To test whether ectopic expression of Msi in mesenchymal cancer cells can Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 13 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Figure 6. Msi levels alter EMT processes breast cancer cell lines. (A) Knockdown of Msi1/Msi2 in BT474 breast cancer cell line using lenti short hairpins (shRNAs). Brightfield images (10x magnification) shown at 24, 72, and 120 hr after Puromycin-selection. Msi2 overexpression in the basal cell layer perturbs mammary ductal branching The association of Msis with the luminal state in breast cancer tumors and their effect on the epithelial- luminal state in breast cancer cell lines prompted us to ask whether Msi proteins play similar roles in the mammary gland in vivo. During maturation, epithelial cells in the mammary gland migrate and form ducts within the mammary fat pad through a process termed mammary ductal branching mor phogenesis. The formation of the mammary ductal system is thought to be a kind of EMT (Chakrabarti et al., 2012; Foubert et al., 2010), making mammary gland an attractive system to study the regula tion of EMT in vivo. The mammary gland Terminal End Buds (TEBs) from which ducts form are organized into discrete layers of cell types, including epithelial luminal and basal cells. The identity of luminal and basal tumors is thought to resemble their mammary gland cell type counterparts. Analysis of RNA-Seq expression analysis of purified mouse mammary luminal (CD24highCD29+) and basal (CD24+CD29high) cells gener ated by dos Santos et al. (2013) revealed enrichment of Msi1 and Msi2 expression in luminal cells (not shown). As predicted by the mRNA expression profile, we observed higher MSI2 protein levels in the luminal cell layer and far lower levels in the basal (K14-positive) cell layer of mouse mammary ducts (Figure 7A). We next examined the effect of Msi overexpression on epithelial cell state in the mammary gland in order to see whether its in vivo effects on epithelial-luminal state are similar to those observed in culture models. We ectopically expressed Msi2 in the basal cell layer, where it is nearly absent normally (Figure 7A), using a basal cell-specific Dox-inducible driver, K14-rtTA. As expected, mice admin istered Dox showed significantly higher levels of MSI2 protein in the basal cell layer (Figure 7—figure supplement 1A) and overall higher levels of Msi2 mRNA in mammary epithelial cells (Figure 7B). Overexpression of Msi2 altered mammary ductal branching morphology (Figure 7C). Overexpression mice showed both a defective and delayed mammary ductal branching pattern. Msi2 overexpression resulted in fewer mammary duct branch points given, after either 4 or 7 weeks of induction with Dox, with the difference between controls and overexpression mice more pronounced after 7 weeks (Figure 7—figure supplement 1B). The TEBs in glands overexpressing Msi2 were smaller relative to controls, following either 4 or 7 weeks of induction (Figure 7C, right inset). Figure 6. Continued mesenchymal-basal line (MDAMB231). Values plotted are fold changes normalized to GAPDH. For BT474 knockdown, cells infected with hairpin against luciferase were used as control (‘Control sh’). For MDAMB231 overexpression, cells infected with tdTomato were used as controls (‘Msi1-tdT’). Msi1 levels were below detection limit in control MDAMB231 cells, therefore Msi1 fold change in MDAMB231 Msi1-overexpression cells (relative to controls) was truncated arbitrarily in plot, indicated by ‘^’. (C) Representative transwell assay image for LM2 control and Msi1-OE breast cancer cells. (D) Quantification of percent of well covered in transwell assay for LM2 control and Msi1-OE cells (4 wells per condition, individual well values plotted as dots.). DOI: 10.7554/eLife.03915.017 The following figure supplement is available for figure 6: Figure supplement 1. Knockdown of Msi1/2 in breast cancer cell lines. DOI: 10.7554/eLife.03915.018 Figure supplement 1. Knockdown of Msi1/2 in breast cancer cell lines. DOI: 10.7554/eLife.03915.018 promote an epithelial state, we overexpressed Msi1 in the mesenchymal cell line MDAMB231, where Msi1 levels are extremely low. Msi1-overexpressing cells had decreased mesenchymal marker expres sion and increased levels of epithelial marker expression (Figure 6B), consistent with promotion of the epithelial state. We conclude that Msi activation promotes the epithelial state in breast cancer cells. We next asked whether the increase in epithelial markers following Msi overexpression is accompa nied by functional changes that reflect the epithelial state. We predicted that ectopic expression of Msi proteins in a mesenchymal cell line would hinder EMT-associated processes such as migration. Msi1 overexpression in the LM2 cell line (an MDAMB231-derivative) resulted in sevenfold reduction in migration in a transwell assay (Figure 6C,D). We were unable to observe this phenotype in the mesen chymal cell lines MDAMB231 or SUM159, where Msi1 overexpression caused no significant change in migration in the same transwell assays (data not shown). In NSCs, overexpression of Msi1 or Msi2 impaired migration as assayed by a scratch assay as well (data not shown), consistent with the phenotype observed in LM2 breast cancer cells. These results show that depending on the cell-type context, Msi activation can decrease the migration capacity of cells, consistent with promotion of an epithelial state and suppression of mesenchymal properties. Msi proteins are required to maintain epithelial-luminal state in breast cancer cells and regulate EMT processes (B) mRNA expressio and mesenchymal markers upon knockdown of Msi1/Msi2 in epithelial-luminal breast cancer cell line (BT474) and overexpression o Figure 6. Continued on next page Figure 6. Msi levels alter EMT processes breast cancer cell lines. (A) Knockdown of Msi1/Msi2 in BT474 breast cancer cell line using lentiviruses carrying short hairpins (shRNAs). Brightfield images (10x magnification) shown at 24, 72, and 120 hr after Puromycin-selection. (B) mRNA expression of epithelial and mesenchymal markers upon knockdown of Msi1/Msi2 in epithelial-luminal breast cancer cell line (BT474) and overexpression of Msi1 in Figure 6. Continued on next page Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 14 of 27 Research article Research article Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Figure 6. Continued Figure 6. Continued Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 Genomics and evolutionary biology \| Human biology and medicine (arrows). Scale bar: 100 μm. (G) qRT-PCR for Slug, Gata3, Twist1, Twist2 in mammary epithelial cells from control and Msi2 overexpressing mice. Slug expression in basal cell layer is reduced upon Dox (arrows). Scale bar: 50 μm. Figure 7. Continued (arrows). Scale bar: 100 μm. (G) qRT-PCR for Slug, Gata3, Twist1, Twist2 in mammary epithelial cells from control and Msi2 overexpressing mice. Slug expression in basal cell layer is reduced upon Dox (arrows). Scale bar: 50 μm. DOI: 10.7554/eLife.03915.019 The following figure supplements are available for figure 7: Figure supplement 1. Msi2 overexpression in mouse mammary gland alters mammary duct morphology. DOI: 10.7554/eLife.03915.020 Figure supplement 1. Msi2 overexpression in mouse mammary gland alters mammary duct morphology DOI: 10.7554/eLife.03915.020 Figure supplement 2. Msi2 overexpression in mouse mammary gland represses Slug and Jag1. DOI: 10.7554/eLife.03915.021 Figure supplement 2. Msi2 overexpression in mouse mammary gland represses Slug and Jag1. DOI: 10.7554/eLife.03915.021 of induction, glands from overexpression mice had shorter ductal lengths relative to controls, but ductal lengths returned to lengths similar to wild type after 7 weeks of induction (Figure 7—figure supplement 1C). These results indicate that Msi2 overexpression resulted in a defect in mammary branching morphogenesis (evidenced by the reduced number of branch points), and a delay in this process, as indicated by the slower rate of branch ductal growth. Since branching morphogenesis requires cells to lose their epithelial identity and undergo migra tion, we hypothesized that the observed defect in branching morphology might result from inability of cells to lose their epithelial identity and/or expansion of an epithelial cell layer. Consistent with this hypothesis, we observed that Msi2 overexpression resulted in expansion of the luminal cell layer (Figure 7D and Figure 7—figure supplement 1D), confirmed by a corresponding increase in expres sion of luminal cell markers and a decrease in basal markers (Figure 7E). Furthermore, Msi2 overex pression led to an increase in epithelial marker E-cadherin and reduction in Slug, a marker of EMT and mesenchymal cells. Expression of EMT regulators Slug, Twist1, and Twist2 decreased upon Msi2 over expression, while expression of the luminal epithelial cell marker Gata3 increased (Figure 7G and Figure 7—figure supplement 2A). Expression of JAG1 protein was also reduced upon Msi2 overex pression, consistent with the results observed in murine NSCs (Figure 7—figure supplement 2B,C). Genomics and evolutionary biology \| Human biology and medicine These results support a model in which ectopic Msi expression leads to expansion of epithelial-luminal cells in the mammary gland, effectively blocking EMT processes required for normal branching mor phogenesis, and resulting in the defective ductal branching pattern described above. The observed functions of Msi proteins in regulation of mammary epithelial cell state mirror the functions we observed in breast cancer cell lines and murine NSCs, and suggest that Msi proteins play similar roles in a healthy in vivo context as in cancer cells. Msi2 overexpression in the basal cell layer perturbs mammary ductal branching In addition, after 4 weeks Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 15 of 27 Research article Research article Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Figure 7. Msi2 activation represses EMT and expands mammary luminal cell layer in vivo. (A) Immunostaining for MSI2, K14, and DAPI in control sections of mammary gland. Scale bar: 50 μm (B) qRT-PCR for Msi2 in mammary epithelial cells from control and Msi2 overexpressing mice (‘Msi2-OE’). (C) Whole mount stain for mammary glands from control and Msi2 overexpressing mice (left: low magnification, right: high magnification.) (D) Immunostaining for K14, K8, and DAPI in mammary gland sections from control and Msi2 overexpressing mice. Scale bar: 100 μm (E) qRT-PCR for luminal markers (K8, K18), basal markers (K14), and smooth-muscle Actin (SMA) in mammary epithelial cells from control and Msi2 overexpressing mice. (F) Staining for E-cadherin (ECAD) (top) and EMT-marker SLUG (bottom) in mammary glands from control and Msi2 overexpressing mice. Luminal cell layer is expanded upon Dox Figure 7. Continued on next page Figure 7. Msi2 activation represses EMT and expands mammary luminal cell layer in vivo. (A) Immunostaining for MSI2, K14, and DAPI in control sections of mammary gland. Scale bar: 50 μm (B) qRT-PCR for Msi2 in mammary epithelial cells from control and Msi2 overexpressing mice (‘Msi2-OE’). (C) Whole mount stain for mammary glands from control and Msi2 overexpressing mice (left: low magnification, right: high magnification.) (D) Immunostaining for K14, K8, and DAPI in mammary gland sections from control and Msi2 overexpressing mice. Scale bar: 100 μm (E) qRT-PCR for luminal markers (K8, K18), basal markers (K14), and smooth-muscle Actin (SMA) in mammary epithelial cells from control and Msi2 overexpressing mice. (F) Staining for E-cadherin (ECAD) (top) and EMT-marker SLUG (bottom) in mammary glands from control and Msi2 overexpressing mice. Luminal cell layer is expanded upon Dox Figure 7. Continued on next page Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 16 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Discussioni For example, the epithelial-specific splicing factors of the ESRP family play important roles in maintenance of epithelial state (Warzecha et al., 2009; Reinke et al., 2012). A recent study presented evidence that the transcription factor Snail can promote the mes enchymal state in part by repressing Esrp1 (Reinke et al., 2012), further highlighting the importance of post-transcriptional control in driving cell state transitions like EMT. Like master transcription factors, master post-transcriptional regulatory factors globally alter gene expression—by affecting RNA splicing, stability, localization, or translation—which makes them suitable for controlling cell identity (Jangi and Sharp, 2014). Our study shows that post-transcriptional regulatory fac tors like Msi proteins can impact both translation and pre-mRNA splicing, utilizing multiple layers of RNA regulation to reshape the transcriptome for a particular cell state. Many of the impacted splicing events are part of an epithelial splicing program, suggesting that effects of Msis on splicing may reinforce the effects of Jag1 repression on maintenance of epithelial cell state. The predominantly cytoplasmic expression of Msis makes it likely that splicing is affected indirectly, e.g., through translational regulation of specific splic ing factors, though our data do not rule out that a small fraction of Msi protein may be nuclear localized and could directly regulate splicing. We have also observed that other RBPs are also enriched in the epithe lial state (Shapiro et al., 2011), suggesting that RBPs as a group may play a broad role in maintenance of this state, and might provide attractive targets for therapeutic efforts to manipulate cell state. Msi proteins are co-expressed with various proliferation markers in a wide variety of stem cell niches, including the breast, stomach, intestine, lung, and brain. This observation suggests the hypo thesis that Msis may act as general epithelial stem cell/progenitor regulators across tissues. Our find ings are consistent with this hypothesis, but further study of Msi in multiple stem cell compartments will be needed to directly test it. The role of Msi in the normal development and transformation of other adult tissues will also be important to understand. For example, our observation that Msi is frequently overexpressed in lung tumors suggests that ectopic expression of Msi proteins in the lung could elucidate their role in lung cancer. Discussioni A model where Msi pro teins repress translation by outcompeting eIF4G for PolyA-binding protein (PABP) was pro posed (Kawahara et al., 2008), but the condi tions under which binding to mRNA results in translational repression are unclear, since only a subset of mRNAs are detectably regulated. It is possible that co-factors are required in vivo for Msi to affect translation following binding to the mRNA. It is also possible that other RNA-binding factors outcompete Msi protein for binding, though MSI1 has relative high RNA-binding affinity. The molecular mechanism underlying Musashi-dependent translational control and the nature of any co- factors involved are not known. Figure 8. Model for Msi roles in regulation of cell state. Model for Msi role in the control of the epithelial state. We show that Msi represses translation of Jag1, a positive regulator of Notch and EMT. We also show that Msi promotes expression of an epithelial-luminal splicing program, which we hypothesize occurs through transla tional regulation of splicing factors. In the model, both the direct regulation of Jag1 and indirect regulation of splicing contribute to maintenance of an epithelial- luminal cell state and inhibition of EMT. DOI: 10.7554/eLife.03915.022 Figure 8. Model for Msi roles in regulation of cell state. Model for Msi role in the control of the epithelial state. We show that Msi represses translation of Jag1, a positive regulator of Notch and EMT. We also show that Msi promotes expression of an epithelial-luminal splicing program, which we hypothesize occurs through transla tional regulation of splicing factors. In the model, both the direct regulation of Jag1 and indirect regulation of splicing contribute to maintenance of an epithelial- luminal cell state and inhibition of EMT. DOI: 10 7554/eLife 03915 022 Figure 8. Model for Msi roles in regulation of cell state. Model for Msi role in the control of the epithelial state. We show that Msi represses translation of Jag1, a positive regulator of Notch and EMT. We also show that Msi promotes expression of an epithelial-luminal splicing program, which we hypothesize occurs through transla tional regulation of splicing factors. In the model, both the direct regulation of Jag1 and indirect regulation of splicing contribute to maintenance of an epithelial- luminal cell state and inhibition of EMT. DOI: 10 7554/eLife 03915 022 This study complements recent reports of the involvement of post-transcriptional regulatory factors in cell state maintenance and EMT. Discussioni Collectively, these studies are con sistent with our working model in which Msi represses Jag1 translationally, in turn altering Notch activity required for EMT. ence of EMT on Notch activation has been observed in normal development as well. During heart devel opment, cardiac valves are generated from endo cardium through EMT, and Notch activity was shown to be required for this process (Timmerman et al., 2004). Collectively, these studies are con sistent with our working model in which Msi represses Jag1 translationally, in turn altering Notch activity required for EMT. The molecular mechanisms by which Msi pro teins regulate translation of a subset of mRNAs like Jag1 remains unclear. Our genome-wide data and in vitro binding assays indicate that Msi proteins act by binding UAG-containing motifs at 3' UTRs of messages. A model where Msi pro teins repress translation by outcompeting eIF4G for PolyA-binding protein (PABP) was pro posed (Kawahara et al., 2008), but the condi tions under which binding to mRNA results in translational repression are unclear, since only a subset of mRNAs are detectably regulated. It is possible that co-factors are required in vivo for Msi to affect translation following binding to the mRNA. It is also possible that other RNA-binding factors outcompete Msi protein for binding, though MSI1 has relative high RNA-binding affinity. The molecular mechanism underlying Musashi-dependent translational control and the nature of any co- factors involved are not known. Figure 8. Model for Msi roles in regulation of cell state. Model for Msi role in the control of the epithelial state. We show that Msi represses translation of Jag1, a positive regulator of Notch and EMT. We also show that Msi promotes expression of an epithelial-luminal splicing program, which we hypothesize occurs through transla tional regulation of splicing factors. In the model, both the direct regulation of Jag1 and indirect regulation of splicing contribute to maintenance of an epithelial- luminal cell state and inhibition of EMT. DOI: 10.7554/eLife.03915.022 The molecular mechanisms by which Msi pro teins regulate translation of a subset of mRNAs like Jag1 remains unclear. Our genome-wide data and in vitro binding assays indicate that Msi proteins act by binding UAG-containing motifs at 3' UTRs of messages. Discussioni The specific expression patterns of Msi proteins in stem and epithelial cells have aroused interest in their functional roles. Here, we show that Msi proteins are associated with the epithelial-luminal cell state in several cancer types, notably breast cancer, where Msi genes are highly enriched in luminal tumors and luminal breast cancer cell lines. We showed that in breast cancer cells, knock down of Msi genes leads to loss of epithelial identity and upregulation of mesenchymal markers, while their ectopic activation promotes the epithelial state and suppresses mesenchymal proper ties such as cell migration. As in cancer cells, overexpression of Msi2 in healthy mammary gland tissue suppressed EMT and resulted in a defective mammary ductal branching pattern. These observations all support a role for Msi proteins in maintenance of a luminal/epithelial cell state and inhibition of EMT (Figure 8). The consistency between our observations in mammary epithelial cells and NSCs and between mouse and human suggests that these functions are shared across cell types and evolutionarily conserved. Our genome-wide data support the hypothesis that Msi proteins are translational regulators. We showed that Msi proteins can translationally repress Jag1, an important regulator of Notch signal ing. However, the role of Notch signaling in cancer remains complex and may vary between cancer types (Dickson et al., 2007; Lobry et al., 2011). The upregulation of Jag1 in the basal state suggests that Notch pathway activity is high in and required for the entry into the mesenchymal state, consistent with previous studies (Zavadil et al., 2004; Dickson et al., 2007). In mammary epithelial cells, Jag1- triggered activation of Notch was shown to reduce E-cadherin expression and increase Slug expres sion (Leong et al., 2007). Furthermore, Jag1 activation in breast cancer cells promotes their metastasis Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 17 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine into the bone in vivo by activating Notch in neigh boring bone cells (Sethi et al., 2011). The depend ence of EMT on Notch activation has been observed in normal development as well. During heart devel opment, cardiac valves are generated from endo cardium through EMT, and Notch activity was shown to be required for this process (Timmerman et al., 2004). Discussioni Furthermore, the systematic downregulation of Msi1/Msi2 and high frequency of Msi1 mutations in kidney tumors suggests that kidney would be an informative model for studying Msi loss-of-function and its consequences in cancer. Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 18 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Culture conditions for human breast cancer lines, shRNA knockdowns and overexpression assays All breast cancer lines were cultured in DME containing 10% FBS, 1% GlutaMAX (Gibco), and Penn/ Strep, except for BT474, which was cultured in RPMI base medium, and SKBR3 which was cultured with McCoy's 5A supplement. Lentiviruses carrying pLKO vectors with hairpins against Msi1, Msi2, or Luciferase (control) were used for knockdowns. Hairpins were obtained from Broad Institute shRNA library. Cells were infected in a centrifuge spin-infection step (1500 RPM, 37°C, 20 min) following a 2-hr incubation with polybrene or protamine sulfate, and viral medium was added to the cells overnight. Cells were subjected to 4–6 day Puromycin selection (2 μg/ml) 48 hr after infection. Msi1-OE vector (Thermo OpenBiosystems) was used for overexpression assays. Virus was prepared was described above and cell lines infected with virus were selected for 4–6 days with Blasticidin (5 μg/ml) 48 hr after infection. Mouse strains and derivation of neural stem cell lines Mouse strains and derivation of neural stem cell lines Inducible overexpression mice (tetO-Msi1/Msi2) were generated as previously described in Beard et al. (2006); Kharas et al. (2010). The generation of Msi2 conditional knockout mice was previ ously described in Park et al. (2014), and the generation of Msi1 conditional knockout mice will be described elsewhere (Yu et al., under review). Mice of the 129SvJae strain were used, and the K14-rtTA strain was obtained from JAX (stock number: 007678). Animal care was in accordance with institutional guidelines and approved by the Committee on Animal Care, Department of Comparative Medicine, Massachusetts Institute of Technology, under animal protocol 1013-088- 16. For derivation of embryonic neural stem cells (NSCs), littermate embryos were used whenever possible. Cortical NSCs were derived from embryos following Kim et al. (2003). Briefly, cortical tissue was isolated from E12.5 embryos (unless otherwise noted) under a light dissection micro scope inside a sterile fume hood and collected by centrifugation. Cortical tissues were dissociated into single cells by trituration in Magnesium/Calcium-free HBSS buffer (Gibco, Woburn MA) fol lowed by 15-min incubation at room temperature. Dissociated tissue was collected by centrifuga tion, resuspended in N2 medium containing growth factors and Laminin (Life Technologies, Woburn MA, Catalog Number: 23017015) and plated onto Polyornithin/Laminin-coated tissue culture dishes as in Okabe et al. (1996). Culture conditions for embryonic neural stem cells NSCs were grown in N2 medium (Okabe et al., 1996) containing EGF (20 ng/ml) and bFGF (20 ng/ml) and Laminin (Life Technologies). Cells were grown on Polyornithin/Laminin-coated dishes. EMT was induced by switching cells to N2 medium containing LIF/FBS as described in Ber et al. (2012). Migration assay in breast cancer cell lines Migration assay was performed using the transwells (Corning 6.5 mm Diameter inserts with 8um pore size, polycarbonate membrane; product #3422, lot #19614003). 50,000 cells were seeded into wells in each condition and allowed to migrate for 9 hr. Cells were stained with Crystal Violet and then percent area covered was calculated using ImageJ. Images were threshold filtered on Hue and Saturation (Hue: 192-255 'pass'; Saturation: 72-255 'pass') and passed to the ‘Analyze Particles’ function with a thresh old size of 2000. Western blotting, immunofluorescence staining, and antibodies used For western blotting, cells were lysed on ice and protein lysates were loaded onto 4-12% gradient Bis-Tris Gel (Life Technologies). Primary antibodies and dilutions used in western blotting on murine NSCs: anti- MSI1/2 (Cell Signaling Technology #2154, 1:800), anti-MSI2 (Abcam #57341, 1:800), anti-Jag1 (Cell Signaling Technology #2620, 1:800), anti-HER2 (Cell Signaling Technologies #2248, 1:1000), anti-phos- HER2 (Cell Signaling Technology #2241, 1:1000), anti-alpha-Tubulin (Sigma-Aldrich T9026, 1:5000), anti- HNRNPA1 (Abcam ab5832, 1:800). Immunofluorescene was performed on cells grown on glass bottom chambers (LabTek II, #1.5), fixed in 4% PFA. Cells were blocked and permeabilized in 5% FBS, .1% Triton in PBS(+). Antibodies were applied in 1% FBS in PBS(+). Immunofluorescence antibodies and dilutions: anti- MSI1 (MBL D270-3, 1:500), anti-HNRNP A2/B1 (Santa Cruz, sc-374052, 1:200). For IHC on murine mam mary glands, anti-Jag1 (Santa Cruz, SC-6011, 1:100) was used. For western on murine mammary glands, anti-Jag1 (Santa Cruz, SC-6011, 1:1000) and anti-Tubulin (Sigma-Aldrich, T5168, 1:4000) were used. Culture conditions for embryonic neural stem cells NSCs were grown in N2 medium (Okabe et al., 1996) containing EGF (20 ng/ml) and bFGF (20 ng/ and Laminin (Life Technologies). Cells were grown on Polyornithin/Laminin-coated dishes. EMT w nduced by switching cells to N2 medium containing LIF/FBS as described in Ber et al. (2012). Immunohistochemistry on human breast cancer sections Paraffin-embedded human breast cancer sections were obtained from Biomax US (BR1505a) an stained using standard protocols with antigen retrieval. Antibodies used: anti-ECAD1 (BD Bioscience 1:50) and anti-MSI1 (MBL D270-3, 1:200). y Paraffin-embedded human breast cancer sections were obtained from Biomax US (BR1505a) and stained using standard protocols with antigen retrieval. Antibodies used: anti-ECAD1 (BD Biosciences, 1:50) and anti-MSI1 (MBL D270-3, 1:200). Confocal imaging for immunofluorescence Confocal imaging was performed using a Perkin–Elmer microscope using oil-immersion 63× objective, imaged with Velocity software. Single confocal stacks or maximum Z intensity projections were obtained using Fiji (Bioformats-LOCI plugin). Computational analysis of RNA-Seq, ribosome profiling and bind-n-seq Ribosome profiling (ribo-seq) analysisi estern blotting, immunofluorescence staining, and antibodies used Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 19 of 27 Research article Research article Ribosome profiling (ribo-seq) analysisi To define a set of translationally regulated targets, we first filtered out genes that had low read counts (5 reads or less) in constitutive CDS exons in either RNA-Seq or Ribo-Seq data. We then further filtered out from this set genes that showed 1.5-fold change or greater in mRNA levels between control and experimental samples, to avoid instances where changes in TE may be confounded by changes in mRNA abundances, and therefore are less likely to be controlled solely at the level of translation. From this set of genes, we defined the subset that had a threefold or higher change in TE as the set of trans lational targets. RNA-seq and ribosome profiling library generation RNA-Seq libraries were prepared from polyA-selected RNA using standard Illumina protocol. Ribosome profiling libraries were prepared following Ingolia et al. (2009) with several modifications. Briefly, cells were collected by centrifugation and immediately flash-frozen. Cells were thawed in lysis buffer (20 mM HEPES [pH 7.0], 100 mM KCl, 5 mM MgCl2, 0.5% Na-Deoxycholate, 0.5% NP-40, 1 mM DTT, Roche mini EDTA-free protease inhibitor tablets [1 tablet/10 ml]) and briefly treated with DNase I and RNAse I. Nuclei and cell debris were removed by centrifugation and lysates were treated with RNase I (NEB) for 75 min at room temperature to generate monosome-protected RNA fragments. Monosomes were collected by ultracentrifugation in a sucrose cushion, denatured in 8 M Guanidium HCl, and protected RNA fragments (footprints) were extracted with Phenol–Chloroform. Footprints were dephosphoryl ated by PNK treatment and size-selected (∼31–35 nt fragments) by purification from a 15% TBE-Urea gel. Subtractive hybridization of ribosomal RNA from footprints was performed as in (Wang et al., 2012). Footprints were then polyA-tailed, and Illumina sequencing adaptors were added in a reverse transcription step to obtain footprint cDNA, which was then isolated by gel purification. cDNA was then circularized, PCR-amplified, and PCR products isolated by gel purification and submitted for sequencing on Illumina Hi-Seq platform. Computational analysis of RNA-Seq, ribosome profiling and bind-n-seq Source code for the pipelines used to analyze RNA-Seq, ribosome profiling and Bind-n-Seq data is available through the open-source library rnaseqlib (available at the git repository: http://www.github. com/yarden/rnaseqlib). Protocols, raw sequencing data and additional information about genomic datasets are available at http://www.musashi-genes.org. Computational analysis of TCGA data p y Publicly available TCGA data sets (Level 2 and Level 3) were downloaded from NIH ‘Bulk Download’ website (RNASeqV2: https://wiki.nci.nih.gov/display/TCGA/RNASeq+Version+2). RNA-Seq analyses were performed using ‘RNASeqV2’ TCGA files. Fold changes for genes were normalized by correction with Lowess-fit of MA-values calculated using raw gene expression estimates. Alternative exon expres sion was quantified using MISO. Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Sequencing data availability All RNA sequencing data was submitted to GEO (accession GSE58423). Computational identification of orthologous exon trios between mouse and human Syntenic regions for exons in mouse alternative exon trios (mm9) were computed using Ensembl Compara Database (Release 66) PECAN multiple genomes alignment, using the Pycogent Python frame work (Knight et al., 2007). Syntenic coordinates in human genome (hg19) were then matched to anno tated hg19 exon coordinates given in TCGA data files. On Numb as a translational target of Msi proteins Early work on mammalian Musashi proteins by the Okano group and colleagues suggested that Numb mRNA is translationally repressed by MSI1 (Okano et al., 2002). A later study by the same group showed that in the gastric system, Msi1 KO mice had lower, not higher, levels of Numb protein, oppo site of the expected change under the translational repression model (Takahashi et al., 2013). Recent work in HSCs (where only Msi2 is expressed) showed a Numb-independent phenotype for Msi2 and found that Msi2 KO HSCs have unchanged levels of Numb protein (Park et al., 2014). Thus, it is unclear if Msi1 or Msi2 directly regulate Numb mRNA translation in all systems and whether such reg ulation always promotes or represses translation of the mRNA. In our data from NSCs, we were unable to detect a large difference in Numb translational efficiency upon Msi1 overexpression as measured by Ribo-Seq, though a small effect cannot be excluded since coverage of the Numb mRNA in our Ribo-Seq data was low. It is possible that Msi1 affects the trans lation of certain Numb mRNA isoforms in a context-specific manner, potentially through alternative mRNA processing of the Numb mRNA, as proposed by Takahashi et al. (2013). Bind-n-seq (RBNS) analysis Bind-n-seq (RBNS) analysis q y To define a set of genes with enriched Msi binding sites, we ranked genes according to the abundance of RBNS-enriched 6mers in their 3' UTR. For each gene g, we calculated the density an RBNS-enriched 6mer k in the gene, Dg,k, as follows: mer k in the gene, Dg,k, as follows: , = 6+1 k g k n D u − where nk is the number of occurrences of the 6mer k in the longest 3' UTR of g, and u is the UTR length. We defined the enrichment density score Sg for each gene g as the sum of densities of all RBNS- enriched 6mers in the gene: , = g g k k S D ∑ We then calculated the distribution of Sg for all genes and ranked each gene by its percentile rank. The score for Jag1 (SJag1) ranked in the 85th percentile of the score distribution. Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 20 of 27 Research article Research article Luciferase reporter assays for protein translation The Jag1 3' UTR was cloned into the pRL-SV40 vector (Promega) downstream of Renilla luciferase using the XbaI and NotI restriction sites creating the Renilla-Jag1-UTR construct. Firefly luciferase expres sion was used as the internal control and expressed from the PGL3 vector (Promega). Renilla and the Firefly luciferase vectors were co-transfected into 293 cells stably expressing hairpins against Msi1, Msi2, or both Msi1 and Msi2, or into mock transfected 293T cells. Cells were harvested between 30–36 hr after transfection and the Renilla and Firefly luciferase signals measured using the Dual-luciferase Reporter Assay System (Promega) according to manufacture's instructions. In vivo overexpression and whole mount mammary gland staining Genomics and evolutionary biology \| Human biology and medicine Inguinal mammary glands were spread on glass slides, fixed in Carnoy's fixative (6:3:1, 100% ethanol: chloroform: glacial acetic acid) for 2 to 4 hr at room temperature, washed Primers and sequences related to RNA Bind-n-Seq RBNS T7 template: CCTTGACACCCGAGAATTCCA(N40)GATCGTCGGACTGTAGAACTCCCTATAGTGAGTCGTATTA T7 oligo: TAATACGACTCACTATAGGG Resulting RNA Pool: GAGTTCTACAGTCCGACGATC(N)40TGGAATTCTCGGGTGTCAAGG Binding site used for validation: GGCUUCUUAAGCGUUAGUUAUUUAGUUCGUUUGUU RBNS RT primer: GCCTTGGCACCCGAGAATTCCA RNA PCR (RP1): AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC Barcoded Primers: CAAGCAGAAGACGGCATACGAGAT–BARCODE-GTGACTGGAGTTCCTTGGCACCCGAGAA TTCCA Jag1 region 1 sequence: UGUCCAGUUAGAUCACUGUUUAGAU Jag1 region 1 mutant: UGUCCAGUUCCAUCACUGUUUCCAU Jag1 region 2 sequence: UCAAAGUAGAAUUUUUGUAUAGUUAUGUAAAUAAU Jag1 region 2 mutant: UCAAAGUCCAAUUUUUGUAUCCUUAUGUAAAUAAU Luciferase reporter assays for protein translation The Jag1 3' UTR was cloned into the pRL-SV40 vector (Promega) downstream of Renilla luciferase using the XbaI and NotI restriction sites creating the Renilla-Jag1-UTR construct. Firefly luciferase expres sion was used as the internal control and expressed from the PGL3 vector (Promega). Renilla and the Firefly luciferase vectors were co-transfected into 293 cells stably expressing hairpins against Msi1, Msi2, or both Msi1 and Msi2, or into mock transfected 293T cells. Cells were harvested between 30–36 hr after transfection and the Renilla and Firefly luciferase signals measured using the Dual-luciferase Reporter Assay System (Promega) according to manufacture's instructions. In vivo overexpression and whole mount mammary gland staining Mice were given Dox (Sigma) via drinking water at 2 g/l. Mice were induced with Dox for 7 weeks unless otherwise indicated. Inguinal mammary glands were spread on glass slides, fixed in Carnoy's fi ti (6 3 1 100% th l hl f l i l ti id) f 2 t 4 h t t t h d NA bind-n-seq protein expression, RNA preparation and binding A streptavidin binding peptide (SBP) tag was added to the pGEX6P-1 vector (GE) after the Presceission protease site. Full-length Musashi (Msi1) was cloned downstream of the SBP tag with infusion (Clontech) using BamHI and NotI cloning sites. Expression of tagged MSI1 was induced with 0.5 mM IPTG at 18° for 4 hr in the Rosetta(DE3)pLysS E. coli strain and subsequently purified on a GST GraviTrap column (GE). MSI1 was eluted from the GST column with PreScission protease (GE) in 4 mL of Protease Buffer (50 mM Tris pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) at 4° C over night (∼16 hr). Protein purity was assayed SDS-PAGE gel electrophoresis and visualized with SimplyBlue SafeStain (Invitrogen). Input random RNA was generated by T7 in vitro transcription: 1 μg T7 oligo was annealed to 1 μg of RBNS T7 template by heating the mixture at 65° C for 5 min then allowing the reaction to cool at room temperature for 2 min. The random RNA was then in vitro transcribed with HiScribe T7 In vitro transcription kit (NEB) according to manufacturer's instructions. The RNA was then gel-purified from a 6% TBE-urea gel. Nine concentrations of purified MSI1 (0 nM, 0.5 nM, 2 nM, 8 nM, 16 nM, 64 nM, 256 nM, 1 μM, and 2 μM) were equilibrated in 250 μl of Binding Buffer (25 mM Tris pH 7.5, 150 mM KCl, 3 mM MgCl2, 0.01% Tween, 1 mg/ml BSA, 1 mM DTT, 30 μg/ml poly I/C [Sigma]) for 30 min at room tem perature. 40 U of Superasin (Ambion) and 1 μM random RNA (final concentration) was added to the MSI1 solutions and incubated for 1 hr at room temperature. During this incubation, Streptavidin magnetic beads (Invitrogen) were washed three times with 1 ml of wash buffer (25 mM Tris pH 7.5, 150 mM KCl, 60 μg/ml BSA, 0.5 mM EDTA, 0.01% Tween) and then equilibrated in Binding Buffer until needed. MSI1 and interacting RNA was pulled down by adding the RNA/protein solutions to 1 mg of washed streptavidin magnetic beads and incubated for 1 hr at room temperature. Supernatant (unbound RNA) was removed from the beads and the beads washed once with 1 ml of Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 21 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Wash Buffer. The beads were incubated at 70° for 10 min in 100 μl of Elution Buffer (10 mM tris pH 7.0, 1 mM EDTA, 1% SDS) and the supernatant was collected. Bound RNA was extracted from the eluate by phenol/chloroform extraction and ethanol precipitation. Half of the extracted RNA from each condition was reverse transcribed into cDNA using Superscript III (Invitrogen) according to manufac turer’s instructions using the RBNS RT primer. To control for any nucleotide biases in the input random library, 0.5 pmol of the RBNS input RNA pool was also reverse transcribed and Illumina sequencing library prep followed by 8–10 cycles of PCR using High Fidelity Phusion (NEB). As Msi1 concentration was increased, decreasing input RT reaction was required in the PCR. For instance, the highest MSI1 condition required 30-fold less input RT product than the no MSI1 condition. All libraries were barcoded in the PCR step, pooled together, and sequenced one HiSeq 2000 lane. p, p g , q q Primers and sequences related to RNA Bind-n-Seq RBNS T7 template: CCTTGACACCCGAGAATTCCA(N40)GATCGTCGGACTGTAGAACTCCCTATAGTGAGTCGTATTA T7 oligo: TAATACGACTCACTATAGGG Resulting RNA Pool: GAGTTCTACAGTCCGACGATC(N)40TGGAATTCTCGGGTGTCAAGG Binding site used for validation: GGCUUCUUAAGCGUUAGUUAUUUAGUUCGUUUGUU RBNS RT primer: GCCTTGGCACCCGAGAATTCCA RNA PCR (RP1): AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC Barcoded Primers: CAAGCAGAAGACGGCATACGAGAT–BARCODE-GTGACTGGAGTTCCTTGGCACCCGAGAA TTCCA Jag1 region 1 sequence: UGUCCAGUUAGAUCACUGUUUAGAU Jag1 region 1 mutant: UGUCCAGUUCCAUCACUGUUUCCAU Jag1 region 2 sequence: UCAAAGUAGAAUUUUUGUAUAGUUAUGUAAAUAAU Jag1 region 2 mutant: UCAAAGUCCAAUUUUUGUAUCCUUAUGUAAAUAAU Luciferase reporter assays for protein translation The Jag1 3' UTR was cloned into the pRL-SV40 vector (Promega) downstream of Renilla luciferase using the XbaI and NotI restriction sites creating the Renilla-Jag1-UTR construct. Firefly luciferase expres sion was used as the internal control and expressed from the PGL3 vector (Promega). Renilla and the Firefly luciferase vectors were co-transfected into 293 cells stably expressing hairpins against Msi1, Msi2, or both Msi1 and Msi2, or into mock transfected 293T cells. Cells were harvested between 30–36 hr after transfection and the Renilla and Firefly luciferase signals measured using the Dual-luciferase Reporter Assay System (Promega) according to manufacture's instructions. In vivo overexpression and whole mount mammary gland staining Mice were given Dox (Sigma) via drinking water at 2 g/l. Mice were induced with Dox for 7 weeks unless otherwise indicated. Genomics and evolutionary biology \| Human biology and medicine in 70% ethanol for 15 min, rinsed through graded alcohol followed by distilled water for 5 min, then stained in carmine alum overnight, washed in 70%, 95%, 100% ethanol for 15 min each, cleared in xylene, and mounted with Permount. in 70% ethanol for 15 min, rinsed through graded alcohol followed by distilled water for 5 min, then stained in carmine alum overnight, washed in 70%, 95%, 100% ethanol for 15 min each, cleared in xylene, and mounted with Permount. Quantitative RT-PCR analysis in mammary glands Mouse mammary epithelial cells were prepared according to the manufacturer's protocol (Stem Cell Technologies, Vancouver, Canada). Briefly, following removal of the lymph node, mammary glands dissected from 10-week-old virgin female mice were digested in EpiCult-B with 5% fetal bovine serum (FBS), 300 U/ml collagenase, and 100 U/ml hyaluronidase for 8 hr at 37°C. After vortexing and lysis of the red blood cells in NH4Cl, mammary epithelial cells were obtained by sequential dissociation of the fragments by gentle pipetting for 1–2 min in 0.25% trypsin, and 2 min in 5 mg/ml dispase plus 0.1 mg/ml DNase I (DNase; Sigma). Total RNA was isolated from mammary epithelial cells. Complementary DNA was prepared using the MMLV cDNA synthesis kit (Promega). Quantitative RT-PCR was performed using the SYBR-green detection system (Roche). Primers were as follows: Msi2 forward primer: ACGACTCCCAGCACGACC; Msi2 reverse primer: GCCAGCTCAGTCCA CCGATA. Msi2 forward primer: ACGACTCCCAGCACGACC; Msi2 reverse primer: GCCAGCTCAGTCCA CCGATA. K8 forward primer: ATCAAGAAGGATGTGGACGAA; K8 Reverse primer: TTGGCAATGTCCT CGTACTG. K14 forward primer: CAGCCCCTACTTCAAGACCA; K14 Reverse primer: AATCTGCAGGA GGACATTGG. K18 forward primer: TGCCGCCGATGACTTTAGA; K18 Reverse primer: TTGCTGAGGTCC TGAGATTTG. Immunofluorescence on mammary gland sections Mammary glands were fixed in 4% PFA, paraffin-embedded and 5-μm sections were used for immuno fluorescence assay. Paraffin sections were microwave pretreated and incubated with primary antibodies, then incubated with secondary antibodies (Invitrogen) and counterstained with DAPI in mounting media. The following antibodies were used: anti-K14 (Abcam), anti-K8 (Abcam), anti-E-cadherin (CST), anti-Msi2 (Novus Biologicals), anti-Hes1 (Abcam), anti-Slug (CST). In vivo overexpression and whole mount mammary gland staining Mice were given Dox (Sigma) via drinking water at 2 g/l. Mice were induced with Dox for 7 weeks unless otherwise indicated. Inguinal mammary glands were spread on glass slides, fixed in Carnoy's fixative (6:3:1, 100% ethanol: chloroform: glacial acetic acid) for 2 to 4 hr at room temperature, washed Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 22 of 27 22 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Additional information Competing interests EMA: Reviewing Editor, eLife. The other authors declare that no competing interests exi Funding Funder Grant reference number Author National Institute of General Medical Sciences R01-GM085319 Christopher B Burge National Cancer Institute U01-CA184897 Christopher B Burge National Cancer Institute RO1-CA084198 Rudolf Jaenisch National Institute of General Medical Sciences R01-GM096193 Edoardo M Airoldi The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. p g EMA: Reviewing Editor, eLife. The other authors declare that no competing interests exist. Quantitative RT-PCR analysis in breast cancer cell lines Quantitative RT-PCR analysis in breast cancer cell lines RNA was extracted using Trizol and cDNA was prepared using SuperScript III (Invitrogen). Primers used are listed below (‘h’ prefix denotes human gene, ‘F’ denotes forward primer, ‘R’ denotes reverse primer): hEcad-F: hEcad-F: TGCCCAGAAAATGAAAAAGG hEcad-R: GTGTATGTGGCAATGCGTTC hTwist-F: GGAGTCCGCAGTCTTACGAG hTwist-R: TCTGGAGGACCTGGTAGAGG hEpCAM-F: CTTTAAGGCCAAGCAGTGCA hEpCAM-R: CGCGTTGTGATCTCCTTCTG hCD24-F: GGTTTGACTAGATGATGGATGCC hCD24-R: TCCATTCCACAATCCCATCCT hMsi1-F: GGGACTCAGTTGGCAGACTAC hMsi1-R: CTGGTCCATGAAAGTGACGAA hMsi2-F: TGCCCAGAAAATGAAAAAGG hEcad-R: TGCCCAGAAAATGAAAAAGG hE d R GTGTATGTGGCAATGCGTTC hTwist-F: GTGTATGTGGCAATGCGTTC h GGAGTCCGCAGTCTTACGAG hTwist-R: TCTGGAGGACCTGGTAGAGG CTTTAAGGCCAAGCAGTGCA hEpCAM-R: Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 23 of 27 23 of 27 Research article Research article Genomics and evolutionary biology \| Human biology and medicine Genomics and evolutionary biology \| Human biology and medicine Acknowledgements We thank V Butty, P Reddien, P Sharp, F Soldner, J Muffat, R Weinberg, L Surface, N Spies, R Friedman, M Kharas and M Lodato for helpful discussions, R Flannery for assistance with mouse colony mainte nance, and D. Fu for assistance processing histology sections. We thank Shmulik Motola and Stuart Levine (MIT BioMicroCenter) for high-throughput sequencing, and Wendy Solomon from Keck Microscopy Facility (Whitehead Institute) for assistance with microscopy. Supported by NIH grants R01-GM096193 (EMA), RO1-CA084198 (RJ), U01-CA184897 and R01-GM085319 (CBB). ZY and FL are supported by the National Basic Research program of China (973 program, 2011CB944103), the National Natural Science Foundation of China (NSFC, 31271584), and the National Transgenic Breeding Project of China (2011ZX08009-001-003). EMA is supported by Alfred P Sloan fellowship, and ESS by an NSF Graduate Research Fellowship (Grant No. 1122374). Author contributions YK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article; FL, NJL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article; ESS, W-LT, Acquisition of data, Drafting or revising the article; AWC, EMA, CJL, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents; PBG, ZY, RJ, CBB, Analysis and interpretation of data, Drafting or revising the article Ethics Animal experimentation: Mice of the 129SvJae strain were used, and the K14-rtTA strain were obtained from JAX (stock number: 007678). Animal care was performed in accordance with institutional guidelines and approved by the Committee on Animal Care, Department of Comparative Medicine, Massachusetts Institute of Technology, under animal protocol 1013-088-16. References d h dl Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R. 2006. 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Major dataset The following dataset was generated: Author(s) Year Dataset title Dataset ID and/or URL Database, license, and accessibility information Katz Y, Burge CB 2014 Transcriptome and translatome analysis of Msi in Mouse Neural Stem Cells http://www.ncbi.nlm.nih. gov/geo/query/acc. cgi?acc=GSE58423 Publicly available at NCBI Gene Expression Omnibus. Additional files Supplementary file • Supplementary file 1. Breast cancer RNA-Seq datasets used in analysis (apart from TCGA). DOI: 10.7554/eLife.03915.023 24 of 27 Katz et al. eLife 2014;3:e03915. DOI: 10.7554/eLife.03915 Research article Research article Major dataset The following dataset was generated: Author(s) Year Dataset title Dataset ID and/or URL Database, license, and accessibility information Katz Y, Burge CB 2014 Transcriptome and translatome analysis of Msi in Mouse Neural Stem Cells http://www.ncbi.nlm.nih. gov/geo/query/acc. cgi?acc=GSE58423 Publicly available at NCBI Gene Expression Omnibus. 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https://openalex.org/W2108392990	https://figshare.com/articles/journal_contribution/Efficient_C-Space_and_Cost_Function_Updates_in_3D_for_Unmanned_Aerial_Vehicles/6554672/1/files/12036857.pdf	English	null	Efficient C-space and cost function updates in 3D for unmanned aerial vehicles	null	2,009	cc-by	6,999	I. INTRODUCTION Unmanned aerial vehicles (UAVs) have many civilian and military applications. However currently such vehicles can only operate at a high altitude because they are not able detect and avoid collisions with obstacles. Our research focuses on UAVs that operate close to obstacles such as buildings, vegetation and power lines. It is necessary to ﬂy low because one can get a better viewpoint, and can avoid manned air trafﬁc. mentation has kept researchers from signiﬁcant progress in this area. The main contribution of this paper is a completely incre- mental framework that enables frequent replanning of paths in 3D for aerial vehicles (Fig. 2) at an order of magnitude lower computation time than current approaches to obstacle expansion. Incremental planning has been well studied [1], [2], however in prior work it has mostly been assumed that planning is performed on a C-space expanded cost map and that calculating this cost map is not difﬁcult. In 2D the number of cells affected to update the cost function based on a new obstacle is a function of the square of the maximum expansion. For example for an expansion of 20 cells about 400 cells are affected. However, in 3D it is not trivial to update the cost function after new sensor data has been received because the number of cells that potentially need to be recomputed changes cubicly. So for the same expansion we have to potentially look at roughly 8000 cells. We assume that the robot (Fig. 1) is given a sequence of sparse waypoints that have to be reached to make ob- servations and that the robot has to ﬁnd its way around obstacles that might be in its path. Flying close to and among obstacles is difﬁcult because of the challenges in sensing small obstacles, and in repeatedly planning a safe path that avoids obstacles in three dimensions. Some aspects of collision avoidance are easier for air vehicles than ground vehicles. Any object close to the intended path of an air vehicle must be avoided as opposed to ground vehicles where the ground is always close and deviations from the nominal ground plane indicate obstacles. The use of rotorcraft, rather than ﬁxed wing aircraft also simpliﬁes the problem because in the worst case it is possible for rotorcraft to come to a hover in front of an obstacle. Sebastian Scherer and Sanjiv Singh are with the Robotics Institute, Carnegie Mellon University. Dave Ferguson is with Intel Research Pitts- burgh. il [b ti dif i h]@ d Efﬁcient C-Space and Cost Function Updates in 3D for Unmanned Aerial Vehicles Sensor data causes an update to the environment map which propagates to changes in the cost function used by the planner. Finally an incremental path planner calculates a new path to the goal with the changed cost values. Fig. 2. Data ﬂow in a completely incremental planning paradigm. Sensor data causes an update to the environment map which propagates to changes in the cost function used by the planner. Finally an incremental path planner calculates a new path to the goal with the changed cost values. Efﬁcient C-Space and Cost Function Updates in 3D for Unmanned Aerial Vehicles Sebastian Scherer, Dave Ferguson, Sanjiv Singh Fig. 1. The autonomous quad-rotor aerial vehicle used for testing. Here, the vehicle is close to a typical pole and wire obstacle in the environment. The system is equipped with a computer, a GPS, an inertial measurement unit, and a ladar scanner to sense the environment. The size of the vehicle with rotors is less than one meter. New Path Limited Incremental Distance Transform Map Update Incremental Planning Algorithm New Sensor Data Updated Obstacle Cells Changed Costs Fig. 2. Data ﬂow in a completely incremental planning paradigm. Sensor data causes an update to the environment map which propagates to changes in the cost function used by the planner. Finally an incremental path planner calculates a new path to the goal with the changed cost values. mentation has kept researchers from signiﬁcant progress in Abstract— When operating in partially-known environments, autonomous vehicles must constantly update their maps and plans based on new sensor information. Much focus has been placed on developing efﬁcient incremental planning algorithms that are able to efﬁciently replan when the map and associated cost function changes. However, much less attention has been placed on efﬁciently updating the cost function used by these planners, which can represent a signiﬁcant portion of the time spent replanning. In this paper, we present the Limited Incremental Distance Transform algorithm, which can be used to efﬁciently update the cost function used for planning when changes in the environment are observed. Using this algorithm it is possible to plan paths in a completely incremental way starting from a list of changed obstacle classiﬁcations. We present results comparing the algorithm to the Euclidean distance transform and a mask-based incremental distance transform algorithm. Computation time is reduced by an order of magnitude for a UAV application. We also provide example results from an autonomous micro aerial vehicle with on-board sensing and computing. Fig. 1. The autonomous quad-rotor aerial vehicle used for testing. Here, the vehicle is close to a typical pole and wire obstacle in the environment. The system is equipped with a computer, a GPS, an inertial measurement unit, and a ladar scanner to sense the environment. The size of the vehicle with rotors is less than one meter. New Path Limited Incremental Distance Transform Map Update Incremental Planning Algorithm New Sensor Data Updated Obstacle Cells Changed Costs Fig. 2. Data ﬂow in a completely incremental planning paradigm. email: [basti, dif, ssingh]@cmu.edu I. INTRODUCTION Still, the availability of appropriate sensors, logistical issues of mounting a vehicle with sufﬁcient sensing, computing and communication gear, and the risk involved in such experi- In this paper, we present the Limited Incremental Distance Transform algorithm (LIDT) to efﬁciently perform this cost function update. In our approach, changes to the environment grid map [3] are propagated via the described limited incre- mental distance transform. The list of changed costs is then used by an incremental planning algorithm [2] to update the current plan. In previous work we have addressed the problem of navigating among obstacles with a layered architecture of a reactive avoidance algorithm that learns parameters by observing operator behavior and a global planning algorithm that operates on an evidence grid [4], [5]. A path planner for a ﬁxed-wing UAV using a grid lattice that operates on C-Space expanded obstacles is proposed by Hwangbo et al. [6]. Grif- ﬁths et al. use an RRT based planner with a grid height map to plan a path through the environment for an autonomous ﬁxed wing UAV in an urban environment [7]. Whalley et al. explore an environment with an autonomous helicopter. Paths among obstacles are planned to use a Voronoi diagram [8]. Andert & Goormann build an occupancy grid for planning with a stereo rig on a UAV [9]. Several interesting cost functions for UAVs depend on the distance to the closest obstacle. For example, the shortest path with a clearance to obstacles: obst(l) = max(0, d2 max −d(l)2 o) (3) (3) A maximum distance d2 max determines a cutoff beyond which the closest obstacle does not inﬂuence the path any- more. In the extreme case if d2 max and γ is large the path found will correspond to the solution of the Generalized Voronoi Graph (GVG) [10] since the path will ﬁrst lead away from the obstacle to get onto the Voronoi graph and then the lowest cost path will be on the graph and ﬁnally will go away from the graph to the goal point. In a natural outdoor environment the separation of obstacles is in many cases unbounded so that planning on that boundary would lead to too long paths. All of the related work described above uses a grid based approach to represent obstacles. III. DISTANCE TRANSFORM ALGORITHMS The distance d(l)2 o is the result that is computed by the distance transform algorithm. There are many possible distance metrics that can be applied, however the squared Euclidean distance is most useful for our application since we want the obstacle expansion and C-space expansion to be spherical. The property of the distance transform that we want can be expressed for a mxnxo grid with boolean obstacles b[i, j, k] as follows: In this paper we assume a spherical robot, a reasonable assumption for a rotor-craft. A spherical expansion is easy to compute if we know the distance to the closest obstacle. If the distance is closer than the radius rv of the UAV one is in contact with an obstacle. We set the cost for such an edge to be inﬁnite cost. We can express the general cost function between two vertices as follows: EDT(x, y, z) = min(b[i, j, k] : (x −i)2 + (y −j)2 + (z −k)2) (5) EDT(x, y, z) = c(k, l) = ∞ if d(l)2 o < r2 v γ · obst(l) + dist(k, l) otherwise (1) (1) (5) This property says that for every coordinate in the distance transform EDT(x, y, z) we determine the minimum of the distance to all the obstacles b[i, j, k]. This would of course not be a very efﬁcient algorithm in most cases however it shows what we need to compute. The Manhattan distance L1 transform can be written like this: This property says that for every coordinate in the distance transform EDT(x, y, z) we determine the minimum of the distance to all the obstacles b[i, j, k]. This would of course not be a very efﬁcient algorithm in most cases however it shows what we need to compute. The Manhattan distance L1 transform can be written like this: where c(k, l) is the cost between position k and l, and the closest obstacle is d(l)2 o. The cost consists of a scale factor γ that scales between the cost of obstacles obst(l) and the cost of the distance dist(k, l). Since we can express the C- space expansion in the cost function as an inﬁnite cost, we will from here on refer to the cost function as the cost of an edge that also includes the C-space expansion. I. INTRODUCTION Since a C-space expansion and cost function have to be computed for planning our approach could be used to update the costs in the map. We begin by describing in Section II how navigation cost functions are typically computed and their relationship to distance transforms. In Section III we discuss several potential approaches on computing such cost functions. Section IV describes our novel algorithm and Section V presents experiments and results, including examples from our autonomous aerial vehicle. Another useful cost function is to stay close to obstacles up to a desired distance ddes but not too close. This can be important for stealth reasons but also one might want to stay closer to obstacles to avoid wind or to stay localized. In this case the cost function can be expressed as follows: II. C-SPACE EXPANSION AND COST FUNCTIONS obst(l) =\| d2 des −min(d2 max, d(l)2 o) \| (4) (4) Incremental replanning using D* or its variants [1], [2] is a general and efﬁcient approach to adapt to a partially-known or dynamic environment. However it is not always easy to determine how the map for planning changed on a 3D grid since it is necessary to plan in the changed free conﬁguration space [10] with changed costs. Note that for both obst(l) functions it is necessary to know d(l)2 o, the distance to the closest obstacle up-to the maximum distance d2 max. Naively computing the distance d(l)2 o is expensive for large d2 max that are typically used in planning for UAV. The contribution of this paper is an efﬁcient algorithm for calculating the changes to d(l)2 o. Even though it is theoretically possible to plan with an arbitrary C-space expansion and cost function, the dominant factor is the distance to the closest obstacle. Therefore we assume that we can calculate the cost for planning around obstacles from distance. III. DISTANCE TRANSFORM ALGORITHMS The algorithm we propose also depends on the number of obstacles that changed however if only a small number of distances changes less work has to be performed by the LIDT algorithm. If an obstacle is removed a similar sweep outward prop- agates the changes to cells whose previous distance values are based on the removed obstacle and updates the distance for those cells since they now have a too close distance value. The cost to each of these cells is then updated based on the closest valid obstacle. Once the removal wavefront terminates each cell that does not have a valid obstacle will be updated with a valid obstacle (up to dmax). Kalra et al. [12] developed an incremental algorithm to reconstruct the Generalized Voronoi Diagram (GVD) in 2D. The GVD is based on a quasi-Euclidean distance transform of the obstacles. The algorithm is the basis of the algorithm presented in this paper, however we have modiﬁed the incremental GVD algorithm to make it suitable for C-space and cost function updates. The presented algorithm also adds another variable to keep track of the changes in the distance transform while it is being computed that can then be used in an incremental planning algorithm (Also see Fig. 2) . Furthermore we have generalized the algorithm to be applicable for different distance metrics (such as the squared Euclidean distance metric) while the original algorithm only allowed a quasi-Euclidean expansion. Also one can control the maximum amount of computation per obstacle in the LIDT algorithm because one controls the maximum distance dmax that needs to be expanded into account. It is important to note that the size of the queue in the wavefront depends on the radius of expansion. The number of cells in the queue is dependent on the radius r of the wavefront and grows linearly with the radius O(r). However since we are calculating the expansion in 3D the number of cells on the surface of the sphere grows with the square of the radius O(r2). Also the maximum radius that has to be expanded depends on the size of the Voronoi region that is affected. One worst case example is an empty grid with one obstacle that is removed. In that example ﬁrst all the cells going outward have to be invalidated and then all cells have to be lowered correctly again. III. DISTANCE TRANSFORM ALGORITHMS In the worst case one therefore has to look at the grid twice for every obstacle removed. III. DISTANCE TRANSFORM ALGORITHMS INITIALIZE() 1 O ←∅ 2 foreach cell s 3 dists ←d2 max 4 distnew s ←d2 max 5 distold s ←d2 max 6 obsts ←∅ SETOBSTACLE(o) 1 if distnew o ̸= 0 2 distnew o ←0 3 obsto ←o 4 UPDATEVERTEX(o) REMOVEOBSTACLE(o) 1 distnew o ←d2 max 2 obsto ←∅ 3 if disto < d2 max 4 UPDATEVERTEX(o) CALCULATEKEY(o) 1 return min(disto, distnew o ) UPDATEVERTEX(o) 1 key ←CALCULATEKEY(o) 2 if o ∈O 3 UPDATE(O, o, key) 4 else 5 INSERT(O, o, key) DISTANCE(n, s) 1 Squared Euclidean: 2 v ←posn −posobsts 3 return v · v DISTANCE(n, s) 1 Quasi Euclidean: 2 v ←posn −poss 3 return v · v + distnew s Fig. 3. The Limited Incremental Distance Transform Algorithm (Helper functions). algorithm scans the grid in three phases. In each phase the grid is scanned along a different axis forward and backward to determine a minimum. Overall the work performed is six passes through the grid for three dimensions. In two dimensions four passes are necessary. A simple incremental approach to update the cost function in a grid is to update the grid with a mask of the distances to the obstacles. We will refer to this algorithm as “mask al- gorithm” in the algorithm evaluation. Every time an obstacle is added a convolution of the surrounding area is performed to check if any of the distances is larger than the distance in the mask. In the case of obstacle removal all non-obstacle cells that are in the mask of the obstacle are set to inﬁnity and a region of two times the size of the mask is scanned for all obstacles. The region inside the removed obstacle is checked for any obstacle and the closest distance is restored. This algorithm serves as an incremental algorithm that one could implement easily. Fig. 3. The Limited Incremental Distance Transform Algorithm (Helper functions). would proceed. Since the values are sorted by increasing distance the cells with the smallest distance get updated ﬁrst. Finally, the wavefront that is moving outwards terminates if the distance has reached a value that is larger than any of the neighboring cells or if the grid boundary has been reached. While the runtime of the Meijster et al. algorithm depends on the size of the grid and is therefore non-incremental, the runtime of the mask algorithm depends on the number of obstacles added and removed. A. Intuition The Limited Incremental Distance Transform algorithm provides an efﬁcient solution to keep an updated distance transform for changes to the cost function in the environment. The algorithm is an incremental version of the brushﬁre algorithm and, as with the original brushﬁre algorithm it propagates a wavefront of distances to update the distance for each cell to its closest obstacle. For a good explanation of the brushﬁre algorithm also see Choset et al. [10]. The open list O keeps track of the wavefront and contains the cells that need to be expanded. Initially if only obstacles are added, the values of cells are lowered from dmax to consistent (or correct) distance values in the same way that brushﬁre IV. LIMITED INCREMENTAL DISTANCE TRANSFORM ALGORITHM For our application we are interested in computing the distance transform only out to a maximum distance dmax. As such the incremental distance transform propagation can be terminated once this distance is reached. This can save a signiﬁcant amount of computation if the Voronoi region that changes is large. III. DISTANCE TRANSFORM ALGORITHMS MDT(x, y, z) = The dist function can be any valid distance metric but one common metric is the squared Euclidean distance: min(b[i, j, k] :\| x −i \| + \| y −j \| + \| z −k \|) (6) (6) dist(k, l) = (kx −lx)2 + (ky −ly)2 + α(kz −lz)2 (2) An efﬁcient non-incremental linear time algorithm to calculate the distance transform was proposed by Meijster et al. [11]. Even though this algorithm is very efﬁcient, we will show in section V that repeatedly recomputing the result takes too long to be useful for navigation on a large grid. The where the x, y, z components are the displacement in the respective axis. If α = 1 going left/right or to climb/sink is equal cost. If α > 1 the robot will prefer to move laterally and if α < 1 it will prefer to move vertically. INITIALIZE() 1 O ←∅ 2 foreach cell s 3 dists ←d2 max 4 distnew s ←d2 max 5 distold s ←d2 max 6 obsts ←∅ SETOBSTACLE(o) 1 if distnew o ̸= 0 2 distnew o ←0 3 obsto ←o 4 UPDATEVERTEX(o) REMOVEOBSTACLE(o) 1 distnew o ←d2 max 2 obsto ←∅ 3 if disto < d2 max 4 UPDATEVERTEX(o) CALCULATEKEY(o) 1 return min(disto, distnew o ) UPDATEVERTEX(o) 1 key ←CALCULATEKEY(o) 2 if o ∈O 3 UPDATE(O, o, key) 4 else 5 INSERT(O, o, key) DISTANCE(n, s) 1 Squared Euclidean: 2 v ←posn −posobsts 3 return v · v DISTANCE(n, s) 1 Quasi Euclidean: 2 v ←posn −poss 3 return v · v + distnew s Fig. 3. The Limited Incremental Distance Transform Algorithm (Helper functions). INITIALIZE() 1 O ←∅ 2 foreach cell s 3 dists ←d2 max 4 distnew s ←d2 max 5 distold s ←d2 max 6 obsts ←∅ SETOBSTACLE(o) 1 if distnew o ̸= 0 2 distnew o ←0 3 obsto ←o 4 UPDATEVERTEX(o) REMOVEOBSTACLE(o) 1 distnew o ←d2 max 2 obsto ←∅ 3 if disto < d2 max 4 UPDATEVERTEX(o) CALCULATEKEY(o) 1 return min(disto, distnew o ) UPDATEVERTEX(o) 1 key ←CALCULATEKEY(o) 2 if o ∈O 3 UPDATE(O, o, key) 4 else 5 INSERT(O, o, key) DISTANCE(n, s) 1 Squared Euclidean: 2 v ←posn −posobsts 3 return v · v DISTANCE(n, s) 1 Quasi Euclidean: 2 v ←posn −poss 3 return v · v + distnew s Fig. 3. The Limited Incremental Distance Transform Algorithm (Helper functions). INCREMENTALDISTANCETRANSFORM(O) C ∅ INCREMENTALDISTANCETRANSFORM(O) ∅ LOWER(s) 1 foreach n ∈Adj(s) 2 if distnew n > distnew s 3 d′ ←DISTANCE(n, s) 4 if d′ < distnew n 5 distnew n ←d′ 6 obstn ←obsts 7 UPDATEVERTEX(n) RAISE(s) 1 foreach n ∈Adj(s) 2 WAVEOUT(n) 3 WAVEOUT(s) WAVEOUT(n) 1 if n ̸= obstn 2 distnew n ←d2 max 3 obstold n ←obstn 4 foreach a ∈Adj(n) 5 if VALID(obsta) 6 d′ ←DISTANCE(n, a) 7 if d′ < distnew n 8 distnew n ←d′ 9 obstn ←obsta 10 if obstn ̸= obstold n 11 UPDATEVERTEX(n) INCREMENTALDISTANCETRANSFORM(O) 1 C ←∅ 2 while O ̸= ∅ 3 s ←POP(O) 4 if distnew s < dists 5 dists ←distnew s 6 LOWER(s) 7 if dists ̸= distold s 8 INSERT(C, s) 9 distold s = dists 10 else 11 dists ←d2 max 12 RAISE(S) 13 if dists ̸= distnew s 14 updateV ertex(s) 15 return C Fig. 4. The Limited Incremental Distance Transform Algorithm (Main functions). 15 return C Fig. 4. The Limited Incremental Distance Transform Algorithm (Main functions). Fig. 5. The virtual campus environment of Carnegie Mellon University, Pittsburgh, PA that is used in the simulation experiments. Buildings that were added to the digital elevation model are shown in white. A hemispher- ical 200m range 3D range sensor is simulated to update the environment map held by the robot. set to zero and the obstacle points to itself. Then the obstacle is added to the queue to be expanded. Similarly a removed obstacles distance is set to d2 max and it is added to the queue with the priority of the old distance it used to have. Since the update to the grid should always be with increasing priority the key is calculated from the smaller of the two distance values in CALCULATEKEY and the heap is updated in UPDATEVERTEX with the new priority unless the element has an inﬁnite priority. Fig. 5. The virtual campus environment of Carnegie Mellon University, Pittsburgh, PA that is used in the simulation experiments. Buildings that were added to the digital elevation model are shown in white. A hemispher- ical 200m range 3D range sensor is simulated to update the environment map held by the robot. Using our algorithm one can calculate a squared Euclidean distance in DISTANCE or a quasi-Euclidean distance that is the shortest distance on a 26-connected grid. INCREMENTALDISTANCETRANSFORM(O) C ∅ It is possible to calculate the squared Euclidean distance because we always keep track of the location of the closest obstacle in obsts. The obsts pointer tells us if a grid cell needs to be updated because it points to an obstacle. If that obstacle changes all cells pointing to that obstacle need to be updated. binary heap there is a fast data structure that can be used in our application because the maximum distance is limited to dmax and we are operating on a grid with integer values of the keys. Since there is only a small range of key values we create a hash table with the distances as key values. On every update we keep track of the lowest distance element. If we pop an element we update the lowest distance if it changes. To insert we just add the element to the list at the appropriate key value. This data structure allows O(1) for INSERT, UPDATE, and POP. The main work of updating the distance transform and keeping track of changed cells happens in INCREMEN- TALDISTANCETRANSFORM which returns a list of updated distances C. If we added or removed obstacles the open list O will not be empty and so we take the ﬁrst element of the list and LOWER the node if it is over-consistent and RAISE it otherwise. Since all nodes have to be made LOWER eventually we can keep track of the changed distances in lines 7-9. V. EXPERIMENTS There are certain tradeoffs between using an incremental and non-incremental algorithm that need to be considered. In this section we examine some parameters that inﬂuence the performance of the LIDT algorithm and compare it to the fastest non-incremental algorithm and a simple incremental algorithm we denote the “mask” approach (described in Section III). A recent survey [13] showed that the algorithm developed by Meijster [11] is fastest in almost all test cases over a variety of problems. We therefore also compare the incremental algorithms to a 3D implementation of this algorithm. LOWER updates the distance of each adjacent node and adds it to the queue if the distance changed. Also we update the associated obstacle if it changed. RAISE on the other hand propagates out a removed ob- stacle in WAVEOUT and so we ﬁrst set the distance to be inﬁnity and try to get a new distance for an adjacent node. If the associated obstacle changed we put the item back on the queue. The algorithm terminates when the open list is empty. At this point all the cells in the grid have consistent distance values and have a valid obstacle pointer if their distance is less than dmax. A list of changed cells is in C. B. Details The algorithm pseudocode is split in two parts the helper functions are shown in Fig. 3 and the main functions are shown in Fig. 4. In INITIALIZE all cell distances are set to d2 max and the obstacle pointer is emptied. As the environment changes obstacles are removed and added with SETOBSTACLE and REMOVEOBSTACLE. If an obstacle is added its distance is A. Simulation experiments The algorithms are run with the same sensor inputs on a grid that was initialized with an elevation model. Meijster is the non-incremental distance transform algorithm by Meijster et al. Mask is a simple incremental algorithm that updates based on a distance mask for each obstacle. Incremental is the limited incremental distance transform algorithm. The environment map changes as the robot discovers new obstacles and removes invalid obstacles from its initial map. dmax = 20. The mean of the number of obstacles added was 370 ± 32.3. The mean of the number of obstacles removed was 14 ± 6.5. The grid size considered is 512x512x80. Box and whisker plot legend: The red line is the median, the blue box extends from the lower quartile to the upper quartile, and the whiskers extend to 1.5 of the interquartile range. Red crosses represent single run outliers. A path tracking algorithm controls the helicopter and regulates speed based on the distance to the closest obstacle. As soon as a sensor measurement is received the evidence grid is updated and changes are given to one of the three distance transform algorithms. The changes to the grid and the cost function are then propagated to a D* Lite [2] planning algorithm. All algorithms run on a 2.5GHz Intel Core 2 Duo processor. We ran an experiment in this environment with the robot avoiding obstacles that it saw within its range sensor and the three algorithms calculating the changes to the cost function for D* Lite. The maximum expansion for the grid was set to dmax = 20 and the environment map was initialized with the prior digital elevation model. During its traverse, a mean of 370 (standard deviation 32.3) obstacles were added and 14 (standard deviation 6.5) obstacles were removed. 0 50 100 150 200 250 0 20 40 60 dmax [cells] time [s] Fig. 7. Initial calculation times for an empty 512x512x80 grid initialized with obstacles from a digital elevation model for different expansions. As the expansion increases so does the initialization time because a lot of cells need to be updated for a large expansion in the limited incremental distance transform. 0 50 100 150 200 250 0 20 40 60 dmax [cells] time [s] Overall the limited incremental distance algorithm per- formed over an order of magnitude better than the competing approaches (Fig. A. Simulation experiments 6 and Table I), because the expansion distance is large and a number of obstacles have to be removed each iteration. As the number of obstacles removed decreases the ‘mask’ algorithm performs better because obstacle removal is an expensive operation for this algorithm. The non-incremental Meijster et al. algorithm has to traverse the grid several times and therefore cannot perform as well as the incremental algorithms which only have to update a small local region. Fig. 7. Initial calculation times for an empty 512x512x80 grid initialized with obstacles from a digital elevation model for different expansions. As the expansion increases so does the initialization time because a lot of cells need to be updated for a large expansion in the limited incremental distance transform. 5 10 30 50 70 90 110130150170190210230250 0 2 4 6 8 10 time [s] dmax [cells] Fig. 8. A box and whisker plot for the limited incremental distance transform of the computation time in seconds for an increasing value of the maximum distance expanded dmax . The computation time spreads out more as dmax increases since a changed obstacle cell can affect a larger Voronoi region however the region affected can also be small if the obstacle added is close to existing obstacles. The grid size considered is 512x512x80. For the legend of the box and whisker plot see Fig. 6. 5 10 30 50 70 90 110130150170190210230250 0 2 4 6 8 10 time [s] dmax [cells] 5 10 30 50 70 90 110130150170190210230250 0 2 4 6 8 10 time [s] dmax [cells] As the expansion distance decreases there is a point at which the mask algorithm becomes more efﬁcient because it can use the processor cache better since it performs lots of sequential accesses. However if dmax increases signiﬁcantly the runtime of the mask algorithm will increase beyond the non-incremental algorithm by Meijster because it must perform double work. Fig. 8. A box and whisker plot for the limited incremental distance transform of the computation time in seconds for an increasing value of the maximum distance expanded dmax . The computation time spreads out more as dmax increases since a changed obstacle cell can affect a larger Voronoi region however the region affected can also be small if the obstacle added is close to existing obstacles. The grid size considered is 512x512x80. A. Simulation experiments To determine the effectiveness of the limited incremental distance transform algorithm for aerial vehicle planning we evaluated it for missions in a simulated environment and compared it with two other distance transform algorithms: the non-incremental distance transform algorithm by Meijster et. al and the simple incremental ‘mask’ algorithm. See Section III for more details on the two competing algorithms. We assume that the open list O has three operations: INSERT inserts an element in the open list with a given priority key, UPDATE updates the key of an element already in the queue, and POP returns and removes the top element from the priority queue. Even though one can implement the open list O as a Meijster Mask Incremental 0 5 10 time [s] Fig. 6. A comparison of running three distance transform algorithms in the environment shown in Fig. 5. The algorithms are run with the same sensor inputs on a grid that was initialized with an elevation model. Meijster is the non-incremental distance transform algorithm by Meijster et al. Mask is a simple incremental algorithm that updates based on a distance mask for each obstacle. Incremental is the limited incremental distance transform algorithm. The environment map changes as the robot discovers new obstacles and removes invalid obstacles from its initial map. dmax = 20. The mean of the number of obstacles added was 370 ± 32.3. The mean of the number of obstacles removed was 14 ± 6.5. The grid size considered is 512x512x80. Box and whisker plot legend: The red line is the median, the blue box extends from the lower quartile to the upper quartile, and the whiskers extend to 1.5 of the interquartile range. Red crosses represent single run outliers. We want to simulate an algorithm load that is similar to a real extended mission of our micro aerial vehicle in a simulated environment of the campus at Carnegie Mellon University, Pittsburgh, PA, USA. See Fig. 5 for a screen shot from our simulation. Meijster Mask Incremental 0 5 10 time [s] The simulation consists of a second order dynamic model of our quad-rotor helicopter shown in Fig. 1, a hemispherical 3D range sensor with a range of 200m, and a geometric model of campus. Incremental Incremental Fig. 6. A comparison of running three distance transform algorithms in the environment shown in Fig. 5. A. Simulation experiments For the legend of the box and whisker plot see Fig. 6. The runtime of the limited incremental distance transform depends on the size of the Voronoi region affected and in many cases if obstacles are close to each other then the affected regions are relatively small. In the case of the simulation and in realistic scenarios the changes to the map will be local and in a neighborhood of existing obstacles. In this case since only a small number of cells need to be updated the limited incremental distance transform has a signiﬁcant advantage. Algorithm Incremental Mean calc. time Std. Dev. Meijster No 13.05s 0.16s Mask Yes 6.61s 2.21s LIDT Yes 0.27s 0.12s TABLE I CALCULATION TIMES OF ONE UPDATE FOR THE ALGORITHM BY MEIJSTER ET AL., THE MASK ALGORITHM AND THE LIMITED INCREMENTAL DISTANCE TRANSFORM ALGORITHM (LIDT). dmax = 20 The standard deviation in Table I indicates that the com- putation time for the LIDT algorithm varies less than for the Mask algorithm. In a second experiment we evaluated the performance of the maximum distance dmax on computation time for our campus environment. Fig. 9. This sequence of images shows our autonomous quadrotor micro aerial vehicle (Fig. 1) avoiding a tree that is in the straight line mission path to its goal. As soon as the robot detects the obstacle it plans a path in 3D with a wide berth around the obstacle. The cost function is set up in such a way that the robot will prefer to move laterally and therefore we only show a top view. Since dmax = 11 the obstacle is avoided by a large margin. The mission path is shown in black and the planned avoidance path is shown in red. The black line shows the path of the vehicle as recorded by GPS. The tree obstacle is shown in green. Fig. 9 shows the quadrotor avoiding a tree in its straight line path to the goal. The cost function used for the planning algorithm in this case is the same as as in Eq. 3 with dmax = 11. Since that expansion is large and we are planning in 3D it is beneﬁcial to use an incremental distance transform algorithm to update the changes to the cost function. VI. CONCLUSION AND FUTURE WORK We have presented a completely incremental framework for planning paths in 3D that enables recomputation of costs an order of magnitude faster than current approaches. This speed up is made possible by using a novel limited incremental distance transform algorithm. This algorithm exploits the local nature of cost function updates when obstacles are added or removed from the map and enables autonomous aerial vehicles to respond to newly observed obstacles (or obstacles that no longer exist) in real-time. We have provided results from simulation demonstrating the beneﬁts of the approach and illustrative examples from a physical implementation on a quad-rotor micro aerial vehicle autonomously navigating in Pittsburgh, PA. We ran a total of 37461 updates with varying maxi- mum expansion values for the limited incremental distance transform. During testing we reset the environment map periodically and then recalculated the incremental distance transform with obstacles based on the digital elevation model which exempliﬁes the overhead cost of the incremental distance transform for starting from scratch. The initial overhead of the incremental distance transform algorithm is signiﬁcant if a large number of obstacles already exist in the environment map since the expansion has to perform more work per cell than the distance transform algorithm. A scatter plot of the overhead is shown in Fig. 7. The initial overhead of the incremental distance transform algorithm is signiﬁcant if a large number of obstacles already exist in the environment map since the expansion has to perform more work per cell than the distance transform algorithm. A scatter plot of the overhead is shown in Fig. 7. After the initial overhead, however, subsequent updates are relatively inexpensive. With an increase in the distance dmax the median computation time increases as well as the overall spread in computation. Since the potential number of cells affected by a change in the environment increases with dmax we also see a larger variation in computation times. Even with a large expansion of 250 cells, however, the limited incremental distance transform algorithm can still outperform the algorithm by Meijster. At such an expansion distance the ‘mask’ algorithm would not be feasible because on every update it essentially needs to check every cell in a 512x512x80 grid. REFERENCES [1] A. Stentz, “The d* algorithm for real-time planning of optimal traverses, Tech. Rep. CMU-RI-TR-94-37, Oct 1994. [1] A. Stentz, “The d* algorithm for real-time planning of optimal traverses, Tech. Rep. CMU-RI-TR-94-37, Oct 1994. p [2] S. Koenig and M. Likhachev, “D* lite,” Eighteenth national conference on Artiﬁcial intelligence, Jul 2002, twocol. ﬁ g [3] M. C. Martin and H. Moravec, “Robot evidence grids, Tech. Rep. CMU-RI-TR-96-06, Mar 1996. [4] S. Scherer, S. Singh, L. Chamberlain, and M. Elgersma, “Flying fast and low among obstacles: Methodology and experiments,” Int. J. Robotics Research, vol. 27, no. 5, pp. 549–574, May 2008. pp y [5] S. Scherer, S. Singh, L. Chamberlain, and S. Saripalli, “Flying fast and low among obstacles,” Robotics and Automation, 2007 IEEE International Conference on, pp. 2023 – 2029, Mar 2007. f pp [6] M. Hwangbo, J. Kuffner, and T. Kanade, “Efﬁcient two-phase 3d motion planning for small ﬁxed-wing uavs,” Robotics and Automation, 2007 IEEE International Conference on, pp. 1035 – 1041, Mar 2007. A. Simulation experiments The C- space expansion is set to 2 meters but since it is expensive to go close to obstacles the path gives the obstacle a wide berth. In this experiment the obstacle is avoided with a speed of 2m/s. Fig. 9. This sequence of images shows our autonomous quadrotor micro aerial vehicle (Fig. 1) avoiding a tree that is in the straight line mission path to its goal. As soon as the robot detects the obstacle it plans a path in 3D with a wide berth around the obstacle. The cost function is set up in such a way that the robot will prefer to move laterally and therefore we only show a top view. Since dmax = 11 the obstacle is avoided by a large margin. The mission path is shown in black and the planned avoidance path is shown in red. The black line shows the path of the vehicle as recorded by GPS. The tree obstacle is shown in green. ACKNOWLEDGMENTS The authors would like to thank Lyle Chamberlain, Wen- fan Shi, and Maggie Scholtz for developing and testing the aerial robot. B. Quad-rotor experiment f pp [7] S. Grifﬁths, J. Saunders, A. Curtis, and T. McLain, “Obstacle and terrain avoidance for miniature aerial vehicles,” IEEE Robotics and Automation Magazine, Jan 2006. Our autonomous quad-rotor robot (Fig. 1) wants to avoid obstacles with a wide berth if possible because it increases the safety of the path and gives a better perspective for sensing. The robot is equipped with a GPS, INS, and a ladar scanner to sense the environment. All computation is performed on-board and a planning cycle is performed at about 3Hz. It can ﬂy missions of up to 20 minutes and can be given a series of waypoints it should reach. Since typically almost all of the waypoints are low the straight line path to the goal is typically obstructed by obstacles. The ladar scanner returns distances to obstacles and the information is processed into a global map as a 3D array in memory and a path is planned using a distance transform expansion. The position of the robot has some uncertainty ( 2m) that is incorporated as part of the C-space expansion. The robot can avoid obstacles in three dimensions since the planning algorithm also operates in three dimensions. g [8] M. Whalley, M. Freed, R. Harris, and M. Takahashi, “Design, integration, and ﬂight test results for an autonomous surveillance helicopter,” Proceedings of the AHS International Specialists’ Meeting on Unmanned Rotorcraft, Jan 2005. [9] F. Andert and L. Goormann, “Combined grid and feature-based occu- pancy map building in large outdoor environments,” Intelligent Robots and Systems, 2007. IROS 2007. IEEE/RSJ International Conference on, pp. 2065 – 2070, Jan 2007. , pp , [10] H. Choset, K. Lynch, S. Hutchinson, G. Kantor, W. Burgard, L. E. Kavraki, and S. Thrun, Principles of Robot Motion: Theory, Algo- rithms, and Implementation, Apr 2005. p p [11] A. Meijster, J. Roerdink, and W. Hesselink, “A general algorithm for computing distance transforms in linear time,” Mathematical Morphology and its Applications to Image and Signal Processing, pp. 331–340, Jan 2000. [12] N. Kalra, D. Ferguson, and A. Stentz, “Incremental reconstruction of generalized voronoi diagrams on grids,” Proc. of the International Conference on Intelligent Autonomous Systems, Mar 2006. f g y [13] R. Fabbri, L. Costa, J. Torelli, and O. Bruno, “2d euclidean distance transform algorithms: A comparative survey,” ACM Computing Sur- veys (CSUR, vol. 40, no. 1, Feb 2008.
https://openalex.org/W3011777273	https://www.e3s-conferences.org/articles/e3sconf/pdf/2020/14/e3sconf_icores2020_02004.pdf	English	null	Torrefaction of oat straw to use as solid biofuel, an additive to organic fertilizers for agriculture purposes and activated carbon – TGA analysis, kinetics	E3S web of conferences	2,020	cc-by	4,022	Torrefaction of oat straw to use as solid biofuel, an additive to organic fertilizers for agriculture purposes and activated carbon – TGA analysis, kinetics Szymon Szufa1, Maciej Dzikuć2 ,Łukasz Adrian3, Piotr Piersa4, Zdzisława Romanowska- Duda5, Wiktoria Lewandowska 6, Marta Marcza7, Artur Błaszczuk8, Arkadiusz Piwowar9 Szymon Szufa1, Maciej Dzikuć2 ,Łukasz Adrian3, Piotr Piersa4, Zdzisława Romanowska- Duda5, Wiktoria Lewandowska 6, Marta Marcza7, Artur Błaszczuk8, Arkadiusz Piwowar9 1 Lodz University of Technology, Faculty of Process and Environmental Engineering, Wolczanska 213, 90-924 Lodz,, Poland, szymon.szufa@p.lodz.pl 2 University of Zielona Góra, Faculty of Economics and Management, ul. Licealna 9, 65-246 Zielona Góra, Poland, m.dzikuc@wez.uz.zgora.pl 2 University of Zielona Góra, Faculty of Economics and Management, ul. Licealna 9, 65-246 Zielo Góra, Poland, m.dzikuc@wez.uz.zgora.pl @ g p 3 University of Kardynal Stefan Wyszyński, Faculty of Biology and Environmental Science, Dewajtis 5, 01-815 Warszawa, Poland, l.adrian@uksw.edu.pl 3 University of Kardynal Stefan Wyszyński, Faculty of Biology and Environmental Science, Dewajtis 5, 01-815 Warszawa, Poland, l.adrian@uksw.edu.pl @ p 4 Lodz University of Technology, Faculty of Process and Environmental Engineering, Wolczanska 213, 90-924 Lodz,, Poland, piotr.piersa@p.lodz.pl 4 Lodz University of Technology, Faculty of Process and Environmental Engineering, Wolczanska 213, 90-924 Lodz,, Poland, piotr.piersa@p.lodz.pl 5 Laboratory of Plant Ecophysiology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha str. 12/16, 90-131 Łódź, Poland, romano@biol.uni.lodz.pl 6 5 Laboratory of Plant Ecophysiology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha str. 12/16, 90-131 Łódź, Poland, romano@biol.uni.lodz.pl 6 University of Lodz, Chemical Faculty, Tamka 13, 91-403 Łódź, Poland, wiktoria.lewandowska.uni.lodz@gmail.com 6 University of Lodz, Chemical Faculty, Tamka 13, 91-403 Łódź, Poland, wiktoria lewandowska uni lodz@gmail com 7 AGH University of Science and Technology, Faculty of Energy and Fuels, al. Mickiewicza 30, 30- 059 Krakow, Poland, mmarczak@agh.edu.pl 7 AGH University of Science and Technology, Faculty of Energy and Fuels, al. Mickiewicza 30, 30- 059 Krakow, Poland, mmarczak@agh.edu.pl 8 Czestochowa University of Technology, Institute of Advanced Energy Technologies, Dabrowskiego 73, 42-200, Czestochowa, Poland, ablaszczuk@is.pcz.pl 8 Czestochowa University of Technology, Institute of Advanced Energy Technologies, Dabrowskiego 73, 42-200, Czestochowa, Poland, ablaszczuk@is.pcz.pl 9 Wroclaw University of Economics, Faculty of Engineering a 53-345 Wrocław, Poland, arkadiusz.piwowar@ue.wroc.pl 9 Wroclaw University of Economics, Faculty of Engineering and Economics, Komandorska 118/120 , 53-345 Wrocław, Poland, arkadiusz.piwowar@ue.wroc.pl 53-345 Wrocław, Poland, arkadiusz.piwowar@ue.wroc.pl Abstract. In this paper authors present research results which are the optimum parameters of the torrefaction process using straw from oats and maize. The most important parameters for the torrefaction process are temperature and residence time. Both parameters are essential to designing and construction of industrial biomass torrefaction installations. E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 https://doi.org/10.1051/e3sconf/202015402004 © 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/). INTRODUCTION In global agriculture and agri-food processing, a huge amount of waste is generated each year. If you take into account straw, husks plus various types of husk and shell, and also biomass from dedicated energy crops, it accounts for 50 billion metric tons per year. Typically, such biomass when it is not suitable for use as fodder is burned or composted and most often, unfortunately, stored in landfills. In the case of storage or composting in open heaps, this biomass in time turns into gas, which escapes into the atmosphere, whose potential as greenhouse gas is higher than that of carbon dioxide. It is obvious, therefore, that one should find an environmentally and economically effective way of converting this biomass into a raw material for the production of useful energy. The main by-product of crop production in agricultural holdings is the straw of cereals and other crops. The amount of straw obtained in Poland is estimated at 25-33 million tonnes per year [1][2][3] p y According to European standards, biochar should be produced in the process of torrefaction or pyrolysis of biomass (or an identical substrate). A external heat source is required to carry out the torrefaction and pyrolysis processes in order to maintain the required temperature in the reaction chamber. Carrying out the torrefaction process with the use of torgas (volatile parts that evaporate during the torrefaction process) is well known on the market: the gas produced - torgas during the torrefaction process is transported to the reactor chamber where they flow in a counter-current exchanger to the biomass fed to the reactor from mountains. The obtained product is cooled and introduced into the screw conveyor from the reactor area in which the torrefaction process takes place. During thermal and chemical conversion of biomass, steam is first produced, followed by process gas. After dedusting the exhaust and volatile mixture, the torrefaction products are directed to the combustion chamber. A portion of the hot combustion gases generated during combustion of the torgas is returned to the torrefaction reactor and the excess is removed to the atmosphere via the outlet channel. Obtaining the right amount of torgas requires the decomposition of a large amount of biomass, and thus the final efficiency of the process is low and the process itself ceases to be profitable from an economic point of view. Torrefaction of oat straw to use as solid biofuel, an additive to organic fertilizers for agriculture purposes and activated carbon – TGA analysis, kinetics Energy crops and waste coming from agricultural production have the most promising perspective from all kind of renewable energy sources in Poland. Currently, intensive studies on the process of biomass torrefaction are being carried out. In this experimental investigation, authors examined the torrefaction process of two types of agriculture biomass, such as: oats, maize. The main overarching objective of the experimental studies described below is the development of various biochar as an additive to agricultural fertilizers resulting from the conversion of biomass from agriculture residues – straw from oats and maize. The last of enumerated biomasses is treated through different conversion processes such as: drying, torrefaction to homogenize their physical and chemical properties. Among many of its areas, it is extremely important to optimize the production of biomass energy plants and its refinement (in the torrefaction process), which will improve the balance and profitability of energy E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 https://doi.org/10.1051/e3sconf/202015402004 production from RES, and reduce the logistics and storage costs of this fuel and improve the efficiency of biomass combustion process. When implementing new technologies indicated in this work and optimizing the harvesting of plant biomass, the negative impact on the environment caused by stored municipal waste can be reduced. This biomass torrefaction process temperature and residence time were necessary for the design and construction of semi-pilot scale biomass torrefaction installations with dryer and torrefaction reactor to perform a continuous biomass torrefaction process using superheated steam Keywords: torrefaction process, straw, oats, biochar MATERIAL CHARACTERIZATION Straw is the most commonly used bedding material, it is food for animals, it is used for fertilizing fields. After deduction of the need for litter and feed as well as the necessary quantity for plowing, there are surpluses for alternative development. The volume of overproduction of straw according to various sources is determined at the level of approx. 8-13 million tonnes per year [4][5]. One of the possibilities is to use straw surpluses in the energy sector. Its heating value is from 14.3 to 15.2 MJ · kg-1, in terms of energy, 1.5 tons of straw is equivalent to about one ton of hard coal. The amount of straw that can be used in the energy sector is equivalent to a calorific value of about 14 million tons of coal, which is about 10% of annual hard coal mining in Poland. The limitation in the widespread use of straw in the energy sector is its scattering, diversification of properties depending on the plant species, variety, fertilization, environmental conditions and weather which causes special requirements in relation to the air regulation in the boilers for its combustion. In addition, straw is a volumetric material, which affects the costs of transport and storage [6]. Tab.1. Technical and elemental analysis of oat straw before and after thermo-chemical conversion Parameters Volatile Moisture Content Carbon Nitrogen Hydrogen Sulphur HHV [MJ/kg] Oat straw 77.8 2.9 49.20 0.64 6.23 0.01 17.68 257.5 ⁰C (8 min) 65.56 1.9 54.21 0.25 5.49 0.01 21.47 300 ⁰C (7min) 49.37 1.6 55.86 0.21 5.39 0.01 22.68 525 ⁰C (6 min) 37.43 0.8 59.06 0.13 4.23 0.01 26.97 The lowest density in the chute and hollow state is characterized by wheat and oat straw. The bulk density for these raw materials is 84.4 kg · m-3 and 84.6 kg · m-3; density in the hollow state 104.1 kg · m-3 and 107.8 kg · m-3. Whereas the highest density in the chute and chilled state was obtained for rape straw and maize straw (bulk density is respectively 110.5 kg · m-3, 108.8 kg · m-3, in the hollow state 137.2 kg · m-3 132, 9 kg · m-3). The mean values of the angle of discharge and repose differ significantly depending on the type of straw. The lowest value of the angle of discharge (37.0 ⁰C) and dump (32.1 ⁰C) is characterized by barley straw. INTRODUCTION In other solutions, heat is generated for the torrefaction process from the combustion of natural gas or propane, which can be used in the torrefaction process, whereas in this case the process is neither environmentally friendly nor economical because it requires the purchase of gases. 2 2 https://doi.org/10.1051/e3sconf/202015402004 E3S Web of Conferences 154, 02004 (2020) E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 Fig.1. The principle of oats straw torrefaction process with process conditions: temperature and optimal mass loss using superheated steam for production of: biofuel, additive for natural fertilizers, activated carbon. Fig.1. The principle of oats straw torrefaction process with process conditions: temperature and optimal mass loss using superheated steam for production of: biofuel, additive for natural fertilizers, activated carbon. Fig.1. The principle of oats straw torrefaction process with process conditions: temperature and optimal mass loss using superheated steam for production of: biofuel, additive for natural fertilizers, activated carbon. MATERIAL CHARACTERIZATION The highest value of the angle of discharge of 41.7 ⁰C was obtained for wheat straw, and the angle of recycle 37.3 ⁰C for oat straw. The fat content 3 https://doi.org/10.1051/e3sconf/202015402004 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 in the tested raw materials is less than 1.6%, and proteins from 4.8%. The average fat content is not significantly different for barley, maize, oat and wheat straw. The fat content ranges from 0.92 % (barley and maize straw) to 1.59 % (rapeseed straw). However, the protein content is from 3.52 % (barley straw) to 4.73% (oat straw). The smallest amount of ash contains wheat straw (4.93 %) and oat straw (4.99 %) and it does not differ significantly for these raw materials. The average ash values for the remaining straws are significantly different. The largest amount of ash amounting to almost 9% has barley straw. The raw materials tested contain: fibers from 35.33 % (corn straw) to 43.14 % (barley straw) and carbohydrates digestible from 35.7 % (barley straw) to 46.76 % (corn straw). The amount of these components in the raw material depends significantly on the type of straw. cited studies show that after burning straw from oats remains the least ash because only 4.99 % which is about 2/3 of ash from barley straw. Oat straw contains the most fat, 1.13 %, which guarantees self-lubrication effects during the granulation process. It is recommended to use the so-called gray straw, that is, after being cut in the field for atmospheric conditions, and then dried. Such straw is characterized by better energy properties and a lower content of chlorine and potassium compounds (partially rinsed out by rain). by rain). Tab. 2. Torrefaction process conditions with different time and temperatures for the production of tree different products: biofuel, additive for fertilizers and activated carbon. MATERIAL CHARACTERIZATION Isothermal conditions [min] 5 6 7 8 9 10 Temperature [˚C] 257,5 80,56% 78,71% 77,78% 74,78% 75,00% 75,79% 0,0108/ g 0,0087 g 0,0108/ g 0,0085 g 0,0108/ g 0,0084 g 0,0107/ g 0,0080 g 0,0108/ g 0,0081 g 0,0095/ g 0,0072 g 300 52,04% 51,02% 48,57% 43,94% 26,04% 21,28% 0,0098/ g 0,0051 g 0,0098/ g 0,0050 g 0,0105/ g 0,0051 g 0,0107/ g 0,0047 g 0,0096/ g 0,0025 g 0,0094/ g 0,0020 g 525 25,86% 25,22% 24,35% 24,35% 24,81% 23,81% 0,0116/ g 0,0030 g 0,0115/ g 0,0029 g 0,0115/ g 0,0028 g 0,0115/ g 0,0028 g 0,0129/ g 0,0032 g 0,0126/ g 0,0030 g ) ab. 2. Torrefaction process conditions with different time and temperatures for the production of tree different products: biofuel, additive for fertilizers and activated carbon. 4 https://doi.org/10.1051/e3sconf/202015402004 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 Fig.2. Diagram shows straw oats torrefaction process conditions: temperature and optimal mass loss for production of: biofuel, additive for natural fertilizers, activated carbon. Fig.2. Diagram shows straw oats torrefaction process conditions: temperature and optimal mass loss for production of: biofuel, additive for natural fertilizers, activated carbon. Oats, as an energy fuel, are gaining more and more supporters. Although there is a very strong mental barrier in Polish society, resulting from the high respect of grain, the economic situation and growing environmental awareness affect the increase in the number of users of installations in which oats are burned. As a result, they enjoy an improvement in the economic situation of the household and the comfort of using a high-performance, automated boiler. Currently, the area of domestic oat cultivation is about 0.5 million hectares. The majority of harvested crop is used as fodder, and as the number of livestock decreases, the demand for grain is constantly decreasing. This year it is 1/4 lower than last year. The food, pharmaceutical or cosmetic industry is not able to receive growing surpluses and so the idea of using oats for energy purposes has arisen. Oats are easy to burn, it affects the contents of the husk and fat, relatively constant humidity (10-15 %) and high calorific value (15-18 MJ / kg). It is easy to dispense fuel into the boiler. 5 https://doi.org/10.1051/e3sconf/202015402004 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 Fig. 3 Thermogravimetric analysis of oat straw for the production of biofuel at temperature 257,5 ⁰C Fig 4. MATERIAL CHARACTERIZATION Thermogravimetric analysis of oat straw for the production of addtive for natural fertilizer at temperature 300 ⁰C Fig. 3 Thermogravimetric analysis of oat straw for the production of biofuel at temperature 257,5 Fig. 3 Thermogravimetric analysis of oat straw for the production of biofuel at temperature 257,5 ⁰C Fig 4. Thermogravimetric analysis of oat straw for the production of addtive for natural fertilizer at temperature 300 ⁰C Fig. 3 Thermogravimetric analysis of oat straw for the production of biofuel at temperature 257,5 ⁰C Fig 4. Thermogravimetric analysis of oat straw for the production of addtive for natural fertilizer at temperature 300 ⁰C 6 6 https://doi.org/10.1051/e3sconf/202015402004 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 Fig. 5 Thermogravimetric analysis of oat straw for the production of activated carbon at temperature 525 ⁰C Fig. 5 Thermogravimetric analysis of oat straw for the production of activated carbon at temperature 525 ⁰C EXPERIMENTAL WORK AND RESULTS EXPERIMENTAL WORK AND RESULTS Materials and methods: The experimental work concentrate on agri-biomass: oat straw. Thermogravimetric analyzes (TGA) was chosen to find the weight loss kinetics of carbonizated agro-biomass before experimental setup with continues working reactor will be design. In this paper, an TG 209 Tarsus Netzsch thermogravimetric analyzer was used to conduct torrefaction process. Installation with special design reactor feed with steam will be design in second stage to obtain the optimal mass to energy loss ratio (highest energy density). The kinetics were calculated using NETZSCH Kinetics Neo software. Also called reaction kinetics or chemical kinetics, this investigates the rates of chemical processes and allows for the determination of reaction rates. The best fit for the experimental data for the oat straw sample was obtained for the n-order reaction model for n = 3.4235. The R2 correlation coefficient was 0.9972, the activation energy 142 kJ/mol, and the pre-exponential coefficient 10.8466. Biomass torrefaction process was conducted using specially designed setup with a batch reactor for thermochemical conversion in inert dry air atmosphere - Figure 1. Author use thermogravimetric analyzes (TGA) was chosen to find the weight loss kinetics of carbonized agro biomass before experimental setup with continuous reactor feed with dry air will be design. Agro-biomasses were subjected to proximate and ultimate analysis in accordance with ISO standard [7-10]. The same analyses were done after the torrefaction process. High heating value of the energy crop after torrefaction were determined in accordance with ISOstandar [11] CONCLUSIONS AND FURTHER IMPROVEMENTS After the oat straw torrefaction processes we received the following primary mass: 73.95% (257.5˚C), 45.11% (300˚C) and 27.28% (525˚C). The actual temperature can vary depending on the initial conditions of the torrefaction process: the heating rate, and the chemical composition of the raw material. In addition, it can be seen that oat straw torrefaction process at this temperature range of 250-350°C has enhanced the solid biofuel qualities of maize and oat straws by the decrease of the O/C ratio and the increase of HHV [16-18]. It was concluded that kinetic analysis methods using multiple heating rate experiments are more efficient compared to the use of a single heating rate [19-20]. The best fit for the experimental data for the oat straw sample was obtained for the n-order reaction model for n = 3.4235. The R2 correlation coefficient was 0.9972, the activation energy 142 kJ/mol, and the pre-exponential coefficient 10.8466. THERMAL GRAVIMETRIC ANALYSIS The work presents the preservation of straw mass from oats after thermogravimetric analysis. This technique made it possible to observe mass changes as a function of temperature. The thermogravimetric analysis (TGA) of oat straw torrefaction process was done using 5 mg samples. To obtain an atmosphere without oxygen, nitrogen was injected 7 https://doi.org/10.1051/e3sconf/202015402004 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 with a flow rate of 20 mL/min. The samples of oat straw was located inside the TG 209 Tarsus Netzsch thermogravimetric analyzer furnace. The electrical furnace containing the samples was calibrated with a precise (0.01 g resolution) electronic balance for the determination of the mass drop during the process. The readings (temperature, and sample mass) were recorded by PC. This method illustrates the change of the sample in different temperature conditions [12-14]. We subjected samples with straw from oats to dynamic and isothermal measurements with 10 K/min heating rate. Thanks to this, we obtained information on the elemental composition and thermal stability of the Isotherms research material, divided into 5 samples, from 5 to 10 minutes. Based on the measurements obtained at three temperatures of 525 ⁰C (with potential use as a activated carbon), 300 ⁰C (which will be use as addtive for natural fertilizer) and 257.5 ⁰C (where the torrefied biomass will be use as a fuel), we can say that the mass loss ratio increases with the length of the isotherm in each case. But we observed a charismatic change in the sample at 300 ⁰C, which shows very large differences between the values before and after the thermo- chemical treatment- maximum (before) 52.04% and minimum (after) 21.28 %. However, attention should be paid to a temperature of 257.5 ⁰C, because the residual masses are the largest from all 3 temperatures this is a very good example showing the decrease in mass with increasing temperature. and the % of remaining biomass at 525⁰C is the lowest of all measurements. At 257.5 and 525⁰C, as the isotherm time increases, a slight increase in weight loss is noted. On the other hand, during extension of the isotherm at 300 ⁰C, the mass of oat straw dropped sharply. That is why we dare say that elongation of the isotherm does not result in a sudden change in mass at all temperatures [15]. References 1. Adamczyk F., Frąckowiak P., Mielec K., Kośmicki Z., 2005. Problematyka badawcza w procesie zagęszczania słomy przeznaczonej na opał. Journal of Research and Application in Agricultural Engineering, 50(4), 5-8. 2. Gradziuk P., 2006. Ekonomiczne i ekologiczne aspekty wykorzystania słomy na cele energetyczne 3. Gradziuk P., Kościk K., 2007. Analiza możliwości i kosztów pozyskania biomasy na cele energetyczne na potrzeby energetycznego wykorzystania w gminie Clomas. Opracowanie na zlecenie Urzędu Gminy Clomas. p ę y 4. Denisiuk W., 2008. Słoma – potencjał masy i energii. Inżynieria Rolnicza, 2(100), 23-30. 5. Grzybek A., Gradziuk P., Kowalczyk K., 2001. Słoma-energetyczne paliwo. Wyd. Wieś Jutra, Warszawa. (in polish) 6. Kowalczyk-Juśko A., 2009. Uciążliwa, ale bardzo atrakcyjna. Agroenergetyka, 4, 17-20. (in polish) 7. ISO 16994, 2016. Solid biofuels - Determination of total content of sulfur and chlorine. 8. ISO 16948, 2015. Solid biofuels - Determination of total content of carbon, hydrogen and nitrogen. 9. ISO 18122, 2015. Solid biofuels - Determination of ash content. . Solid biofuels - Determination of ash conte 10. ISO 18123, 2015. Solid biofuels - Determination of the content of volatile matter. 11. ISO 18125, 2017. Solid biofuels - Determination of calorific value. 12. Romanowska-Duda Z., Piotrowski K., Wolska B., Dębowski M., Zieliński M., Dziugan P., Szufa S., Stimulating effect of ash from Sorghum on the growth of Lemnaceae – a new source of energy biomass – Springer Nature Switzerland AG 2019, M. Wróbel et al. (eds.), Renewable Energy Sources: Engineering, Technology, Innovation, Springer Proceedings in Energy, ISBN 978-3-030-13887- 5, https://doi.org/10.1007/978-3-030-13888-2_34 , pp. 341-349. 13. Szufa S., Adrian Ł., Piersa P., Romanowska-Duda Z., Ratajczyk-Szufa J., Torrefaction process of millet and cane using batch reactor - Springer Nature Switzerland AG 2019, M. Wróbel et al. (eds.), Renewable Energy Sources: Engineering, Technology, Innovation, Springer Proceedings in Energy, ISBN 978- 3-030-13887-5, https://doi.org/10.1007/978-3-030-13888-2_37 , pp. 371-379 14. Adrian Ł., Szufa S., Piersa P., Kurowski K.: Experimental research and simulation of computer processes of heat exchange in a heat exchanger working on the basis of the principle of heat pipes for the purpose of heat transfer from the ground, 4th Renewable Energy Sources 15. Adrian Ł., Szufa S., Piersa P., Romanowska-Duda Z., Grzesik M., Cebula A., Kowalczyk S., Ratajczyk-Szufa J., Thermographic analysis and experimental work using laboratory installation of heat transfer processes in a heat pipe heat exchanger utilizing as a working fluid R404A and R407A - Springer Nature Switzerland AG 2019, M. Wróbel et al. Acknowledgements The studies presented were financed by the National Center of Research and Development (NCBR) Poland under the research program LIDER. The research and development project is entitled "BIOCARBON - Modern technology biomass torrefaction process to produce fuel mixtures, biocoal as additives for fertilizers, activated carbon for energy sector, agriculture, civil engineering and chemical industry”, „LIDER IX” NCBiR 2014-2020 (0155/L-9/2017). 8 8 https://doi.org/10.1051/e3sconf/202015402004 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 References (eds.), Renewable Energy Sources: Engineering, Technology, Innovation, Springer Proceedings in Energy, ISBN 978- 3-030-13887-5, https://doi.org/10.1007/978-3-030-13888-2_77 , pp. 799-807 p g pp 16. Szufa S., Adrian Ł., Piersa P., Romanowska-Duda Z., Grzesik M., Cebula A., Kowalczyk S., Experimental studies on energy crops torrefaction process using batch reactor to estimate torrefaction temperature and residence time, SPRINGER PROCEEDINGS IN ENERGY, (Web of Science Core Collection), ISBN 978-3-319-72370-9, 2018, https://doi.org/10.1007/978-3-319-72371-6_35 , pp. 365-373, 9 E3S Web of Conferences 154, 02004 (2020) ICoRES 2019 https://doi.org/10.1051/e3sconf/202015402004 17. Adrian Ł., Szufa S., Piersa P., Romanowska-Duda Z., Grzesik M., Cebula A., Kowalczyk S., Experimental research and thermographic analysis of heat transfer processes in a heat pipe heat exchanger utilizing as a working fluid R134A, SPRINGER PROCEEDINGS IN ENERGY, (Web of Science Core Collection), ISBN 978-3-319-72370-9, Chapter, 2018 https://doi.org/10.1007/978-3-319- 72371-6_40 , pp. 413-421, pp 18. Szufa S., Romanowska-Duda B. Z., Grzesik M., Torrefaction proces of the Phragmites Communis growing in soil contaminated with cadmium, 20th European Biomass Conference and Exibition, Milan, 18-22 June 2014, Italy, ISBN: 978-88-89407-54-7, pp. 628 – 634, https://doi.org/10.5071/20thEUBCE2012-1DV.2.63 19. Halina Kruczek, Mateusz Wnukowski, Lukasz Niedzwiecki Guziałowska-Tic, Torrefaction as a Valorization Method Used Prior to the Gasification of Sewage Sludge, January 2019, Energies 12(1):175, DOI: 10.3390/en12010175 20. Ewa Syguła, Jacek A. Koziel, Andrzej Białowiec, Proof-of-Concept of Spent Mushrooms Compost Torrefaction—Studying the Process Kinetics and the Influence of Temperature and Duration on the Calorific Value of the Produced Biocoal, August 2019Energies 12(16):3060, DOI: 10.3390/en12163060 10
https://openalex.org/W2527823477	https://europepmc.org/articles/pmc5059748?pdf=render	English	null	Spontaneous assembly of chemically encoded two-dimensional coacervate droplet arrays by acoustic wave patterning	Nature communications	2,016	cc-by	11,296	ARTICLE Received 31 May 2016 \| Accepted 31 Aug 2016 \| Published 6 Oct 2016 1 Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK. 2 Faculty of Engineering, Queens Building, University of Bristol, Bristol BS8 1TR, UK. 3 School of Physics, HH Wills Physics Laboratory, University of Bristol, Bristol BS8 1TL, UK. Correspondence and requests for materials should be addressed to S.M. (email: s.mann@bristol.ac.uk). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 M Using an acoustic standing wave trap, we demonstrate the spontaneous assembly and organization of polydiallydimethy- lammonium chloride (PDDA)/adenosine 5/-triphosphate (ATP) coacervate micro-droplets into defect-free arrays with controllable lattice spacing and droplet size. We show that individual PDDA/ATP droplets of near uniform size, typically 50–100 mm in diameter, are produced in the acoustic ﬁeld by in situ coalescence of sub-micrometer-sized droplets that aggregate speciﬁcally at the Gor’kov potential energy minima (acoustic pressure nodes) of the standing wave in the early stages of pattern formation. Signiﬁcantly, coalescence between the primary droplets can be curtailed by adjusting the composition of the coacervate droplets such that localized aggregates of closely packed droplets are produced at each node in the acoustic pressure ﬁeld. The localized clusters exhibit collective responses to modulations in the acoustic standing wave to produce arrays with reversible dynamical properties based on transformations in droplet shape and exchange of matter between adjacent nodes in the acoustic ﬁeld. M iniaturization of ﬂuid compartments in the form of liquid micro-droplets is important in diverse scientiﬁc areas1 such as chemical and biochemical analysis2,3, protein crystallization4 and micro-reactor technology5. Many of these applications require high-throughput analyses of spatially addressable arrays of liquid micro-droplets over a range of timescales and chemical/physical environments. Typically, arrays of droplets with a uniform size have been prepared by microﬂuidics6,7, microfabrication8–10, printing11,12 and by application of electrical13 or magnetic ﬁelds14. The droplets are stabilized by immersion in an appropriate continuous phase (water droplets in oil for example) or exposure on a dry surface, which lead to patterns of physically isolated droplets, which can then be exploited as independent micro-reactors that are essentially free from cross-contamination. On the other hand, isolation of the droplets within the arrays is not compatible with dynamical interactions such as triggering chemical signals between the droplets or enabling the droplets to communicate with and respond to time-dependent changes in their external environment. To achieve these dynamical interactions, new technologies are required that provide the production and organization of liquid micro-droplets with similar polarity to the associated continuous phase, such as the formation and patterning of water-rich droplets in a continuous aqueous phase. Such systems are characterized by a relatively low surface tension between the droplets and continuous phase, and remain technically challenging. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 In this regard, recent studies have described the formation of aqueous coacervate micro- droplets in a water continuous phase using a parallel-ﬂow focusing microﬂuidic device15, and the printing of water-rich droplets in an aqueous phase using a dextran/polyethylene glycol (PEG)-based aqueous two-phase system16. However, the spontaneous organization of water-rich droplets in an aqueous phase into two-dimensional (2D) arrays with non-close packed lattices remains a major challenge. Our methodology is applicable to a wide range of complex coacervate systems involving proteins, DNA, polysaccharides, nucleotides and synthetic polyelectrolytes. Coacervates exhibiting strong interactions with the underlying substrate remained spatially patterned when the acoustic ﬁeld is switched off, while those showing reduced surface pinning produce arrays of single micro-droplets that display spatially conﬁned dynamic motions such as localized spinning. Moreover, periodic arrays of chemically encoded single droplets containing sequestered dye molecules, proteins, enzymes, nanoparticles or microparticles can be readily produced in situ during droplet assembly or partitioned into the patterned arrays post-assembly. By adjusting the ratio of PDDA and ATP to limit local molecular diffusion we demonstrate that acoustically patterned populations of coacervate droplets containing different chemical information can be spatially positioned within the sample chamber of the trapping device. Finally, we show that it is possible to transit a reaction wavefront through an array of acoustically trapped enzyme-containing coacervate micro-droplets by establishing an appropriate chemical gradient within the sample chamber of the device. p j g In this paper, we demonstrate the spontaneous assembly and spatial organization of water-rich molecularly crowded micro-droplets to form 2D arrays in aqueous media. Droplet assembly is achieved by a spontaneous process of complex coacervation17–19, which is a liquid–liquid phase separation phenomenon driven by attractive electrostatic interactions usually between counter-charged polyelectrolytes, and entropic gains from the release of small, bound counter-ions and restructuring of water molecules. The resultant micron-sized coacervate droplets comprise a dense, component-enriched viscoelastic phase, dispersed in a chemically deﬁcient aqueous continuous phase. Coacervate droplets have been used for storage of food additives19,20, drug delivery21,22, protein puriﬁcation23, and more recently, exploited as membrane-free protocells24–26 capable of enhanced enzymatic activity27, electric ﬁeld-induced energization28, and in vitro gene expression29. Herein, we show that the in situ generation of coacervate micro-droplets and their spatial organization into 2D periodic lattices in water can be achieved without direct contact by acoustic trapping methods. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Acoustic radiation forces depend on the acoustic contrast generated by compositional differences between media30, and although acoustic beams and standing waves have been exploited for multi-dimensional trapping, patterning and manipulation of micron-sized particles, aqueous droplets in oil and intact cells31–39, generating defect-free uniform patterns with a single particle positioned at each acoustic pressure node has only been achieved at a highly speciﬁc ratio of particle size to acoustic standing wavelength36. Signiﬁcantly, acoustic trapping has not been used to generate arrays of water-rich droplets dispersed in an aqueous medium, principally because of the low interfacial tension of the system. Spontaneous assembly of chemically encoded two-dimensional coacervate droplet arrays by acoustic wave patterning Liangfei Tian1, Nicolas Martin1, Philip G. Bassindale2,3, Avinash J. Patil1, Mei Li1, Adrian Barnes3, Bruce W. Drinkwater2 & Stephen Mann1 The spontaneous assembly of chemically encoded, molecularly crowded, water-rich micro-droplets into periodic defect-free two-dimensional arrays is achieved in aqueous media by a combination of an acoustic standing wave pressure ﬁeld and in situ complex coacervation. Acoustically mediated coalescence of primary droplets generates single-droplet per node micro-arrays that exhibit variable surface-attachment properties, spontaneously uptake dyes, enzymes and particles, and display spatial and time-dependent ﬂuorescence outputs when exposed to a reactant diffusion gradient. In addition, coacervate droplet arrays exhibiting dynamical behaviour and exchange of matter are prepared by inhibiting coalescence to produce acoustically trapped lattices of droplet clusters that display fast and reversible changes in shape and spatial conﬁguration in direct response to modulations in the acoustic frequencies and ﬁelds. Our results offer a novel route to the design and construction of ‘water-in-water’ micro-droplet arrays with controllable spatial organization, programmable signalling pathways and higher order collective behaviour. 1 Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK. 2 Faculty of Engineering, Queens Building, University of Bristol, Bristol BS8 1TR, UK. 3 School of Physics, HH Wills Physics Laboratory, University of Bristol, Bristol BS8 1TL, UK. Correspondence and requests for materials should be addressed to S.M. (email: s.mann@bristol.ac.uk). 1 NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 g PZT Trapping chamber Water a b f c d e /2 /2 Figure 1 \| Acoustic patterning of coacervate micro-droplet arrays. (a) Schematic representation of the acoustic trapping device. Four piezoelectric transducer (PZT) elements (purple cuboids) are arranged around a central square sample chamber, and driven as two pairs. Coacervate micro-droplets are spontaneously assembled and patterned into 2D arrays within the chamber due to the periodic acoustic standing wave pressure ﬁeld. Additional chambers behind each of the PZTs are ﬁlled with water to provide cooling. (b) Simulation of the acoustic pressure distribution in the acoustic trapping device; high pressure (red), low pressure (blue). Gradients in acoustic pressure cause the coacervate micro-droplets to be forced towards the acoustic pressure nodes (blue). (c) Simulation showing the Gor’kov potential distribution in the acoustic trapping device. The separation distance between the nodes is half of the acoustic wavelength (l). Inset shows single anti-node with local directions of the acoustic radiation force (arrows). (d,e) Optical microscopy images of acoustically patterned PDDA/ATP droplets produced using transducer pairs operated at 6.76/6.78 MHz (10 V) by addition of ATP (100 ml, 50 mM) to PDDA (1 ml, 5 mM monomer, 100–200 kDa) contained in the sample holder (d), or at 4.99/5.00 MHz (10 V) (ATP, 200 ml, 50 mM; PDDA, 2 ml, 5 mM monomer) (e). The lattice spacing is increased at the lower acoustic frequency. Mean size of the droplets (ca. 110 mm) in e is larger because of the increased amounts of PDDA and ATP used in the preparations. (f,g) Fluorescence microscopy images of TNP-ATP (0.1 mol%) (f) and RITC-PAH-doped (10 mol%) (g) PDDA/ATP droplets showing the presence of TNP-ATP and PAH throughout the interior of the coacervate phase. Images shown in d,f,g and e were recorded at 45 and 30 min, respectively, after mixing the PDDA and ATP solutions. Scale bars, 150 mm. ng er b PZT Trapping chamber Water a b c /2 /2 a c Trapping chamber d d e e e f f g Figure 1 \| Acoustic patterning of coacervate micro-droplet arrays. (a) Schematic representation of the acoustic trapping device. Four piezoelectric transducer (PZT) elements (purple cuboids) are arranged around a central square sample chamber, and driven as two pairs. Coacervate micro-droplets are spontaneously assembled and patterned into 2D arrays within the chamber due to the periodic acoustic standing wave pressure ﬁeld. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Vigorous mixing of the solutions gave rise to spontaneous liquid–liquid phase separation and the formation of molecularly crowded PDDA/ATP coacervate droplets that exhibited sufﬁcient acoustic contrast to become trapped by the acoustic radiation force. Migration and trapping of the droplets at the pressure nodes of the acoustic ﬁeld was consistent with the increased density and bulk modulus of the coacervate micro-droplets compared with the aqueous continuous phase. Moreover, coalescence of the trapped coacervate droplets in the pressure node was accelerated due to the second acoustic radiation force that acts over short distances32. As a consequence, the micro-droplets although initially trapped in the three-dimensional ﬁeld, slowly sedimented under gravity onto the PEG functionalized glass substrate to produce a defect-free square array of uniform-sized droplets with a lattice spacing that was determined by half of the acoustic wavelength. Typically, centre-to-centre spacings of 110 and 150 mm were observed for arrays produced under acoustic frequencies of 6.76/6.78 and 4.99/5.00MHz, respectively (Fig. 1d,e). Doping of the polymer/ nucleotide mixtures with rhodamine isothiocyanate (RITC)-tagged polyallylamine hydrochloride (PAH; polycationic polymer) or trinitrophenol-ATP (TNP-ATP) produced acoustically ordered arrays of ﬂuorescent micro-droplets (Fig. 1f,g), and conﬁrmed the presence of the complex coacervate phase within the droplets. primary droplets that accumulated around each node in the early stages of pattern formation (Fig. 2a and Supplementary Movie 1). Accumulation and coalescence of multiple primary droplets at each acoustic node was predominant up to 5 min after mixing PDDA (1 ml, 5 mM monomer, 100–200 kDa) and ATP (100 ml, 50 mM), after which the trapped single droplets grew slowly over a period of 45 min to attain a near uniform size with a mean diameter of approximately 70 mm. When all the nodes were ﬁlled with droplets (typically after 3 min), a plot of the average droplet diameter against time was ﬁtted to an exponential function (R2 ¼ 0.991) (Fig. 2b), and measurements of the droplet polydispersity index showed a marked increase in particle size homogeneity within the ﬁrst 9 min of acoustic trapping (Fig. 2c). Thus, it was possible to control the size of the droplets while maintaining a constant lattice spacing by removing the supernatant from the sample chamber after various time intervals to quench the coalescence process. For this, we used the pseudo-kinetic plots shown in Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Typically, centre-to-centre spacings of 110 and 150 mm were observed for arrays produced under acoustic frequencies of 6.76/6.78 and 4.99/5.00MHz, respectively (Fig. 1d,e). Doping of the polymer/ nucleotide mixtures with rhodamine isothiocyanate (RITC)-tagged polyallylamine hydrochloride (PAH; polycationic polymer) or trinitrophenol-ATP (TNP-ATP) produced acoustically ordered arrays of ﬂuorescent micro-droplets (Fig. 1f,g), and conﬁrmed the presence of the complex coacervate phase within the droplets. primary droplets that accumulated around each node in the early stages of pattern formation (Fig. 2a and Supplementary Movie 1). Accumulation and coalescence of multiple primary droplets at each acoustic node was predominant up to 5 min after mixing PDDA (1 ml, 5 mM monomer, 100–200 kDa) and ATP (100 ml, 50 mM), after which the trapped single droplets grew slowly over a period of 45 min to attain a near uniform size with a mean diameter of approximately 70 mm. When all the nodes were ﬁlled with droplets (typically after 3 min), a plot of the average droplet diameter against time was ﬁtted to an exponential function (R2 ¼ 0.991) (Fig. 2b), and measurements of the droplet polydispersity index showed a marked increase in particle size homogeneity within the ﬁrst 9 min of acoustic trapping (Fig. 2c). Thus, it was possible to control the size of the droplets while maintaining a constant lattice spacing by removing the supernatant from the sample chamber after various time intervals to quench the coalescence process. For this, we used the pseudo-kinetic plots shown in Fig. 2b as a calibration curve to guide the preparation of 2D arrays with ﬁxed spacing and geometry but variable droplet size. Increasing the initial PDDA and ATP concentrations accelerated droplet growth for the same acoustic frequencies by increasing the rate of coalescence at the acoustic nodes. For example, increasing the PDDA and ATP concentrations to 7.5 and 75 mM, respectively, produced droplets with a larger mean diameter (83 mm) within a shorter time period (18 min) (Supplementary Fig. 2). Merging of single droplets located at adjacent positions in the 2D array was also more apparent at higher polymer concentrations (Supplementary Fig. 3). Conversely, decreasing the initial concentrations in the acoustic trapping device reduced the rate of droplet coalescence at 4.99/5.00MHz with corresponding wavelengths of 219/218 mm and 297/296 mm, respectively (see Methods). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Additional chambers behind each of the PZTs are ﬁlled with water to provide cooling. (b) Simulation of the acoustic pressure distribution in the acoustic trapping device; high pressure (red), low pressure (blue). Gradients in acoustic pressure cause the coacervate micro-droplets to be forced towards the acoustic pressure nodes (blue). (c) Simulation showing the Gor’kov potential distribution in the acoustic trapping device. The separation distance between the nodes is half of the acoustic wavelength (l). Inset shows single anti-node with local directions of the acoustic radiation force (arrows). (d,e) Optical microscopy images of acoustically patterned PDDA/ATP droplets produced using transducer pairs operated at 6.76/6.78 MHz (10 V) by addition of ATP (100 ml, 50 mM) to PDDA (1 ml, 5 mM monomer, 100–200 kDa) contained in the sample holder (d), or at 4.99/5.00 MHz (10 V) (ATP, 200 ml, 50 mM; PDDA, 2 ml, 5 mM monomer) (e). The lattice spacing is increased at the lower acoustic frequency. Mean size of the droplets (ca. 110 mm) in e is larger because of the increased amounts of PDDA and ATP used in the preparations. (f,g) Fluorescence microscopy images of TNP-ATP (0.1 mol%) (f) and RITC-PAH-doped (10 mol%) (g) PDDA/ATP droplets showing the presence of TNP-ATP and PAH throughout the interior of the coacervate phase. Images shown in d,f,g and e were recorded at 45 and 30 min, respectively, after mixing the PDDA and ATP solutions. Scale bars, 150 mm. 4.99/5.00MHz with corresponding wavelengths of 219/218 mm and 297/296 mm, respectively (see Methods). Vigorous mixing of the solutions gave rise to spontaneous liquid–liquid phase separation and the formation of molecularly crowded PDDA/ATP coacervate droplets that exhibited sufﬁcient acoustic contrast to become trapped by the acoustic radiation force. Migration and trapping of the droplets at the pressure nodes of the acoustic ﬁeld was consistent with the increased density and bulk modulus of the coacervate micro-droplets compared with the aqueous continuous phase. Moreover, coalescence of the trapped coacervate droplets in the pressure node was accelerated due to the second acoustic radiation force that acts over short distances32. As a consequence, the micro-droplets although initially trapped in the three-dimensional ﬁeld, slowly sedimented under gravity onto the PEG functionalized glass substrate to produce a defect-free square array of uniform-sized droplets with a lattice spacing that was determined by half of the acoustic wavelength. NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications Results Acoustic patterning of coacervate micro-droplet arrays. The spontaneous assembly and patterning of molecularly crowded PDDA/ATP coacervate micro-droplets in water was undertaken using a custom-made acoustic trapping device that was fabricated from polyethylene terephthalate and consisted of a central square chamber and four piezoelectric transducers arranged around the periphery (Fig. 1a and Supplementary Fig. 1). Opposing transducers were wired in parallel and driven as a pair with a sinusoidal voltage supply by a signal generator. Each of the two orthogonally arranged pairs was operated at slightly different frequencies such that the total acoustic radiation force ﬁeld tended to the sum of the force ﬁelds of the individual pairs at times greater than 8 ms (see Methods). As the response time of the droplets was considerably greater than 8 ms, this simple summation approach was valid for all the experiments undertaken. Simulations of the resultant acoustic standing wave pressure ﬁeld showed a periodic array of nodes and anti-nodes in a 2D grid-like pattern that were associated with the minima (pressure nodes) and maxima (pressure antinodes), respectively, of the Gor’kov potential energy distribution (Fig. 1b,c). An aqueous solution of PDDA (Mw ¼ 100–200kDa) was placed into the central chamber and aqueous ATP then added in the presence of an acoustic standing wave ﬁeld generated from orthogonally arranged opposing transducer pairs typically operating at 6.76/6.78MHz or NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 2b as a calibration curve to guide the preparation of 2D arrays with ﬁxed spacing and geometry but variable droplet size. Increasing the initial PDDA and ATP concentrations accelerated droplet growth for the same acoustic frequencies by increasing the rate of coalescence at the acoustic nodes. For example, increasing the PDDA and ATP concentrations to 7.5 and 75 mM, respectively, produced droplets with a larger mean diameter (83 mm) within a shorter time period (18 min) (Supplementary Fig. 2). Merging of single droplets located at adjacent positions in the 2D array was also more apparent at higher polymer concentrations (Supplementary Fig. 3). Conversely, decreasing the initial concentrations in the acoustic trapping device reduced the rate of droplet coalescence at Optical microscopy video monitoring of the formation of single droplets within the acoustic trap operating at 6.76/6.78 MHz (10 V) indicated that they were produced simultaneously by in situ coalescence of sub-micrometer-sized 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 the acoustic nodes (Supplementary Fig 4) In general the ratio Dynamical behaviour in coacervate micro-droplet arrays a 0 3 5 10 15 45 b 0 9 18 27 36 45 Time (min) 0 9 18 27 36 45 Time (min) 75 55 15 35 Diameter (µm) 0.18 0.08 c 0.13 PDI Figure 2 \| Growth of acoustically trapped coacervate micro-droplets. (a) Time-dependent optical microscopy images showing the growth of PDDA/ATP coacervate droplets at the nodes of an acoustic standing wave pressure ﬁeld generated by transducer pairs operating at 6.76/6.78MHz (10V). The images were recorded at t ¼ 0, 3, 5, 10, 15 and 45min after addition of ATP (100 ml, 50mM) to a PDDA solution (1 ml, 5mM monomer, 100–200kDa) contained within the sample chamber of the acoustic trapping device. Inset at t ¼ 3 min: high-magniﬁcation image showing localized aggregate of coalescing primary droplets; sub-micrometre-sized droplets are observed as indistinct areas of higher optical contrast surrounding the two larger droplets undergoing coalescence; scale bars, 20mm (inset) and 50mm. (b) Plot showing change in mean diameter of PDDA/ATP coacervate micro-droplets against time during growth within the acoustic ﬁeld under the above conditions. An induction time of ca. 3min was observed during which the primary sub-micrometer-sized droplets sedimented onto the glass substrate. Error bars represent the standard deviation of the size of coacervate micro-droplets at different time intervals in the same device. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Inset at t ¼ 3 min: high-magniﬁcation image showing localized aggregate of coalescing primary droplets; sub-micrometre-sized droplets are observed as indistinct areas of higher optical contrast surrounding the two larger droplets undergoing coalescence; scale bars, 20mm (inset) and 50mm. (b) Plot showing change in mean diameter of PDDA/ATP coacervate micro-droplets against time during growth within the acoustic ﬁeld under the above conditions. An induction time of ca. 3min was observed during which the primary sub-micrometer-sized droplets sedimented onto the glass substrate. Error bars represent the standard deviation of the size of coacervate micro-droplets at different time intervals in the same device. (c) Corresponding time-dependent plot of the polydispersity index (PDI) for PDDA/ATP micro-droplets shown in b after mixing PDDA and ATP. Dynamical behaviour in coacervate micro-droplet arrays. Given the propensity for coacervates to adopt a range of physical and chemical properties depending on their composition, surface charge, dehydration and complexation strength17–23, we developed experimental procedures for controlling the dynamical behaviour of the acoustically patterned droplets when immersed in aqueous media. Binding of the droplets to the underlying PEGylated glass substrate was modulated by changes in composition and exploited to induce droplet rotation and provide a method for immobilizing or dispersing the arrays in water when the acoustic ﬁeld was switched off. Acoustically trapped PDDA (100–200 kDa)/ATP single droplets were so strongly attached to the substrate that they adopted a hemispherical morphology and exhibited minimal rotational movement in the presence of the acoustic ﬁeld (Supplementary Fig. 10). As a consequence, they remained spatially ﬁxed at the lattice positions for extended periods of time (tZ12 h) when the acoustic pressure was switched off. Acoustically trapped PDDA (8.5kDa)/CM-D single droplets in contrast were less ﬁrmly attached to the PEGylated glass substrate and displayed spatially conﬁned localized spinning at the potential energy minima of the pressure ﬁeld possibly due to an acoustic streaming force Similar procedures were used to self-assemble, trap and acoustically pattern PDDA-containing coacervate micro-droplets prepared from a range of components including proteins (bovine serum albumin, BSA), polysaccharides (carboxymethyldextran, CM-D), polynucleotides (double stranded DNA (dsDNA)) and anionic polymers (polyacrylic acid, PAA), as well as coacervates formed by complexation of polyethylenimine (PEI) and CM-D. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 (c) Corresponding time-dependent plot of the polydispersity index (PDI) for PDDA/ATP micro-droplets shown in b after mixing PDDA and ATP. a a 0 3 5 10 15 45 10 15 45 b 0 9 18 27 36 45 Time (min) 0 9 18 27 36 45 Time (min) 75 55 15 35 Diameter (µm) 0.18 0.08 c 0.13 PDI Figure 2 \| Growth of acoustically trapped coacervate micro-droplets. (a) Time-dependent optical microscopy images showing the growth of PDDA/ATP coacervate droplets at the nodes of an acoustic standing wave pressure ﬁeld generated by transducer pairs operating at 6.76/6.78MHz (10V). The images were recorded at t ¼ 0, 3, 5, 10, 15 and 45min after addition of ATP (100 ml, 50mM) to a PDDA solution (1 ml, 5mM monomer, 100–200kDa) contained within the sample chamber of the acoustic trapping device. Inset at t ¼ 3 min: high-magniﬁcation image showing localized aggregate of coalescing primary droplets; sub-micrometre-sized droplets are observed as indistinct areas of higher optical contrast surrounding the two larger droplets undergoing coalescence; scale bars, 20mm (inset) and 50mm. (b) Plot showing change in mean diameter of PDDA/ATP coacervate micro-droplets against time during growth within the acoustic ﬁeld under the above conditions. An induction time of ca. 3min was observed during which the primary sub-micrometer-sized droplets sedimented onto the glass substrate. Error bars represent the standard deviation of the size of coacervate micro-droplets at different time intervals in the same device. (c) Corresponding time dependent plot of the polydispersity index (PDI) for PDDA/ATP micro droplets shown in b after mixing PDDA and ATP 5 0 9 18 27 36 45 Time (min) 0.18 0.08 c 0.13 PDI b 0 9 18 27 36 45 Time (min) 75 55 15 35 Diameter (µm) b c Figure 2 \| Growth of acoustically trapped coacervate micro-droplets. (a) Time-dependent optical microscopy images showing the growth of PDDA/ATP coacervate droplets at the nodes of an acoustic standing wave pressure ﬁeld generated by transducer pairs operating at 6.76/6.78MHz (10V). The images were recorded at t ¼ 0, 3, 5, 10, 15 and 45min after addition of ATP (100 ml, 50mM) to a PDDA solution (1 ml, 5mM monomer, 100–200kDa) contained within the sample chamber of the acoustic trapping device. NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 (Supplementary Movie 2). Attachment of the PDDA/CM-D droplets was further reduced by addition of high molecular weight ﬂuorescein isothiocyanate (FITC)-CM-D (25 mol%) such that the arrays slowly dispersed into bulk solution when the acoustic ﬁeld was removed. aggregates that were only loosely attached to the underlying substrate. As a consequence, modulations in the acoustic radiation force could be used to generate fast (o1 min) and reversible changes in the shape and spatial conﬁguration of the droplet aggregates to produce dynamical patterns capable of exchanging matter between adjacent nodes in the acoustic ﬁeld. For example, by exciting transducer pairs that operated in-phase at the same frequency (6.76 MHz) we were able to transform the localized aggregates into new spatial conﬁgurations (Fig. 3c,d and Supplementary Movie 3)41. In addition, patterns of discrete spherical aggregates could be reversibly transformed in aqueous media into vertically or horizontally oriented elliptical shapes, further distorted into continuous fringes of micrometre-sized coacervate droplets, and then recapitulated by switching on/off the corresponding pairs of piezoelectric transducers (Fig. 3e–g and Supplementary Movie 4). (Supplementary Movie 2). Attachment of the PDDA/CM-D droplets was further reduced by addition of high molecular weight ﬂuorescein isothiocyanate (FITC)-CM-D (25 mol%) such that the arrays slowly dispersed into bulk solution when the acoustic ﬁeld was removed. Using a similar strategy, changes in composition, PDDA molecular weight or charge ratio between the coacervate components were exploited to promote or inhibit single droplet formation at the nodes of the acoustic standing wave pressure ﬁeld by inﬂuencing the interfacial tension between the coalescing primary droplets. Whereas 2D arrays of polymer-protein PDDA/BSA single droplets were produced using a low molecular weight PDDA (8.5 kDa), replacing the polymer with a 100–200 kDa PDDA gave rise to a square grid comprising aggregates of non-coalescing PDDA/BSA droplets that remained trapped at the acoustic nodes (Supplementary Fig. 11). We attributed these differences to increases in the relaxation time and viscosity of the coacervate droplets prepared with high molecular weight PDDA (ref. 40). Aggregates of non-coalescing primary droplets with a mean diameter of 310 nm and net negative surface charge ( 20 mV) (Supplementary Fig. 12) were also patterned in acoustically trapped arrays prepared from mixtures of PDDA (8.5 kDa), FITC-tagged CM-D and CM-D (Fig. 3a,b). ARTICLE Signiﬁcantly, by using FITC-CMD in the above preparations, we were able to generate ordered arrays of Molecular uptake and spatially positioned enzyme activity. We prepared acoustically trapped arrays of chemically encoded single droplets by spontaneously sequestering water-soluble organic dyes of different charge, proteins, enzymes, polystyrene nanoparticles or silica microparticles into the molecularly crowded PDDA/ATP droplets during or after the assembly process in aqueous media (Fig. 4a–f and Supplementary Fig. 13). a c b d e f g 0 s 5.2 s 23.4 s 27.3 s 32.5 s 0 s 3.9 s 26.0 s 29.5 s 44.2 s Figure 3 \| Dynamical behaviour of acoustically trapped coacervate micro-droplets. (a) Optical microscopy image showing acoustically patterned 2D array comprising discrete spherical aggregates of non-coalescing sub-micrometre-sized PDDA/FITC-CM-D/CM-D coacervate droplets. The optical contrast originates from the collection of micro-droplets within each aggregate; individual droplets are too small to be clearly resolved. The droplets were prepared by adding PDDA (100ml, 50mM monomer, 8.5 kDa) to 1 ml of a CM-D (36 mM)/FITC-CM-D (9mM) mixture contained within the sample chamber of an acoustic trapping device using two orthogonal transducer pairs operating at different frequencies (6.76/6.78MHz, 10V); scale bar, 100 mm. (b) Simulation of the acoustic ﬁeld shown in a showing the Gor’kov potential distribution in the acoustic trapping device; high pressure (red), low pressure (blue). (c) Optical microscopy image of sample shown in a but using orthogonal transducer pairs operating in-phase at the same frequency (6.76 MHz, 10V) to produce a 2D array of non-spherical coacervate micro-droplet aggregates; scale bar, 100 mm. (d) Simulation of the acoustic ﬁeld shown in c showing the Gor’kov potential distribution in the acoustic trapping device; high pressure (red), low pressure (blue). (e,f) Time-dependent series of ﬂuorescence microscopy images showing acoustically induced reversible transformation of a square lattice of discrete spherical aggregates of non-coalescing sub-micrometre PDDA/FITC-CM-D/ CM-D coacervate droplets into elliptical arrangements and continuous vertical fringes (e) followed by recapitulation of the 2D array and subsequent reversible manipulation of the square lattice into horizontal lines of non-interacting coacervate micro-droplets (f); t ¼ time in seconds after switching on/off the corresponding pairs of piezoelectric transducers (PZTs); scale bar, 100 mm. (g) Simulation of the acoustic pressure distribution in the acoustic trapping device operating with only one pair of PZTs; high pressure (red), low pressure (blue). NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 In general, the PDDA-containing coacervates were prepared by placing the above solutions into the sample chamber of the device followed by addition of PDDA (Mw ¼ 8.5 kDa) in the presence of two orthogonal acoustic standing waves (6.76/6.78 MHz, 10 V) (see Methods). In each case, 2D arrays of spatially organized single micro-droplets could be produced in aqueous media via coalescence of primary droplets at the acoustic nodes (Supplementary Figs 5–9). NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 For example, uptake of FITC-tagged glucose oxidase (FITC-GOx) was achieved by injecting an ATP solution into a pre-mixed PDDA/FITC-GOx solution contained within the device under an acoustic radiation force ﬁeld (Supplementary Fig. 14). Measure- ments of the time-dependent changes in FITC-GOx mean ﬂuorescence intensity at the acoustic pressure nodes and antinodes showed progressive increases and decreases, respectively, over the initial 30 min period (Fig. 4g), indicating that FITC-GOx was spontaneously sequestered into the assembling coacervate droplets. Corresponding mean ﬂuorescence line intensity measurements recorded over the initial 30 min across a row of FITC-GOx-containing PDDA/ATP coacervate droplets showed the emergence of a periodic one-dimensional proﬁle with a spacing of ca. 110 mm (Fig. 4h) and highly uniform local intensity distributions (Fig. 4i), indicating that droplets with comparable enzyme concentrations could be readily produced by acoustic trapping. was contained within the sample chamber and a small volume of PDDA along with an enzyme then gently injected at a localized position and the mixture left undisturbed in the acoustic ﬁeld. Consequently, the ATP: PDDA monomer molar ratio was 100: 1 rather than 1: 1, and coacervation occurred immediately around the point of injection to produce primary droplets that coalesced in the acoustic ﬁeld to generate a highly localized array of enzyme-containing single droplets (Supplementary Fig. 15). By repeating this procedure but using three different enzymes with relatively high partition constants (K) (GOx, K ¼ 60; amyloglucosidase (AGx, K ¼ 355); or horseradish peroxidase (HRP), K ¼ 62)) in the injected PDDA solutions, we were able to prepare distinct spatial domains of functional droplet arrays within the same device (Fig. 5a). g Given the above observations, we reasoned that it should be possible to develop acoustic trapping protocols for the preparation of water-based droplet arrays capable of sensing speciﬁc chemicals in the environment. As proof-of-principle we acoustically patterned an array of HRP-containing PDDA/ATP coacervate micro-droplets at a spacing of ca. 110 mm, and exposed the pattern to a gradient of H2O2 and o-phenylenediamine (o-PD) by diffusing the substrate mixtures speciﬁcally from one side of the device. Conversion of non-ﬂuorescent o-PD to ﬂuorescent cationic 2,3-diaminophenazine (2,3-DAP) by the coacervate droplet-sequestered enzyme was recorded by ﬂuorescence microscopy42,43. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Within a few minutes of injecting Based on the capability of the acoustically trapped coacervate droplets to sequester a range of water-soluble molecules during pattern formation, we developed a method for preparing spatially positioned populations of different functional arrays within the same sample chamber. For this, we inhibited the formation of a single extended uniform array throughout the chamber by limiting local molecular diffusion on mixing PDDA (100–200 kDa) and ATP. This was achieved by reversing the standard location of the PDDA and ATP solutions such that ATP 13 15 17 19 21 23 Intensity (a.u.) 0 6 12 18 24 30 Time (min) 135 90 45 0 i Count 16 11 6 Intensity (a.u.) Intensity (a.u.) Postion across the droplet (mm) 0 0.3 0.6 0.9 1.2 1.5 19 16 13 10 e a b c d h g f Figure 4 \| Chemical encoding of acoustically patterned coacervate micro-droplets. (a–f) Fluorescence microscopy images of acoustically patterned PDDA/ATP coacervate droplets containing methylene blue (a), calcein (b), sulforhodamine B (c), nile red (d), FITC-labelled 100 nm-sized polystyrene particles (partition constant ¼ 4,000) (e) or FITC-GOx (f); scale bars, 100 mm. (g) Plots of time-dependent increase and decrease in FITC-GOx mean ﬂuorescence intensity measured at the acoustic nodes (red curve) and antinodes (blue curve), respectively, after addition of ATP (50 ml, 25 mM) to a premixed solution of PDDA (1 ml, 2.5 mM, monomer, 8.5 kDa) and FITC-GOx (1 mg ml 1) solution under an acoustic standing wave pressure ﬁeld (6.76/6.78 MHz, 10 V). Error bars represent the standard deviation of the mean ﬂuorescence intensities at acoustic nodes (red) or antinode (blue) at different time intervals in the same device. (h) Mean ﬂuorescence line intensity proﬁles recorded across FITC-GOx-containing PDDA/ATP coacervate droplets prepared as in g after 0 (black line), 10 (blue line) and 30 min (red line) of exposure in the acoustic trapping ﬁeld. (i) Statistical counts of mean ﬂuorescence intensity of FITC-GOx-containing PDDA/ATP droplets after 30 min. Sample was prepared as in g. Images shown in a–e and f were recorded at 45 and 30 min, respectively, after mixing. a.u., arbitrary unit. ARTICLE b d a c a d c e f g 0 s 5.2 s 23.4 s 27.3 s 32.5 s 0 s 3.9 s 26.0 s 29.5 s 44.2 s e f 0 s 5.2 s 23.4 s 27.3 s 32.5 s 0 s 3.9 s 26.0 s 29.5 s 44.2 s e g e g Figure 3 \| Dynamical behaviour of acoustically trapped coacervate micro-droplets. (a) Optical microscopy image showing acoustically patterned 2D array comprising discrete spherical aggregates of non-coalescing sub-micrometre-sized PDDA/FITC-CM-D/CM-D coacervate droplets. The optical contrast originates from the collection of micro-droplets within each aggregate; individual droplets are too small to be clearly resolved. The droplets were prepared by adding PDDA (100ml, 50mM monomer, 8.5 kDa) to 1 ml of a CM-D (36 mM)/FITC-CM-D (9mM) mixture contained within the sample chamber of an acoustic trapping device using two orthogonal transducer pairs operating at different frequencies (6.76/6.78MHz, 10V); scale bar, 100 mm. (b) Simulation of the acoustic ﬁeld shown in a showing the Gor’kov potential distribution in the acoustic trapping device; high pressure (red), low pressure (blue). (c) Optical microscopy image of sample shown in a but using orthogonal transducer pairs operating in-phase at the same frequency (6.76 MHz, 10V) to produce a 2D array of non-spherical coacervate micro-droplet aggregates; scale bar, 100 mm. (d) Simulation of the acoustic ﬁeld shown in c showing the Gor’kov potential distribution in the acoustic trapping device; high pressure (red), low pressure (blue). (e,f) Time-dependent series of ﬂuorescence microscopy images showing acoustically induced reversible transformation of a square lattice of discrete spherical aggregates of non-coalescing sub-micrometre PDDA/FITC-CM-D/ CM-D coacervate droplets into elliptical arrangements and continuous vertical fringes (e) followed by recapitulation of the 2D array and subsequent reversible manipulation of the square lattice into horizontal lines of non-interacting coacervate micro-droplets (f); t ¼ time in seconds after switching on/off the corresponding pairs of piezoelectric transducers (PZTs); scale bar, 100 mm. (g) Simulation of the acoustic pressure distribution in the acoustic trapping device operating with only one pair of PZTs; high pressure (red), low pressure (blue). 5 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 0 6 12 18 24 30 Time (min) 16 11 6 Intensity (a.u.) g b b a ( ) Intensity (a.u.) Postion across the droplet (mm) 0 0.3 0.6 0.9 1.2 1.5 19 16 13 10 h d d d f i 13 15 17 19 21 23 Intensity (a.u.) 135 90 45 0 i Count Postion across the droplet (mm) f e Figure 4 \| Chemical encoding of acoustically patterned coacervate micro-droplets. (a–f) Fluorescence microscopy images of acoustically patterned PDDA/ATP coacervate droplets containing methylene blue (a), calcein (b), sulforhodamine B (c), nile red (d), FITC-labelled 100 nm-sized polystyrene particles (partition constant ¼ 4,000) (e) or FITC-GOx (f); scale bars, 100 mm. (g) Plots of time-dependent increase and decrease in FITC-GOx mean ﬂuorescence intensity measured at the acoustic nodes (red curve) and antinodes (blue curve), respectively, after addition of ATP (50 ml, 25 mM) to a premixed solution of PDDA (1 ml, 2.5 mM, monomer, 8.5 kDa) and FITC-GOx (1 mg ml 1) solution under an acoustic standing wave pressure ﬁeld (6.76/6.78 MHz, 10 V). Error bars represent the standard deviation of the mean ﬂuorescence intensities at acoustic nodes (red) or antinode (blue) at different time intervals in the same device. (h) Mean ﬂuorescence line intensity proﬁles recorded across FITC-GOx-containing PDDA/ATP coacervate droplets prepared as in g after 0 (black line), 10 (blue line) and 30 min (red line) of exposure in the acoustic trapping ﬁeld. (i) Statistical counts of mean ﬂuorescence intensity of FITC-GOx-containing PDDA/ATP droplets after 30 min. Sample was prepared as in g. Images shown in a–e and f were recorded at 45 and 30 min, respectively, after mixing. a.u., arbitrary unit. NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications 6 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 GOx HRP AGx 0 0.18 0.36 0.54 Relative positions along Y (mm) 0.09 0.27 0.45 8 5 2 –1 Intensity (a.u.) y x Time (s) 40 130 220 310 400 Time (s) 40 130 220 310 400 13 7 1 Intensity (a.u.) 4 10 c e d b a Time y y Figure 5 \| Spatial positioning of mixed populations and enzyme activity. (a) Fluorescence microscopy image showing three spatially positioned domains of enzyme-containing PDDA (100–200 kDa)/ATP droplet arrays with sequestered GOx (green), AGx (blue) or HRP (red) (see arrows). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 The ensemble was prepared by sequentially adding premixed solutions of PDDA (1 ml, 25 mM, monomer)/FITC-GOx (0.2 mg ml 1), PDDA (1 ml, 25 mM monomer)/AGx (0.2 mg ml 1), or PDDA (1 ml, 25 mM, monomer)/RITC-HRP (0.2 mg ml 1) to ATP (1 ml, 2.5 mM) contained within the acoustic trapping device operating under an acoustic standing wave pressure ﬁeld (6.76/6.78 MHz, 10 V). (b) Fluorescence microscopy image of an acoustically patterned 2D array of 110 mm-spaced HRP-containing PDDA (100–200 kDa)/ATP coacervate droplets recorded 250 s after diffusion of H2O2/o-PD into one side of the reaction chamber along the y direction (arrows). Formation of the ﬂuorescent 2,3-DAP product is observed in the form of a chemical wave-front that transits across the droplet array; scale bars in a and b, 100 mm. (c) Plots of time-dependent changes in 2,3-DAP ﬂuorescence mean intensity associated with patterned HRP-containing PDDA/ATP coacervate micro-droplets positioned 110 mm apart along a single row aligned parallel to the direction of H2O2/o-PD diffusion (y axis in b). Plots for three droplets positioned at alternate lattice points lying parallel to the diffusion direction (y axis) and at increasing distances from the advancing wave-front (redogreenoblue) are shown. A time lag of ca. 12 s is observed between droplets position on adjacent lattice points. (d) Fluorescence line intensity proﬁles recorded across a single row of HRP-containing PDDA/ATP droplets aligned along the direction of diffusion (y axis in b) 50, 100, 150, 200, 225, 250, 275 and 300 s after injection of H2O2/o-PD into the reaction chamber. (e) Similar plots as in c but for three droplets positioned at alternate lattice points in a row lying perpendicular to the diffusion front showing minimal differences in their enzymatic activity at a given time. a.u., arbitrary unit. Time (s) 40 130 220 310 400 Time (s) 40 130 220 310 400 13 7 1 Intensity (a.u.) 4 10 c e d y y GOx HRP AGx a a c y x b b d 0 0.18 0.36 0.54 Relative positions along Y (mm) 0.09 0.27 0.45 8 5 2 –1 Intensity (a.u.) Time (s) Time (s) d Time Figure 5 \| Spatial positioning of mixed populations and enzyme activity. (a) Fluorescence microscopy image showing three spatially positioned domains of enzyme-containing PDDA (100–200 kDa)/ATP droplet arrays with sequestered GOx (green), AGx (blue) or HRP (red) (see arrows). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 The ensemble was prepared by sequentially adding premixed solutions of PDDA (1 ml, 25 mM, monomer)/FITC-GOx (0.2 mg ml 1), PDDA (1 ml, 25 mM monomer)/AGx (0.2 mg ml 1), or PDDA (1 ml, 25 mM, monomer)/RITC-HRP (0.2 mg ml 1) to ATP (1 ml, 2.5 mM) contained within the acoustic trapping device operating under an acoustic standing wave pressure ﬁeld (6.76/6.78 MHz, 10 V). (b) Fluorescence microscopy image of an acoustically patterned 2D array of 110 mm-spaced HRP-containing PDDA (100–200 kDa)/ATP coacervate droplets recorded 250 s after diffusion of H2O2/o-PD into one side of the reaction chamber along the y direction (arrows). Formation of the ﬂuorescent 2,3-DAP product is observed in the form of a chemical wave-front that transits across the droplet array; scale bars in a and b, 100 mm. (c) Plots of time-dependent changes in 2,3-DAP ﬂuorescence mean intensity associated with patterned HRP-containing PDDA/ATP coacervate micro-droplets positioned 110 mm apart along a single row aligned parallel to the direction of H2O2/o-PD diffusion (y axis in b). Plots for three droplets positioned at alternate lattice points lying parallel to the diffusion direction (y axis) and at increasing distances from the advancing wave-front (redogreenoblue) are shown. A time lag of ca. 12 s is observed between droplets position on adjacent lattice points. (d) Fluorescence line intensity proﬁles recorded across a single row of HRP-containing PDDA/ATP droplets aligned along the direction of diffusion (y axis in b) 50, 100, 150, 200, 225, 250, 275 and 300 s after injection of H2O2/o-PD into the reaction chamber. (e) Similar plots as in c but for three droplets positioned at alternate lattice points in a row lying perpendicular to the diffusion front showing minimal differences in their enzymatic activity at a given time. a.u., arbitrary unit. the small molecule substrates the ﬂuorescence images showed a clear response to the environmental stimulus in the form of a chemical wavefront that propagated through the coacervate micro-droplet array (Fig. 5b). Measurements of the ﬂuorescence associated with HRP-mediated 2,3-DAP production in individual coacervate droplets positioned along a single row lying parallel to the direction of diffusion showed a lag time of ca. 12 s between adjacent droplets (Fig. 5c). Line proﬁles recorded along the direction of diffusion at different time intervals showed that the output signals of each droplet were strongly dependent on their spatial positions (Fig. 5d). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Moreover, although the droplets were activated at different times depending on their proximity to the diffusion front, the rate of change in mean ﬂuorescence intensity was similar for each droplet (Fig. 5c), suggesting that the enzymatic response was effectively unchanged across the 2D array. In contrast, only marginal differences in activation were observed between adjacent droplets positioned in single rows lying perpendicular to the diffusion front (Fig. 5e). liquid droplet periodic arrays comprising selective chemicals, biomolecules and catalysts. Patterning functional water-based droplets in aqueous media is challenging because of the minimal acoustic contrast, structural instability associated with low interfacial tension, and difﬁculty of sequestering solutes into the droplets due to the small values of the equilibrium partitioning constants. These challenges have been circumvented by combining an acoustic standing wave force ﬁeld with in situ complex coacervation to generate defect-free arrays of single coacervate droplets or droplet aggregates arranged in 2D lattices with controllable spacing, variable surface-attachment properties and reversible dynamical behaviour. The ﬁnal size of the droplets can be controlled by changes in the chemical concentrations or by quenching coalescence of the primary droplets to produce 2D arrays with ﬁxed spacing and geometry but variable droplet size. While our studies have focused on the generation of square grids, it should be straightforward to extend the methodology to more complex droplet patterns through appropriate geometrical recon- ﬁguration of the piezoelectric transducers and sample chamber44. A key advantage of the described methodology is associated with the diverse physical and chemical properties of coacervates, which can be readily tailored by changes in composition, binding constants, dehydration and surface charge17–23. As a consequence, coacervate micro-droplets exhibiting variable viscosities, different levels of molecular crowding and surface adherence, and diverse NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications Discussion l Acoustically patterned arrays of PDDA/ATP droplets were prepared as above (6.76/6.78 MHz, 10 V) in the presence of methylene blue (10 ml, 1 mM), nile red (10 ml, 1 mM), sulforhodamine B (10 ml, 1 mM) or calcein (1 ml, 1 mM), and ﬂuorescence images recorded after 15min to determine the level of molecular uptake. Sequestration of polymer or inorganic particles into the acoustically trapped droplets was undertaken by injecting 100ml of premixed polystyrene nanoparticles (100 nm, 5 10 2 wt.%) or silica microparticles (2.5mm, 2.5 10 2 wt.%) and aqueous ATP (100ml, 50 mM) into a PDDA solution (1 ml, 5 mM monomer; PDDA monomer: ATP ¼ 1, pH ¼ 7) contained with the device and subjected to two orthogonal acoustic standing waves (6.76/6.78 MHz, 10V). The mixtures were stirred to ensure homogeneous formation of the coacervate dro- plets in the square chamber. Three-dimensional confocal microscopy construction of the silica microparticle-containing PDDA/ATP droplets was undertaken by adding sulforhodamine B (10 ml, 1 mM) to the coacervate micro-droplet array after 45 min of acoustic processing. In situ sequestration of FITC-GOx into PDDA/ATP acoustically trapped micro-droplet arrays was achieved by injecting aqueous ATP (100 ml, 50 mM) into 1 ml of PDDA (8.5kDa, 5 mM monomer) containing FITC-GOx (1mgml 1) in the presence of two orthogonal acoustic standing waves (6.76/6.78MHz, 10 V; PDDA monomer: ATP¼ 1, pH¼ 7). Partition coefﬁcients (K) for dye molecules and enzymes were determined by measuring the concentrations (C) in the supernatant (super) and coacervate (coac) phase, and given as K ¼ Ccoac/Csuper. Concentrations were determined from characteristic absorption/emission spectra according to previously reported methods25. Spatial organization of acoustically trapped PDDA/ATP droplet arrays. A 1 ml volume of a pre-mixed solution of PDDA (25 mM monomer, 100–200 kDa) and FITC-GOx (0.2 mg ml 1) were gently added to an ATP solution (1 ml, 2.5 mM) contained within the chamber of an acoustic trapping device in the presence of two orthogonal acoustic standing waves (6.76/6.78 MHz, 10 V). After injection, the device was left undisturbed so that enzyme-containing coacervate micro-droplets were formed speciﬁcally at the nodal regions within a localized area close to the point of injection of the PDDA/FITC-GOx mixture. Methods A ti t Acoustic trapping. A custom-built acoustic trapping device based on a square arrangement of four piezoelectric transducers with a thickness of 1 mm (Noliac, NCE 51, L15 W2 mm) or 0.4 mm (Noliac, NCE 51, L15 W5 mm) was used. The opposing transducer pairs were wired in series, driven by two signal generators (Agilent 33220a-001), and connected to an oscilloscope (Agilent DSOX2014A). The orthogonal transducer pairs were run at slightly different frequencies during the trapping process. Transducer pairs with a thickness of 1 or 0.4 mm were operated at the third harmonic frequency (6.76/6.78 MHz) or at their fundamental frequency (4.99/5.00 MHz), respectively. The corresponding wavelengths were 219/218 mm (6.76/6.78 MHz) and 297/296 mm (4.99/5.00 MHz). Acoustic-mediated assembly and geometric patterning. Neutrally charged PDDA/ATP coacervate micro-droplets were prepared in situ within a custom-built acoustic trapping device based on a square arrangement of four piezoelectric transducers. Typically, an aqueous solution of PDDA (1 ml, 5 mM monomer, 100–200 kDa) was placed into the device chamber and aqueous ATP (100 ml, 50 mM) then added in the presence of two orthogonal acoustic standing waves generated from opposing transducer pairs operating at 6.76/6.78 MHz (10 V). Experiments were also undertaken under the same ﬁeld conditions but at different polymer and nucleotide concentrations (PDDA; 1 ml, 2.5 and 7.5 mM monomer, 100–200 kDa; ATP; 100 ml , 25 and 75 mM). Solution volumes were doubled (PDDA, 2 ml, 5 mM monomer; ATP, 200 ml, 50 mM) for trapping experiments undertaken with transducer pairs operating at lower frequencies of 4.99/5.00 MHz. In all cases, the PDDA monomer: ATP molar ratio was approximately 1: 1, mixtures were stirred to ensure homogeneous formation of the coacervate droplets in the square chamber, and the pH was adjusted to 7. The composition of the trapped coacervate micro-droplets was elucidated using ﬂuorescence microscopy by doping the polymer/nucleotide mixtures with a ﬂuorescent derivative of ATP (TNP-ATP) or a rhodamine-tagged cationic polymer (RITC-PAH). Samples of doped PDDA/ATP droplets were prepared with a TNP-ATP: ATP or PAH: PDDA monomer molar ratios of 1:1000, or 1:9, respectively. Enzyme reactions in acoustically trapped droplets. A mixture (1 ml) of PDDA (25 mM monomer, 100–200 kDa) and RITC-HRP (0.2 mg ml 1) was added to 1 ml of ATP (2.5 mM) in the presence of two orthogonal acoustic standing waves (6.76/6.78 MHz, 10 V). Discussion l Our results indicate that structured acoustic radiation forces are a powerful, versatile and inexpensive tool to manipulate the spatial assembly of uniform-sized coacervate micro-droplets in aqueous media to produce functional water-based molecularly crowded 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 sequestration of small molecules and macromolecules can be easily prepared using a wide range of components and conditions. Acoustic patterning of such droplets could, therefore, provide steps towards new types of aqueous-based arrays for use in the storage and spatially ordered release of drugs such as antimicrobials from 2D platforms, as ordered substrates for the patterning of micro- reactors based on DNA/enzyme sensing, and as organized platforms for biomolecule puriﬁcation and protein crystallization. In each case, the high sequestration potential and relatively low dielectric constant of the molecularly crowded coacervate droplets speciﬁcally enable the selective uptake, storage and potential communication of a wide range of functional components capable of operating dynamically in a continuous aqueous media. pH 7), CM-D (1 ml, 45 mM monomer, pH 8), 4/1 (mol/mol) CM-D/FITC-CM-D mixtures (1 ml, 45 mM total monomer, pH 8), dsDNA (1 ml, 5 mg ml 1, Tris buffer (10 mM, pH 8)) or PAA (100 ml, 200 mM monomer, pH 8) were placed in the chamber of the acoustic device operating with two orthogonal acoustic standing waves (6.76/6.78 MHz, 10 V), and the following amounts of PDDA with a molecular weight of 8.5 kDa added: BSA (62 ml, 50 mM monomer, pH 7), CM-D (75 ml, 100 mM monomer, pH 8), 4/1 (mol/mol) CM-D/FITC-CM-D mixtures (75 ml, 100 mM monomer, pH 8), dsDNA (100 mM, 150 ml, 8.5 kDa, monomer, Tris buffer (10 mM, pH ¼ 8)) or PAA (1 ml, 20 mM monomer, pH 8). Typically, a PDDA: BSA weight ratio of 1: 10, and PDDA: CM-D, PDDA: DNA base, and PDDA: PAA monomer molar ratios of approximately 1: 6, 1: 1 and 1: 1, respectively, were used. PEI/CM-D coacervate droplets were prepared by injecting an aqueous solution of PEI (50 ml, 300 mM monomer) into a CM-D solution (1 ml, 15 mM monomer) housed in the chamber of the acoustic device operating with two orthogonal acoustic standing waves (6.76/6.78 MHz, 10 V) to give a ﬁnal monomer molar ratio of 1: 1 and pH 8. In each case, the mixtures were stirred to ensure a homogeneous formation of coacervate droplets in the square chamber. Discussion l Finally, we describe a rudimentary demonstration of using chemically encoded droplet arrays for the enzyme-mediated sensing of substrate molecules ﬂowing through the sample chamber, suggesting that it should be possible to develop devices capable of sustaining chemical signals between the droplets as well as enabling spatial and temporal responses to changing conditions in the external environment. While this is a challenging prospect, the ability to spatially position multiple populations of functionally correlated droplets under water within the same device, such as three members of an enzyme cascade reaction as shown in Fig. 5a, control their surface attachment, and acoustically regulate their inter-droplet edge distance to control the diffusion length of signalling molecules could together provide the basis for increased operational complexity. In this regard, we note that coacervate micro-droplets have been recently exploited as models of membrane-free protocells24–29, suggesting that the combination of acoustic patterning and in situ coacervation could provide a novel route towards the design and construction of protocell communities with controllable spatial organization, programmable signalling pathways and chemical circuitry, and higher order collective behaviour. Uptake studies in PDDA/ATP droplet arrays. Acoustically patterned arrays of PDDA/ATP droplets were prepared as above (6.76/6.78 MHz, 10 V) in the presence of methylene blue (10 ml, 1 mM), nile red (10 ml, 1 mM), sulforhodamine B (10 ml, 1 mM) or calcein (1 ml, 1 mM), and ﬂuorescence images recorded after 15min to determine the level of molecular uptake. Sequestration of polymer or inorganic particles into the acoustically trapped droplets was undertaken by injecting 100ml of premixed polystyrene nanoparticles (100 nm, 5 10 2 wt.%) or silica microparticles (2.5mm, 2.5 10 2 wt.%) and aqueous ATP (100ml, 50 mM) into a PDDA solution (1 ml, 5 mM monomer; PDDA monomer: ATP ¼ 1, pH ¼ 7) contained with the device and subjected to two orthogonal acoustic standing waves (6.76/6.78 MHz, 10V). The mixtures were stirred to ensure homogeneous formation of the coacervate dro- plets in the square chamber. Three-dimensional confocal microscopy construction of h ili i i l i i PDDA/ATP d l d k b ddi Uptake studies in PDDA/ATP droplet arrays. Discussion l A mixture (1 ml) of PDDA (25 mM monomer) and RITC-HRP (0.2 mg ml 1) was then injected under the same acoustic standing wave ﬁeld at a different location in the ATP-ﬁlled chamber so that two spatially separated arrays of PDDA/ATP droplets containing either FITC-GOx or RITC-HRP were obtained. 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Using this approach the acoustic radiation force, ~F, can be found from 19. de Kruif, C. G., Weinbreck, F. & de Vries, R. Complex coacervation of proteins and anionic polysaccharides. Curr. Opin. Colloid Interface Sci. 9, 340–349 (2004). 20. Schmitt, C. & Turgeon, S. L. Protein/polysaccharide complexes and coacervates in food systems. Adv. Colloid Interface Sci. 167, 63–70 (2011). ~F ¼ rU; ð2Þ U ¼ 4p 3 a3 f1 1 2r0c2 0 p j j2 f2 3 4 r0 ~vj j h i2 ð3Þ f1 ¼ 1 r0c2 0 rpc2 p and f2 ¼ 2 rp=r0 1 2rp=r0 þ 1 ; ð4Þ ~F ¼ rU; ð2Þ U ¼ 4p 3 a3 f1 1 2r0c2 0 p j j2 f2 3 4 r0 ~vj j h i2 ð3Þ f1 ¼ 1 r0c2 0 rpc2 p and f2 ¼ 2 rp=r0 1 2rp=r0 þ 1 ; ð4Þ ~F ¼ rU; ð2Þ 21. Johnson, N. R. & Wang, Y. D. Coacervate delivery systems for proteins and small molecule drugs. Expert Opin. Drug Deliv. 11, 1829–1832 (2014). ð3Þ 22. MacEwan, S. R. & Chilkoti, A. Applications of elastin-like polypeptides in drug delivery. J. Control. Release 190, 314–330 (2014). 23. Xu, Y. S., Mazzawi, M., Chen, K. M., Sun, L. H. & Dubin, P. L. 23. Xu, Y. S., Mazzawi, M., Chen, K. M., Sun, L. H. & Dubin, P. L. Protein puriﬁcation by polyelectrolyte coacervation: inﬂuence of protein charge anisotropy on selectivity. Biomacromolecules 12, 1512–1522 (2011). ð4Þ 24. Koga, S., Williams, D. S., Perriman, A. W. & Mann, S. Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model. Nat. Chem. 3, 720–724 (2011). where p j j2 and ~vj j2 are the mean squared pressure and particle velocity respectively at the object, a is the radius of the spherical object, r is the density and the subscripts denote the particle, ‘p’, or host, ‘o’ properties. Note also, for a harmonic sound ﬁeld, ~v ¼ 1 ior0 rp. 25. Williams, D. S. et al. Polymer/nucleotide droplets as bio-inspired functional micro-compartments. ARTICLE ARTICLE ARTICLE References Tavana, H. et al. Nanolitre liquid patterning in aqueous environments for spatially deﬁned reagent delivery to mammalian cells. Nat. Mater. 8, 736–741 (2009). 17. 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Methods A ti t After 45 min, the acoustic ﬁeld was switched off and then the supernatant was carefully removed and exchanged with Milli-Q water three times without disturbing the array of coacervate micro-droplets. 20 ml of a mixture of H2O2 (50 mM) and o-phenylenediamine (o-PD, 25 mM) was then added at the bottom of the device, and left undisturbed. Fluorescence microscopy (lex ¼ 355–425 nm, lem ¼ 455 nm) was used to detect the HRP-mediated conversion of non-ﬂuorescence o-PD to ﬂuorescence cationic 2,3-DAP. Simulation methods. Each opposed transducer pair is excited with a sinusoidal voltage and after an initial transient period an acoustic standing wave is established in the chamber. If reﬂections are ignored, the standing wave can be thought of as the sum of two counter-propagating plane waves according to a previous approach45. The total acoustic pressure in the devices is then given by the sum of the two standing waves created by the orthogonal pairs: p ¼ p1ðeik1x þ e ik1xÞeio1t þ p2ðeik2y þ e ik2yÞeio2t: ð1Þ Similar procedures were used to self-assemble, trap and acoustically pattern coacervate PDDA-containing coacervate micro-droplets prepared from a range of components including proteins (BSA), polysaccharides (CM-D), polynucleotides (dsDNA) and anionic polymers (PAA), as well as coacervates formed by complexation of PEI and CM-D. In each case, solutions of BSA (1 ml, 5 mg ml 1, ð1Þ where k1 ¼ o1 c0 and o1 is the angular frequency in rad/s and c0 is the speed of sound in the host ﬂuid. The numerical subscript denotes the orthogonal pair under consideration, and is required as the transducer pairs were often operated at where k1 ¼ o1 c0 and o1 is the angular frequency in rad/s and c0 is the speed of sound in the host ﬂuid. The numerical subscript denotes the orthogonal pair under consideration, and is required as the transducer pairs were often operated at 8 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13068 Paul Dinham for assistance with the fabrication of acoustic trapping devices. P.G.B. is supported by EPSRC through the Bristol Centre for Functional Nanomaterials. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ How to cite this article: Tian, L. et al. Spontaneous assembly of chemically encoded two-dimensional coacervate droplet arrays by acoustic wave patterning. Nat. Commun. 7, 13068 doi: 10.1038/ncomms13068 (2016). Author contributions L.T. and S.M. conceived the experiments; L.T. fabricated the acoustic trapping device together with P.G.B., performed confocal microscopy together with N.M., and undertook all other experiments; B.W.D. contributed to the simulation; L.T., N.M., A.J.P., M.L., A.B., B.W.D. and S.M. undertook the data analysis; N.M., A.J.P., M.L. and B.W.D contributed to the preparation of the manuscript; L.T. and S.M. wrote the manuscript. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Acknowledgements 18. van der Gucht, J., Spruijt, E., Lemmers, M. & Stuart, M. A. C. Polyelectrolyte complexes: Bulk phases and colloidal systems. J. Colloid Interface Sci. 361, 407–422 (2011). We thank the Engineering and Physical Sciences Research Council (EPSRC, UK) for ﬁnancial support, Dr Pavan Kumar Bosukonda for fruitful discussions, Dr Hao Liu for assistance with data processing, and Maddy Nichols, Paul Chappell, Peter J. Rowe and NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications 9 NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications ARTICLE ARTICLE r The Author(s) 2016 Additional information Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. 10 NATURE COMMUNICATIONS \| 7:13068 \| DOI: 10.1038/ncomms13068 \| www.nature.com/naturecommunications
https://openalex.org/W2589198380	https://dergipark.org.tr/tr/download/article-file/90346	Turkish	null	Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değerlendirilmesi	International journal of social sciences and education research	2,015	cc-by	6,626	International Journal of Social Sciences and Education Research Online, http://dergipark.gov.tr/ijsser Volume: 1(1), 2015 International Journal of Social Sciences and Education Research Online, http://dergipark.gov.tr/ijsser Volume: 1(1), 2015 ISSN: 2149-5939 Abstract Hotel animation services are important for both management and tourists. Tourist satisfaction with recreational and animation services can contribute to increase food and beverage sales during show programs at hotels and resorts in the coastal regions. Animation services include three types of animation programs such as, sports activ- ities, evening entertainment/shows and entertainment programs for children. The purpose of this study is to eval- uate hotel animation services from managers and tourist perspective. This paper which qualitative research method was used in, firstly, a literature review that related to recreational and animation services at hotels were conducted and then data were collected by researchers in face-to-face interviews with 10 managers and 22 tourists at hotels and resorts in Antalya and Muğla. The results indicate that managers and tourists believe in hotel animation pro- grams and people have a crucial role on customer satisfaction. Keywords: Animation services, Hotel, Managers, Tourists Received Date: 01 / 01 / 2015 Öz Otel animasyon hizmetleri hem yönetim hem de turistler açısından önemsenmektedir. Kıyı bölgelerindeki resort otellerde rekreasyon ve animasyon hizmetlerinden turistlerin memnuniyeti yiyecek-içecek satışlarının artmasına da katkı sağlayabilmektedir. Bu animasyonlar, spor aktiviteleri, akşam eğlence/gösterileri ve çocuklar için eğlence programları olmak üzere üç animasyon hizmetlerini içerir. Bu çalışmanın amacı otel yöneticileri ve turistlerin bakış açısıyla animasyon hizmetlerini değerlendirmektir. Nitel araştırma yöntemi kullanılan bu çalışmada önce- likle otellerde rekreasyon ve animasyon hizmetleriyle ilgili alan yazın taraması gerçekleştirilmiştir. Sonra Antalya ve Muğla Bölgelerindeki otellerde 10 yönetici ve 22 turist ile yüz yüze mülakatlar yapılarak veriler toplanmıştır. Sonuçlar otel yönetici ve turistlerin müşteri memnuniyetinde otel animasyonları ve personelinin önemli bir rolü olduğuna inandıklarını göstermektedir. Anahtar sözcükler: Animasyon hizmetleri, Otel, Yöneticiler, Turistler Anahtar sözcükler: Animasyon hizmetleri, Otel, Yöneticiler, Turistler g g ç ş g ş ş 2Sorumlu yazar: Doç. Dr., Süleyman Demirel Üniversitesi, Eğirdir Turizm ve Otelcilik Yüksekokulu, mde- mir1@gmail.com. 3 1Bu çalışma 31 Ekim-3 Kasım 2013 tarihlerinde Kuşadası’nda gerçekleştirilen II. Rekreasyon Araştırmaları Ko gresinde sunulan “Otel Animasyon Faaliyetlerinin Yönetici ve Turistlerin Bakış Açısıyla Değerlendirilmesi” isim bildirinin gözden geçirilmiş ve genişletilmiş halidir. 2 The evaluation of hotel animation services from managers and tourists’ perspective Mahmut Demir2 Şirvan Şen Demir3 Received Date: 01 / 01 / 2015 Accepted Date: 01 / 02 / 2015 @g 3Doç. Dr., Süleyman Demirel Üniversitesi, İİBF, Turizm İşletmeciliği Bölümü, sirvansendemir@gmail.com. bildirinin gözden geçirilmiş ve genişletilmiş halidir. 2Sorumlu yazar: Doç. Dr., Süleyman Demirel Üniversitesi, Eğirdir Turizm ve Otelcilik Yüksekokulu, mde- mir1@gmail.com. 3Doç. Dr., Süleyman Demirel Üniversitesi, İİBF, Turizm İşletmeciliği Bölümü, sirvansendemir@gmail.com. Copyright © 2015 by IJSSER 1Bu çalışma 31 Ekim-3 Kasım 2013 tarihlerinde Kuşadası’nda gerçekleştirilen II. Rekreasyon Araştırmaları Kon- gresinde sunulan “Otel Animasyon Faaliyetlerinin Yönetici ve Turistlerin Bakış Açısıyla Değerlendirilmesi” isimli bildirinin gözden geçirilmiş ve genişletilmiş halidir. 2Sorumlu yazar: Doç. Dr., Süleyman Demirel Üniversitesi, Eğirdir Turizm ve Otelcilik Yüksekokulu, mde- mir1@gmail.com. 3Doç. Dr., Süleyman Demirel Üniversitesi, İİBF, Turizm İşletmeciliği Bölümü, sirvansendemir@gmail.com. 1. Giriş Dünya'da endüstri devrimiyle yaşanan değişim ve gelişmelere bağlı olarak çalışma saatlerinin azaltılması ve ücretli tatil hakkı verilmesi sonucunda çalışanların serbest zamanlarının arttığı gö- rülmektedir. Serbest zamanlarını çeşitli rekreatif etkinliklerle doldurma çabası içindeki insanların 1Bu çalışma 31 Ekim-3 Kasım 2013 tarihlerinde Kuşadası’nda gerçekleştirilen II. Rekreasyon Araştırmaları Kon- gresinde sunulan “Otel Animasyon Faaliyetlerinin Yönetici ve Turistlerin Bakış Açısıyla Değerlendirilmesi” isimli bildirinin gözden geçirilmiş ve genişletilmiş halidir. Copyright © 2015 by IJSSER ISSN: 2149-5939 36 Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. International Journal of Social Sciences and Education Research, 1 (1), 35-48. yöneldikleri etkinliklerden birisi de turizmdir. Turizm, genlikle dinlenme, eğlenme amacı taşıyan, serbest zamanları değerlendirme gereksiniminden kaynaklanmaktadır (Borhan & Erkmen, 2009). Özellikle ikinci dünya savaşından sonra atıl kalan savaş uçaklarının turizm amacı ile kullanılması çok uzak mesafelere insanların daha kısa sürede ulaşabilmelerini sağlamıştır. Daha sonraki tek- nolojik gelişmelerle birlikte daha büyük uçakların yapılması, daha fazla sayıda insanın bu ulaşım aracından daha ucuza yararlanmasını sağlamıştır. Artık daha fazla sayıda insan çok daha ucuza tatil yapabilmektedir. Özellikle soğuk iklime sahip ülkede yaşayanların sıcak denizlere kolay ulaşması beraberinde kitle turizmini getirmiştir. Kitle turizminin ortaya çıkardığı deniz/kum/güneş ve kar/kayak odaklı resort oteller misafir- lerini tesis içinde daha fazla tutmak amacıyla farklı organizasyonlara yönelmektedir. Bu tür tesis- lerde turistlerin konaklama dışındaki yeme-içme, alışveriş, spor gibi birçok gereksinimi de aynı alan içerisinde karşılamaya başlamıştır. Ancak otel içerisinde bir kaç gün geçirdikten sonra sıkıl- maya başlayan turistlerin eğlenme gereksinimlerini karşılayacak olan animasyon programları ilk kez 1980'li yıllarda İspanya, İtalya ve Yunanistan'da başlamıştır (Glinia, Costa & Drakou, 2004). Türkiye'de ise, ilk kez Club Med ile başlayan animasyon hizmetinin müşteri memnuniyetine olumlu etki etmesi ile birlikte, 1990'lı yıllarda diğer resort oteller ve tatil köyleri tarafından da uygulanmaya başlanmıştır. Ancak o dönemde Türkiye'de animasyonu gerçekleştirebilecek yeterli personel olmadığı için bu tür eğlence programları yabancı tur operatörlerinin turla birlikte getir- dikleri kendi animatörleri tarafından yapılmaktadır (Koçak, 2001). Günümüzde, Türk animatör- lerin yetişmesinde oldukça önemli bir yere sahip olan yabancı animatörlerin ilk yıllara göre oran- larının daha düşük olduğu ifade edilebilir. 2. Otellerde animasyon hizmetleri Otel animasyonu canlı eğlence hizmetlerinin yanı sıra, serbest zaman, fitness ve spor faaliyet- lerinin de yer aldığı rekreasyon hizmetlerini de içermektedir. Zaten "animate" sözcüğünden gelen animasyon, aktif hale gelme, canlanma, aktif bir yaşam sunma anlamı yanında, canlandırma ve yenileme anlamına gelen "recreating" kelimesini de içermektedir (Glinia, Costa & Drakou, 2004). Otelde turistlerin zamanlarını en iyi şekilde geçirmelerini sağlama sorumluluğu yüklenen animas- yon hizmetleri resort otel ürün kalitesinde oldukça önemli bir rol oynamaktadır (Vogt & Fesen- meier,1995). Animasyon hizmeti özellikle herşey dahil sistemi uygulayan işletmelerde turistlerin tatil süresince otel içinde daha fazla kalmalarını sağlarken, onları eğlendirerek, canlandırarak, spor yapmalarını sağlayarak keyifli bir tatil geçirmesine katkı sağlamaktadır. Otellerdeki animas- yon hizmeti turistlerin diğer turistlerle sosyal ilişkiler kurmalarını kolaylaştırmanın yanı sıra yerel kültür hakkında bilgi edinmelerini de sağlama gibi bir misyonu bulunmaktadır (Shportko, 2012). Ancak animatörlerin yeterli eğitimi almamış olmaları ve yeteri kadar Türk halk kültürü ile ilgili bilgiye sahibi olmamaları nedeniyle çoğu kez gösteriler bu misyonu yerine getirememektedir. Otel animasyon hizmetleri spor faaliyetleri, gece gösterileri ve çocuk animasyonu (mini kulüp) olarak sınıflandırılabilir (Mikulic & Prebezac, 2011). Spor faaliyetleri genellikle gündüz saatle- rinde yapılan sabah jimnastiği, su topu, plaj voleybolu, dart, bocce gibi basit, grup halinde yapı- labilecek oyunları ve yarışmaları kapsamaktadır. Spor faaliyetleri hem turistlerin spor yaparak canlanmalarını, hem de birbirleri ile iletişim kurmalarını sağlamaktır. Spor faaliyetlerini turist- lerle birlikte yapacak olan animatörlerin yeterli düzeyde bilgi sahibi olmasının yanı sıra onları Copyright © 2015 by IJSSER ISSN: 2149-5939 37 Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. zorlamadan bu oyunlara katılmaları sağlayacak ikna yeteneğine sahip olmaları da oldukça önem- lidir. Animatör-turist ilişkisinin diğer insanları rahatsız edecek düzeyde ve şekilde olmaması ol- dukça önemlidir. zorlamadan bu oyunlara katılmaları sağlayacak ikna yeteneğine sahip olmaları da oldukça önem- lidir. Animatör-turist ilişkisinin diğer insanları rahatsız edecek düzeyde ve şekilde olmaması ol- dukça önemlidir. Gece gösterileri, akşam yemeğinden sonraki zamanlarını nasıl değerlendireceğini bilmeyen turistlere yardımcı olmak amacıyla yapılmaktadır. Bu gösteriler skeçler, playback showlar, kaba- reler, müzikaller, dans gösterileri, çeşitli yarışmalar, değişik ülkelere ait geceleri (Türk gecesi, Latin gecesi gibi) kapsamaktadır. Gösteriler genellikle turistlerin oteldeki kalış süreleri dikkate alınarak belli aralıklarla, örneğin 7 veya 15 günde bir olmak üzere, tekrarlanmaktadır. Gece gös- terilerinin gerek kostüm, gerekse dekor bakımından zengin olması, ayrıca herkesçe bilinen basit gösteriler olmaması oldukça önemlidir. 2. Otellerde animasyon hizmetleri International Journal of Social Sciences and Education Research, 1 (1), 35-48. büyük çoğunluğunun otelin animasyon şefinden aldıkları birkaç haftalık eğitimlerle bu işe başla- dıkları da bilinen bir gerçektir (Koçak, 2001). Alınan bu eğitimler genellikle sezon içinde yapıla- cak gösterilere yönelik olmakta ve uzun süreli çalışma programlarını kapsamamaktadır. Animasyon hizmetlerini gerçekleştirecek personeli işe alırken dikkatli seçim yapılması, kali- teli eğlence hizmeti açısından da oldukça önemlidir. Animatörler birçok beceriyi bir arada bulun- duran kişiler olarak algılanmaktadır. İyi bir animatör cömert, neşeli, konuşkan, yaratıcı, ilgili, dürüst, kültürlü ve enerjik olması yanı sıra pedagoji, spor, pazarlama, yönetim tecrübesi, gelişmiş iletişim becerisi ve birden fazla yabancı dil bilgisine de sahip olan bir kişi olarak bilinmektedir (Jakovlev, Koteski & Bardarova, 2013; Medlik, 2003; Thibal, 1985). Bir animatörün dans, şarkı söyleme, resim yapma gibi konularda yetenekli olması işin kolaylaşmasını sağlar. Ayrıca kendi ülkesinin kültürünü en iyi şekilde temsil ettiği gibi turistlerin geldikleri ülkelerin kültürleri hak- kında da bilgi sahibi olması oldukça önemlidir. Turistler arasında yaş, meslek, eğitim, kişilik özelliklerine bağlı olarak farklılıklar olduğunun bilincinde olma, animatörlere etkin iletişim kur- mada önemli katkılar yaratabilmektedir (Ivkov ve Stamenkovic, 2008). Bu yetenekte ve bilinçte olmayan animatörlerin yaptıkları hatalar turistlerin tatilden duydukları memnuniyetleri olumsuz yönde etkileyerek bir daha aynı oteli tercih etmemelerine neden olabilmektedir (Demir, 2010). Yapılan araştırmalarda da animasyon hizmetlerinin turistlerin oteli tekrar tercih etmelerinde etkili olduğu sonucu ortaya konulmuştur (Costa, Glinia, Goudas & Antoniou, 2004; Demir & Demir, 2014a,b; Shportko, Lehto & Ghiselli, 2013). Turizmin bir hizmet sektörü olması, turist ile üreticinin aynı anda üretim ve sunum yerinde olmasını gerektirmektedir (Rowley, 1995). Doğal olarak bu durum çalışanlar ile tatil amaçlı ko- naklayanlar arasında yüksek oranda etkileşime neden olmaktadır (Güler, 2009; Vallaster ve Cher- natony, 2005). Özellikle animasyon hizmeti direk turistle iletişimi gerektirdiğinden animatörler diğer çalışanlardan çok daha fazla müşteri-çalışan etkileşimi yaşamaktadır. Tüketicilerin anima- törler ile yakın teması, onların olumlu veya olumsuz davranış ve performanslarının turistler tara- fından anında değerlendirilmesinin (Tütüncü, 2009) yanı sıra işletme ile ilgili her türlü sorunlarını onlarla paylaşmalarına ve çözümleri onlardan beklemelerine neden olmaktadır. Animatörlerin tu- riste yönelik yaklaşımı, davranışları ve tutumu müşterilerin hizmetten memnuniyet duymalarını sağlayabileceği gibi sadakatlerini de sağlayabilmektedir. Yapılan araştırmalar, genel olarak çalı- şanların müşteri memnuniyetinin dolayısıyla da sadakatinin sağlanmasında etken bir faktör oldu- ğunu doğrulamaktadır (Demir & Günaydın, 2013). Özellikle animasyon ekibinin diğer çalışanlara göre daha fazla müşterilerle iletişim halinde olması, daha samimi ilişkilerin kurulmasına olanak sağlamaktadır. 2. Otellerde animasyon hizmetleri Gece gösterilerinde görev alan animatörlerin dans ve ti- yatro yeteneklerinin olması yanı sıra müşterilerin milliyetlerine göre birkaç dili konuşabiliyor ol- ması önemlidir. Çünkü animatörler gösteri sırasında söyledikleri cümleleri bir kaç farklı dilde tekrarlamak zorundadır. Çocuk animasyon hizmeti ise hem çocuklara hem de onların ailelerine hitap etmektedir. Bazı aileler çocuklarına günlük yaşamlarında zaman ayıramadığı için tatilde onlarla birlikte değişik etkinliklerde bulunarak hoş vakit geçirmek isterken bazıları da tatilde kendilerine zaman ayıra- bilmek için çocukları ile ilgilenecek, onları eğlendirme hizmetini yapabilecek bir yer aramaktadır (Mikulic & Prebezac, 2011). Çocuk animasyonunda gündüz değişik faaliyetler, yarışmalar yaptı- rılmakta, onların birbiriyle kaynaşmaları ve oyun oynamaları sağlanmakta, akşam ise, gece gös- terisinden önce çocuklar ve isteyen ailelerle birlikte mini club dansları yapılmaktadır. Özellikle bu bölümde çocuk psikolojisinden anlayan, sabırlı, sorumluluk sahibi ve müşteri özelliklerine bağlı olarak farklı yabancı dilleri bilen animatörlerin istihdam edilmesi oldukça önemlidir. Yapı- lan araştırmalar çocukların tatil seçiminde ailelerin kararında oldukça önemli bir yere sahip oldu- ğunu göstermektedir (Costa & Glinia, 2004; Mikulić & Prebežac, 2011; Pompl, 1983). Müşteri- lerin tercihleri ve memnuniyetlerinde artış göz önüne alındığında işletmelerin çocuk animasyon hizmetine önem vermeleri gerekliliği ortaya çıkmaktadır. Özellikle herşey dahil pansiyon türünü uygulayan otellerin animasyon ekipleri ve gösterileri- nin kaliteli ve orjinal olması müşteri memnuniyeti açısından oldukça önemlidir (Demir & Demir, 2001). Günümüzde animasyonun otel tatil paketinin içinde öneminin giderek artmasına rağmen, işletmelerin animasyon birimi ve faaliyetlerine yeterli bütçe ayırmadıkları da bilinmektedir. Bu durum gösterilerin ve animatörlerin kalitesine, dolayısıyla da müşteri memnuniyetsizliğine yan- sımaktadır. İşletmeler maliyetleri azaltmak için işe aldıkları animatörlerin deneyim ve yetenekle- rini dikkate almamakta, daha önce hiç animatörlük yapmamış, bu konuda herhangi bir eğitim almamış personeli istihdam edebilmekte ya da bir animasyon şirketi ile anlaşarak düşük maliyetli eğlence faaliyetleri ile hizmet verebilmektedir. Özellikle deniz/kum/güneş odaklı tatillere yönelik hizmet veren büyük ve zincir resort otelle- rin sayısındaki artış ile birlikte profesyonel animatörlere daha fazla gereksinim olmaktadır (Gli- nia, Costa & Drakou, 2004). Ancak sadece Türkiye'de değil, diğer birçok turizm ülkesinde de kalifiye animatör bulmak önemli bir sorundur. Costa ve arkadaşları (2004) Yunanistan'da profes- yonel animatör eksikliğinden dolayı bu departmanın hizmet kalitesinin standartların altında kal- dığını belirtmektedir. Yapılan bu çalışmada animasyon personelinin eğitiminin son derece yeter- siz olduğu belirtilerek, animatörlerin sadece %57'sinin yüksek öğrenimli, bunlarında büyük ço- ğunluğunun (%63) turizm dışındaki okullardan mezun olduğu belirtilmektedir. Animatörlerin Copyright © 2015 by IJSSER ISSN: 2149-5939 38 Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. 3.1. Araştırmanın amacı Otel işletmelerinin animasyon hizmetlerine ilişkin alan yazında ve uygulamada farklı değer- lendirmeler yer almaktadır. Buradan hareketle gerçekleştirilen bu araştırmanın amacı, otellerde sunulan animasyon hizmetlerinin yöneticiler ile turistler açısından değerlendirmesini yapmaktır. Bununla birlikte, her iki grupla yapılan görüşmelerden elde edilen bilgilerin karşılaştırılarak, ani- masyon hizmetlerinin otel işletmeleri için olduğu kadar turistler için de önemini ortaya koymaktır. Copyright © 2015 by IJSSER ISSN: 2149-5939 39 Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. 3.2. Veri toplama Bu çalışmada nitel araştırma yöntemi seçilmiştir. Antalya ve Muğla Bölgesinde bulunan 4 ve 5 yıldızlı otellerde animasyon hizmetlerinden yararlanan turistlerle ve bu işletmelerin yöneticileri ile derinlemesine mülakatlar yapılarak gerçekleştirilmiştir. İşletme genel müdürlerinden randevu alınarak başlayan görüşmeler, bazı genel müdürlerin çeşitli nedenlerle görüşmeye katılmak iste- memesi nedeniyle yönlendirmiş oldukları diğer yöneticilerle gerçekleştirilmiştir. Katılımcılara önceden belirlenen yarı yapılandırılmış sorular yöneltilmiştir. Yarı-yapılandırılmış görüşme (mü- lakat), yapılandırılmış görüşmelerle yapılandırılmamış görüşmeler arasında kalan ve en çok kul- lanılan görüşme tekniğidir. Bu yöntem katılımcıların seçilen konulardaki genel ve teknik bilgi, düşünce, yorum, tutum ve davranışları kadar bunların neden ve sonuçlarının öğrenilmesinde en etkin yol olarak bilinmektedir. Bu araştırmada nicel yöntem yerine nitel bir yöntem seçilmiştir. Çünkü araştırma hem turist- lere hem de otel yöneticilerine yönelik yapılmaktadır ve her iki gruba da aynı ölçeği uygulamanın zorluğu nedeniyle derinlemesine mülakat yapılması tercih edilmiştir. İz sürme yöntemi kullanıla- rak 10 otel yöneticisi ve 22 turist olmak üzere toplam 32 katılımcıya ulaşılmıştır. Bu katılımcıların özellikleri tablo 1 ve tablo 2’de yer almaktadır. Her iki gruptan ilk katılımcı ile başlayarak yapılan görüşmeden sonra elde edilen bilgiler ışığında, bir diğerine geçilerek görüşmeler gerçekleştiril- miştir. Toplanan bilgilerin birbirini tekrar etmeye başlaması, araştırmada doyum noktasına ula- şıldığını göstermekte olup bu aşamadan sonra görüşmelere son verilmiştir. Katılımcıların her biri ile yaklaşık 20 dakika görüşülmüş olup toplam 10 soru yöneltilmiştir. Katılımcılardan alınan ce- vaplar, araştırmacıların analiz ve sentezleriyle deneyim ve mantık süzgecinden geçirildikten sonra alan yazındaki bilgilerle de bütünleştirilmiş ve yönetici ve turistlerin vermiş oldukları bazı cevap- lar gruplandırılarak ortaya konulmuştur. 3.3. Araştırma sınırlılıkları Bu araştırma, belirlenen birtakım sınırlılıklar içerisinde yapılmıştır. Öncelikle araştırma An- talya ve Muğla bölgesindeki 4 ve 5 yıldızlı otellerde gerçekleştirilmiştir. Otellerin seçiminde “ani- masyon ekibi ve kalitesi” bakımından çok büyük farklılıklar olacağı düşüncesi ile çok lüks zincir oteller yerine müşteri profili ve fiyat bakımından birbirine yakın olan oteller tercih edilmiştir. İkinci olarak, işletme genel müdürleri ya da müşterilerle daha yakın temasta olan departman yö- neticileri ile görüşülmüştür. Üçüncüsü görüşme yapmak için seçilen turistlerin daha öncede Tür- kiye’ye gelmiş ve birçok kez farklı otellerde animasyon hizmetlerinden yararlanmış kişiler olma- sına dikkat edilmiştir. 4. Bulgular Araştırmada yönetici ve turistlerden elde edilen veriler bir arada karşılaştırılarak verilmiş ve birlikte değerlendirilmiştir. Veriler, hem otel yöneticileri hem de aynı otellerde tatil yapan müş- terilerden elde edilmiştir. Yöneticiler tablo 1’den de anlaşılacağı gibi, genel müdür, önbüro mü- dürü, satış müdürü ve misafir ilişkileri müdürlerinden oluşurken, turistler Türk, İngiliz ve Alman- lardan oluşmaktadır (tablo 2). Otel yöneticileri ve turistlerle yapılan görüşmelerde aynı soru for- matı kullanılmıştır. Sorulara verilen cevaplarda, hizmeti sunan ile yararlananların algılamaların- daki farklılıkların yarattığı bakış açısı açıkça görülmektedir. Aynı şekilde yerli turistler ile yabancı turistlerin de farklı değerlendirmeleri olmuştur. Copyright © 2015 by IJSSER ISSN: 2149-5939 40 Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Tablo 1. Katılımcılar: Yöneticiler Katılımcı Görevi Otel sınıfı Şehir GM-1 Genel Müdür 4* Bodrum GM-2 Genel Müdür 5* Alanya GM-3 Genel Müdür 5* Side GM-4 Genel Müdür 5* Alanya ÖM-1 Önbüro Müdürü 5* Bodrum ÖM-2 Önbüro Müdürü 4* Fethiye SM-1 Satış Müdürü 5* Bodrum SM-1 Satış Müdürü 5* Bodrum MİY-1 Müşteri İlişkileri Yöneticisi 4* Alanya MİY-2 Müşteri İlişkileri Yöneticisi 5* Marmaris Tablo 1. Katılımcılar: Yöneticiler Bir otelde animasyon hizmetlerinin varlığı, hem müşteriler hem de yöneticiler açısından olumlu etki ve sonuçlar ortaya koyabilmektedir. Yöneticilerin satış gücünü arttırabilme yönün- deki çalışmalarına destek olarak görülebilecek animasyon hizmetleri, müşteriler açısından da bir otelin tercih edilmesine olumlu etki edebilmektedir. Nitekim animasyon hizmetlerinin bir otel için önemine ilişkin değerlendirmede, yöneticilerin tamamı “özellikle çocuklu aileler için önemli bir satış avantajı yarattığını” belirtirken, hem yerli hem de yabancı turistler, “otelde iyi bir animas- yon hizmetinin olmasının otel tercihlerinde etkili olacağını” ifade etmektedir. SM-1’in “otele gelen bir misafir öncelikle animasyon olup olmadığını soruyor” şeklinde ifadesini destekleyen bir başka görüşte GM-2’nin “müşteri otelde eğlence ister ve bu eğlence şeklide turistin milliyetine göre değişir” ifadesidir. Görülüyor ki, animasyon hizmetleri otel için önemli olduğu kadar müş- teriler içinde önemlidir. Tablo 2. 4. Bulgular Katılımcılar: Turistler Katılımcı Milliyet Cinsiyet Tatil süresi (Gün) TR-1 Türk Erkek 5 TR-2 Türk Kadın 7 TR-3 Türk Kadın 7 TR-4 Türk Kadın 5 TR-5 Türk Erkek 7 TR-6 Türk Erkek 6 TR-7 Türk Kadın 10 TR-8 Türk Kadın 10 TR-9 Türk Erkek 7 TR-10 Türk Kadın 10 ALM-1 Alman Erkek 14 ALM-2 Alman Erkek 14 ALM-3 Alman Kadın 7 ALM-4 Alman Kadın 14 ALM-5 Alman Kadın 7 ALM-6 Alman Kadın 7 İNG-1 İngiliz Kadın 7 İNG-2 İngiliz Erkek 14 İNG-3 İngiliz Erkek 14 İNG-4 İngiliz Erkek 14 İNG-5 İngiliz Kadın 14 İNG-6 İngiliz Kadın 14 Copyright © 2015 by IJSSER ISSN: 2149-5939 41 Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Animasyon departmanı otel için önemli işlevlere sahip bir birimdir. Yönetim ile müşteriler arasında bir köprü, otelin gülen yüzü, müşteriye en yakın otel personeli, hoş vakit geçirmenin kaynağıdır. Animasyon departmanının otel içinde aynı zamanda müşterilerin birbiriyle olan ileti- şimi ve ilişkisinin de sağlanmasında önemli bir rolü bulunmaktadır. Katılımcıların oteldeki ani- masyon departmanı ile ilgili düşüncelerinin ortak noktası “animatörler turistler arasındaki ileti- şimi, kaynaşmayı sağlar” ve “animasyon departmanı otel içinde önemli bir görev üstlenir” şek- linde hem yerli ve yabancı turistler hem de yöneticilerce aynı şekilde algılanmasıdır. Otellerde animasyon departmanının verdiği hizmetler gece ve gündüz farklılık göstermektedir. Gündüz daha çok yarışmalar ve spor etkinlikleri olurken gece ağırlıklı olarak skeçler, dans gös- terileri, müzikaller ve playbacklerden oluşan bir program turistlere sunulmaktadır. Ancak çoğu kez özgünlükten uzak, yıllardır tekrar eden gösteri ve yarışmalar turistlere sunulmaktadır. Ani- masyon departmanının sunmuş olduğu programların otellere göre farklılık gösterip göstermedi- ğinin belirlenmesi amacıyla hem yöneticiler hem de turistlere aynı soru yöneltilmiştir. Yönetici- lerin ve katılımcıların hali hazırda bulundukları otelden başka tesis deneyimine sahip olması, kar- şılaştırma yapabilmesi açısından önemlidir. Çalışmaya katılan turistlerin büyük çoğunluğu (% 82) otel animasyon programlarının özgün olduğunu belirtmişlerdir. Yabancı turistlerin ilk kez Türkiye’ye gelmiş olmaları (Türkiye dışında başka ülkelerdeki otel animasyon programlarını sey- retmişlerdir) ve Türk turistlerin de otelde ilk kez farklı amaçla (bir kısmı çocukları nedeniyle ilk kez çocuk animasyonlarına katılmışlar, bir kısmı da çocuklarının büyümesi nedeniyle yetişkin programlarına katılmışlardır) animasyon programlarına katılmalarından kaynaklanabilir. Türki- ye'ye üç yıldır geldiklerini belirten ALM-1, ALM-2 ve İNG-3 "diğer otellerde geçen yıllarda izlediğim skeçlerin biraz daha farklılaştırılmış haliydi" diyerek gösterilerin yenilenmediğine dik- kat çekmektedir. 4. Bulgular İNG-5 "daha önceki yıllarda Türkiye'de bulunduğumuzdan gece gösterilerini daha önce bir kaç kez izlemiştik bu nedenle gece gösterilerine pek fazla katılmıyoruz” ALM-3 “gündüz tanıtımlarda özgün bir gösterinin olacağı bilgisini alırsak o zaman gösteriyi izlemeye gidiyoruz" şeklinde görüşlerini bildirmiştir. Yöneticilerin tamamı animasyon hizmetlerinin lüks oteller dışında birbirinin aynı olduğunu ifade etmişlerdir. SM-2 "Otelimizde sıradan bilinen programların yanında özgün yenilikçi prog- ramlarda yer almaktadır. Sıradan bilinen programlarla başarılı olma şansınız çok azdır. Ne ka- dar yenilik o kadar memnuniyet söz konusudur" şeklinde yeni programların olmasının gereklili- ğini vurgulamıştır. GM-3 ise, "özgün gösteriler (revü showları, özel müzikaller, ünlü sanatçılar gibi) çok yüksek maliyetlidir ve her otelin bu maliyeti karşılaması mümkün değildir. Ancak büyük ve lüks oteller bu maliyeti karşılayabilir" diyerek konunun bir başka boyutuna dikkat çekmiştir. Turistlerin yöneticilere göre gösterilerin daha özgün olduğunu düşünmelerinde geçmiş yıllardaki deneyimlerinin azlığı önemli bir rol oynamaktadır. Yöneticiler yıllardır bu sektörün içinde olduk- larından hangi otelin nasıl bir animasyon hizmeti verdiği konusunda daha deneyimlidir. Tüketicinin bir ürün veya hizmetten beklentisi geçmiş yıllardaki deneyimlerinden, tanıdıkları ve arkadaşlarının anlattıklarından, işletmelerin reklam ve tanıtımlarında vaat ettikleri hizmetler- den oluşmaktadır. Turistlere; otel animasyon hizmeti beklentilerinizi karşıladı mı? sorusuna tu- ristlerin büyük çoğunluğunun (%77) animasyon hizmetlerinin beklentilerini karşıladığını ifade ederken yalnızca 5 kişi (%23) animasyonların genelde oteldeki müşterilerin çoğunlukta olduğu millete göre hazırlandığını ifade etmiştir. Buna karşın, aynı soru, bir başka bakış açısıyla, yöneti- cilere animasyon hizmetinizin müşteri beklentilerini karşıladığını düşünüyor musunuz? şeklinde Copyright © 2015 by IJSSER ISSN: 2149-5939 42 Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. International Journal of Social Sciences and Education Research, 1 (1), 35-48. yöneltilmiştir. Görüşmeye katılan yöneticilerin yarısı animasyon hizmetlerinin turistlerin beklen- tilerini karşıladığı, yarısı da karşılamadığı görüşündedir. SM-2 "animasyon program açısından %70 oranında turistlerin beklentilerini karşılamaktadır, ancak turistle animatörün karşılıklı di- yaloglarında %100 oranında turistlerin beklentileri karşılanmaktadır", ÖBM-1 ise "beklentilerin karşılanması animatöre bağlıdır, animatör mesleğinde iyi ise müşteri beklentileri karşılanır" şeklindeki yorumlar aslında gösterilerden daha önemlisinin animatörlerin müşterilere karşı ilgisi ve iletişim becerileri olduğunu göstermektedir. Bu ifadelerden anlaşıldığı üzere otellerde kalifiye animatör istihdam edilmesi animasyon hizmetlerinin başarısında oldukça önemli bir rol oynamak- tadır. Bir otelden memnun ayrılan turist iki tür davranış göstermektedir. Bunlardan birincisi, bir son- raki tatil için yine aynı otele gelmek, ikincisi ise kendisi gelmese bile arkadaş ve tanıdıklarına oteli tavsiye etmektir. Otellerin sadık müşterilerinin fazla olması, karlılığı arttırmada, risklerle karşılamada, diğer otellerle rekabette oldukça avantajlı olmalarını sağlar. 4. Bulgular Turistlere animasyon hizmetlerinden dolayı tekrar bu otele gelir misiniz? diye sorulduğunda yerli turistlerin ikisi (TR- 3 ve TR-5) dışındaki büyük çoğunluğu "evet tekrar gelmek isterim" diye cevaplandırırken, ya- bancı turistler otel tercihinde animasyon hizmetlerini doğrudan önemli bir etken olarak görmeye- rek büyük çoğunluk "hayır, animasyon için bir daha aynı otele gelmem" demişlerdir. Aynı soru yöneticilere müşterilerinizin otelinizi yeniden tercih etmelerinde animasyon programları etkili olur mu? ifadesi ile sorulmuş bir yönetici dışındaki diğer yöneticilerin ortak görüşü “eğer turist otelin animasyon hizmetinden memnun kalmışsa bu onların tekrar işletmeyi tercih etmesinde di- ğer unsurlar ile birlikte etkili olacaktır” şeklindedir. GM-3 "tahminen % 30 oranında etkili olur. Kimi turiste göre animasyon hizmeti otelin olmazsa olmazıdır, kimine göre ise olsa da olur ol- masa da olur" şeklinde aşırı bir etkisinin olmayacağı yönünde görüş bildirmiştir. Animasyon hizmeti turistlerin oteldeki eğlence arayışlarından doğmuştur. Gündüz saatlerini deniz veya havuzda geçiren turist için akşam saatlerinde yapacak çok fazla alternatif yoktur. Özel- likle tatilleri boyunca çok fazla otel dışına çıkmayan turistler her tür eğlenceyi de otelden bekle- mektedir. Animasyon hizmeti olmazsa otel sıkıcı olur mu? sorusuna yerli turistlerin tamamı ke- sinlikle sıkıcı olur şeklinde cevap verirken, yabancı turistlerin bir kısmı sıkıcı olmayacağını, bir kısmı ise kesinlikle sıkıcı olacağını belirtmiştir. Özellikle bu tür gösterileri gürültü olarak gören ve tatilinde sakinlik arayan turistler için animasyon hizmetinin olmaması daha iyidir. Nitekim İNG-6 "keşke hiç animasyon hizmeti olmasa da gürültü azalsa" diyerek animasyon hizmetini ge- reksiz bir hizmet olarak görmektedir. TR-4 ise, "gündüz yarışmalarına katılmaktan büyük zevk alıyorum. Özellikle yabancılarla rekabet edeceğimiz yarışmaları daha çok seviyorum. Eğer ani- masyon hizmeti olmasa bütün gün havuza ve denize girmekten çok sıkılırdım" diyerek animasyon hizmetinin gerekliliğine işaret etmiştir. Aynı soru yöneticilere sorulduğunda genel olarak bütün yöneticiler animasyon olmazsa turistin otelde sıkılabileceğini belirtmiştir. SM-1 "otellerin birço- ğunda relax diye tabir ettiğimiz kısımlar bulunur plaj ve relax havuz gibi zaten animasyon iste- meyen misafirlerimiz oradan faydalanarak tatillerini geçirirler. Diğer misafirlerimiz içinse ani- masyon olmazsa olmazlardandır" şeklinde yorumda bulunmuştur. GM-1 ise, "turistler akşam dı- şarı çıkmadıkları için otel içinde eğlence istemektedir. Animasyon hizmeti olmazsa misafirlerimiz sıkılabilir" diyerek animasyon hizmetinin bir otelde iyi veya kötü mutlaka bulunması gerektiği vurgulanmıştır. Copyright © 2015 by IJSSER ISSN: 2149-5939 43 Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Bir oteldeki animasyon hizmetleri genel olarak gündüz programı, gece gösterileri ve mini ku- lüpten oluşmaktadır. 4. Bulgular Animasyon programlarının çeşitliliği (mini kulüp, su sporları, gece ve gün- düz showları) otel tercihinizde etkili oldu mu? şeklindeki bir soruya ALM-6 "otele gelmeden önce sadece animasyon hizmetinin olup olmadığını sorduk, ancak ne tür animasyon hizmeti verileceği ile ilgili ayrıntılı bilgi almadık, daha çok diğer faktörler (otelin yeri, yıldızı, pansiyon türü gibi) otel tercihimizde etkili oluştur". ALM-5 "çocuğumuz olduğu için sadece mini kulübün olup olma- dığını sorduk" diye belirtmiştir. Otel yöneticileri arasında animasyon hizmetlerinin çeşitliliğinin kesinlikle turistlerin otel tercihinde etkisi olacağını özellikle çocuklu aileler için mini kulübün olmasının önemli olduğunu belirtenlerde olmuştur. "Otel tercihlerinde animasyon hizmetleri et- kili olur ama lokasyon kadar değil" diyen ya da "ne yazık ki genel anlamda misafirlerimiz ani- masyon için değil de herşey dahil olsunda ne olursa olsun düşüncesi ile otel tercihlerini yapıyor. Bunun yanında animasyonun olması tatlısı şerbeti oluyor" diye görüş bildirenlerin de olması tu- ristlerin tatil tercihlerinde farklı faktörlerin etkili olduğunu göstermektedir. Otelde animasyon hizmeti hiç olmaması sizi nasıl etkiler? sorusuna, görüşmeye katılan hem yerli hem de yabancı turistler şaşırtıcı bir şekilde otelde animasyon hizmeti olmasa da önemli olmadığını belirtmişlerdir. Diğer sorulara verilen cevaplarla çelişkili gibi görünen bu cevap as- lında turistlerin iyi bir animasyon hizmeti istediklerini, eğer böyle olmayacaksa animasyon hiz- meti olmasa da onlar için bunun çokta bir öneminin olmadığı anlamı çıkmaktadır. Yöneticilere aynı soru oteli, acentayı ve müşterileri etkilemesi bakımından sorulmuştur. ÖBM-2 "animasyon otelde farklı kültür ve milletlerden konaklayan misafirlerin kaynaşması, birbirlerini daha yakın- dan tanıması ve iyi zaman geçirmelerini ayrıca otel yönetimi ile misafirler arasında bir köprü oluşturmaktadır. Animasyon ekibinin olmaması bu köprü ve ilişkilerin sağlanamaması dolayı- sıyla otel yönetiminin misafir istek ve ihtiyaçlarının sadece resepsiyon ve halkla ilişkiler depart- manlarından alacağı bilgiler doğrultusunda hareket edeceği bunun sonucunda misafirlerin tam anlamıyla istek ve ihtiyaçlarına karşılık verememesine neden olacaktır. Halkla ilişkiler ve resep- siyon misafir ile formal iletişim kurmaktadır. Animasyon departmanı ise hem formal hem de in- formal bir iletişim içerisindedir. Misafirler kendi istek ve ihtiyaçlarını bu departmana daha sa- mimi bir şekilde ifade etmektedir. Animasyon departmanının olması konaklayan misafirlere tam anlamıyla ulaşılmasını hedefleyen işletmelerin olmazsa olmazıdır" şeklinde otel için animasyon hizmetinin olmasının önemini açıklamıştır. SM-1'de "bölgedeki otellerde olması animasyon hiz- meti olmayan oteli satış ve kalite yönünden etkiler" şeklinde cevaplandırmıştır. Görüşmeye katılan turistlere, animasyon hizmetlerinden rahatsızlık duyduğunuz yönleri neler- dir? diye sorulduğunda, istisnasız tüm katılımcılar animatörlerin etkinliklere katılım konusundaki ısrarlarından rahatsız olduklarını belirtmişlerdir. Ayrıca Türk müşteriler gece gösterilerinin müs- tehcen olmasını bu nedenle çocuklarına izletmek istemediklerinden dolayı rahatsızlıklarını bildir- miştir. 4. Sonuç ve tartışma Araştırmada otel animasyon hizmetlerinin birbirine benzer olduğu ve genellikle aynı hizmet- leri kapsadığı görülmektedir. Gündüz her otelde yapılan yarışmalar, spor faaliyetleri ve diğer et- kinlikler hemen hemen aynıdır. Gece gösterileri de genellikle basit kostüm ve sahne dekoru ile hazırlanmış bilinen skeçlerin tekrarı şeklindedir. Bunda tur operatörlerini paket turlarla birlikte getirdikleri daha çok Tunuslu ve Faslı olan ilk animatörlerin etkisi çok fazla olmuştur. Bu anima- törlerin yetiştirdiği bu günkü animatör şefleri o zaman öğrendikleri ve yaptıkları gösterilerin üze- rine pek fazla yenilik katamamışlardır. Türk geceleri kapsamında yapılan gösteriler dışında ülke kültürünü yansıtmamaktadır. Arap-Hint kültürüne özgü Fakir show veya Afrikalı dansçıların yap- tığı gösterilerin Türk kültürüne özgü eğlenceler gibi turistlere sunulduğu görülmektedir. Özellikle herşey dahil sistemi uygulayan otellerden hiç çıkmayan ve yerel halkla hiç temasa geçmemiş olan turistler ülke kültürünü hiç tanımadan ülkeden ayrılmak zorunda kalmaktadır. Oysaki o ülke kül- türünü yansıtan animasyon gösterileri ile turist sadece yerel ortama aşina olmakla kalmaz, hem de aktif olarak sürecin içinde yer alır ve animatörlerin yardımıyla ziyaret edilen ülke ile ilgili kendi deneyimlerini yaratabilirler. Araştırmada animasyon hizmetlerinin bir otelde olmasının en önemli yararlarının müşterilerin birbirleri ile daha iyi iletişim kurabilmelerinin sağlanması yanında müşterilerin işletme ile ilgili şikayet veya isteklerini animatörlere çok daha kolaylıkla iletebilmeleridir. Turistlere doğru yak- laşan ve onlarla iyi diyaloglar kurabilen tecrübeli animatörler sayesinde işletmedeki bazı aksak- lıklar müşteriler tarafından hoş görülebilecek ya da daha kolay bir şekilde müşteri şikayetleri gi- derilebilecektir. Bu durum turistlerin otel memnuniyetlerine yansıyarak tekrar işletmeyi tercih etmelerine ya da arkadaşlarına ve tanıdıklarına işletmeyi önermelerine neden olabilecektir. Ancak animatörlerin özelliklede bu işe yeni başlamış olanların, turistleri rahatsız edici yaklaşımları, on- ları etkinliklere katılmaya zorlamaları, müşteri memnuniyetinin azalmasına neden olabilmektedir. Diğer bir sonuç otellerin animasyon departmanlarına yeteri kadar önem vermediklerini gös- termektedir. Bu departmanı diğer departmanlardan farklı görmemekte, otelde mutlaka olması ge- rektiğini düşünmekte ancak verilen hizmetin kalitesine çok da önem vermemektedir. Otel yönetici ve sahipleri için önemli olan maliyetlerin ne kadar düşürülebildiğidir. Bu nedenle animasyon de- partmanı için yeterli bütçe ayrılmamaktadır. Animatöre çok yüksek maaşlar ödememek için bu işin inceliklerini bilmeyen acemi personel veya stajyerlerle günü kurtarmaya çalışmaktadır. Sahne dekoru, kostüm, spor malzemesi gibi animasyon departmanının kullandığı ekipmanlara da çok para harcanmasını istememektedir. Ancak unutulmamalıdır ki personel ve malzeme yeterli ol- mazsa en iyi animatör şefinin bile müşteri memnuniyetini sağlamak için yapabileceği bir şey kal- mamaktadır. Müşteri istek ve beklentilerinin karşılanmasında, şikayetlerin değerlendirilmesinde ve otel-müşteri ilişkilerinin kurulmasında animasyon departmanı önemi oldukça fazladır. 4. Bulgular TR-9 "animasyonun gece gösterileri kısmına çocuklar mini discoya katılsınlar diye gidi- yoruz. Skeçler başladığında oldukça müstehcen olduğu için hemen odamıza dönüyoruz. Çocuklar ısrarla kalmak istiyorlar ama biz seyretmelerini uygun bulmuyoruz. Keşke insanları güldürmek ve eğlendirmek için belden aşağı espri yapılması gerekmese daha farklı yollarla da insanlar eğ- lendirilebilir" diyerek animasyonun bu kadar kısır bir eğlence şekli olmaması gerektiğine vurgu yapmaktadır. TR-7 "bizim kültürümüzde yer almayan bazı gösterilerin sanki bize aitmiş gibi gös- terilmesinden rahatsızlık duydum" diyerek Türk kültürüne özgü gibi gösterilen, ancak Arap ve Afrika kültürüne ait bazı gösterilerin yapılmasının Türk müşterileri rahatsız ettiğine işaret etmek- Copyright © 2015 by IJSSER ISSN: 2149-5939 44 Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. International Journal of Social Sciences and Education Research, 1 (1), 35-48. tedir. Yabancı turistler ise daha çok gece gösterilerinin ve sahne dekorunun basitliğinden rahat- sızlık duyduklarını belirtmişlerdir. Özellikle daha önce daha lüks otellerde animasyon gösterisi izlemiş olan bazı turistler animatörlerin gidip o otellerdeki gösterileri izlemelerini önermişlerdir. 4. Sonuç ve tartışma Otel işletmelerinin Türk kültürünü yansıtabilecek özgün gösterilere yönelik çalışmaların yapılması, kalifiye animatörlerin istihdamı ve yetiştirilmesi, kaliteli hizmet sunumu ve memnun müşteri ya- ratılması için animasyon hizmetlerine ekip, araç-gereç malzeme, program, nitelik ve niceliksel olarak önem vermeleri gerekmektedir. Copyright © 2015 by IJSSER ISSN: 2149-5939 45 Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Kaynakça Borhan, O. & Erkmen, N. (2010). Antalya’nın Kemer ilçesinde tatil yapan turistlerin rekreasyon ve ani- masyon faaliyetleri hakkındaki görüşlerinin incelenmesi. Turkish Journal of Sport and Exercise, 11(3), 21-26. Costa, G., & Glinia, E. (2004). Sport tourism in Greece. Journal of Sport & Tourism, 9(3), 283-286. Costa, G., Glinia, E., Goudas, M. & Antoniou, P. (2004). Recreational services in resort hotels: Customer satisfaction aspects. Journal of Sport & Tourism, 9(2), 117-126. Demir, M. & Demir, Ş.Ş. (2001). Herşey Dahil (All Inclusive) Pansiyon Türü Uygulamasının Konaklama İşletmeleri, Personel, Müşteriler, Seyahat Acentaları ve Bölgedeki Diğer İşletmeler Açısından Olumlu ve Olumsuz Yönlerinin Analizi. Maltepe Üniversitesi Turizm Araştırmaları Dergisi, 1 (1), 67-100. Demir, Ş.Ş. & Günaydın, Y. (2013). İşgörenlerin müşteri sadakati üzerindeki etkileri: Konaklama işletme- leri örneği. The Journal of Academic Social Science Studies, 6(6), 1039-1059. Demir, Ş.Ş. (2010). Tatil satınalma sürecinde itici faktörler, bilgi arama ve memnuniyet ilişkisi: Yerli tu- ristler üzerine bir araştırma. İşletme ve Ekonomi Araştırmaları Dergisi, 1 (4), 119-132. Demir, Ş.Ş. & Demir, M. (2014). Kamu Kesimindeki Yöneticilerin Serbest Zaman Etkinliklerini Algıla- ması ve Değerlendirmesi. Manas Sosyal Araştırmalar Dergisi 3 (11), 61-76 Demir, Ş.Ş. & Demir, M. (2014). İşgörenlerin Serbest Zaman Gereksinimi ve Serbest Zaman Doyumunu Etkileyen Faktörler. İşletme ve İktisat Çalışmaları Dergisi 2 (3), 74-84 Glinia, E., Costa, G. & Drakou, A. (2004). Hotel animation and professional perspectives in Greece. Tou- rism Today Tourism Today. www.cothm.ac.cy Güler, E.G. (2009). Otel işletmelerinde değer yaratma ve müşteri değeri algılaması üzerine bir araştırma: Edirne’deki oteller örneği. Anatolia: Turizm Araştırmaları Dergisi, 20 (1), 61-76. Hornby, P. ve Symon, G (1994). “Tracer studies”, (Eds: C. Casselland G. Symon). Qualitative methods in Organisational Research: A Practical Guide. 167-186, London: Sage, Ivkov, A., & Stamenkovic, I. (2008). The implementation of the “Bologna Process” into the subject of animation in tourism, as a significant part of the hotel industry products promotion. Tourism & Hospi- tality Management, 14 (1), 129-140. Jakovlev, Z., Koteski, C., & Bardarova, S. (2013). Animator roles in enriching the content of tourist stay. In Collection of works of The Third International Scientific Congress-Biennale ICON BEST 2013. Karasar, N. (2002). Bilimsel Araştırma Yöntemi. Ankara: Nobel Yayınları. Koçak, N. (2001). Konaklama işletmelerinde animasyonun önemi ve animasyon personeline yönelik bir araştırma. Dokuz Eylül Üniversitesi İktisadi ve İdari Bilimler Fakültesi Dergisi, 16(1), 61-79. Luborsky, M. R. (1994). Qualitative Research in Ageing Research. Thousand Oaks: Sage. Medlik, S. (2003). Dictionary of travel, tourism and hospitality. Routledge. Medlik, S. (2003). Extended abstract in English Animations at resort hotels include mini club, sports, evening entertainment/shows and enter- tainment programs as different indoor and outdoor activities. This study evaluates the hotel ani- mation activities from both managers and tourist perspective. Purpose and Significance: In tourism industry, hotel animation programs and activities are a generally used term to describe entertainment and recreational services offered by resorts facilities in tourist destinations. Because animation services include three types of animation programs such as, sports activities, evening entertainment/shows and entertainment programs for children. And animation term also includes dance, shows, mini club activities, sports activities in swim- ming pool and/or outdoor for tourists who desire active recreation, have fun and social interaction with each other. It stated that hotel animations are important for both management and tourists. Tourist satisfaction with recreational and animation services can contribute to increase food and beverage sales during show programs at hotels and resorts in the coastal regions. The resort hotels offer different animation shows and programs as services free of charge, aiming at high sales of food and beverages which increase their profits. This paper reviews the nature of animation ser- vices. Therefore, the purpose of this study is to evaluate hotel animation services from managers and tourist perspective. Literature: One of the attributes and attractiveness is animation activities which hotel present. It is stated that indoor and outdoor animation activities is a great opportunity to improve market- ing public relations. Tourists have a better opinion of hotels which stayed with animation activi- ties and they come back again in the future. It is explained that lots of reasons to be important at a hotel; a) the effects on a positive image of the hotel, b) can provide to increase repeat guest to the hotel in the future, c) cause to increase consumption in the restaurant, bar, swimming pool and increase total profit of the hotel, d) take care of children, allowing parents to relax and e) other positive impacts both tourist and hotel management. In effective animation shows, the ability, performance, communication skills and cultural knowledge of animators are important and have crucial role on tourists’ expectations. Neverthe- less, there have been contradictory findings regarding the qualification of animators and the vari- ety of animation activities at resort hotels. According to tourists, the quality and variety of ani- mation activities are not sufficient level. Kaynakça Dictionary of travel, tourism and hospitality. Routledge. Mikulić, J. & Prebežac, D. (2011). Evaluating hotel animation programs at Mediterranean sun-and-sea re- sorts: An impact-asymmetry analysis. Tourism Management, 32(3), 688-696. Pompl, W. (1983). The concept of animation: Aspects of tourism services. Tourism Management, 4(1), 3- 11. Rowley, J.E. (1995). From storekeeper to salesman: Implementing the marketing concept in libraries. Lib- rary Review, 44 (1), 24-35. Copyright © 2015 by IJSSER ISSN: 2149-5939 46 Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Shportko, A. (2012). The Effect of Animation Services on the Guests’ Perception of a Hotel and Intention to Visit it (Doctoral dissertation, Purdue University). Shportko, A., Lehto, X., & Ghiselli, R. (2013). Investigating guest experience and satisfaction with hotel animation. In The 18 th Annual Graduate Education and Graduate Student Research Conference in Hospitality and Tourism. Thibal, S. (1985). A changing career in tourism. Espaces, (75), 31-34. Thibal, S. (1985). A changing career in tourism. Espaces, (75), 31-34. Tütüncü, Ö. (2009). Ağırlama Hizmetlerinde Kalite Sistemleri. Ankara: Detay Yayınevi. Vallaster, C. & De Chernatony, L. (2005). Internationalisation of service brands: The role of leadership during internal brand building process. Journal of Marketing Management, 13, 181-203. Vogt, C. A., & Fesenmaier, D. R. (1995). Tourists and retailers' perceptions of services. Annals of Tourism Research, 22(4), 763-780. Copyright © 2015 by IJSSER ISSN: 2149-5939 47 Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Demir, M. & Demir, Ş.Ş. (2015). The evaluation of hotel animation services from managers and tourists’ per- spective. International Journal of Social Sciences and Education Research, 1 (1), 35-48. Copyright © 2015 by IJSSER ISSN: 2149-5939 Extended abstract in English Although the animation activities at hotels are important, hotel managers do not allocate higher budget for animation team, equipment and other require- ments. Due to cheaper, animation activities include only Indian, Arabic, Russian or African shows and are presented by foreign animators as Turkish culture. Methodology: This paper which qualitative research method was used in, firstly, a literature review that related to recreational and animation services at hotels were conducted and then data were collected by researchers in face-to-face interviews with 10 managers (4-General Manager, 2-Front Office Manager, 2-Sales and Marketing Manager and 2-Customer Relation Manager) and 22 tourists (10-Turkish, 6-German and 6-English) at five stars hotels and resorts in Antalya region (Alanya, Kemer and Manavgat) and Muğla region (Fethiye, Marmaris, Bodrum and Dalaman). Results and Conclusion: The results indicate that managers and tourists believe in hotel ani- mation programs and people have a crucial role on customer satisfaction. Animation activities provide the most satisfaction for the family with children. Because children’s expectation and desire differs from their family and the “variety of activities” in animation is the most influential Copyright © 2015 by IJSSER ISSN: 2149-5939 48 Demir, M. & Demir, Ş.Ş. (2015). Otel animasyon hizmetlerinin yöneticiler ve turistlerin bakış açısıyla değer- lendirilmesi. International Journal of Social Sciences and Education Research, 1 (1), 35-48. attribute in explaining satisfaction of their holiday. The findings of the evaluation of hotel man- agers about animation activities have a positive and significant effect on tourists’ holiday satis- faction which creating their loyalty. Hotel managers stated that tourists purchase different good and services at hotel if they stay longer and spend more time in facilities. Both hotel managers and tourists agreed that the animation activities facilitate the sales efforts of hotel services. This study explored the evaluation of animation activities at resort hotels from managers and tourists’ perspective. Hotel animation activities is the tool that can provide managers to increase the sale of hotel gadgets and services in facilities and satisfy tourists' needs for social interaction, relax- ation and having a good time during holiday. This study presented that tourists’ evaluation of hotel animation activities are different from managers’ evaluation. The quality of animation programs and animators can affect both hotel goals and tourist expectations. Animators who plays with kids, organizes sports activities and presents evening shows for tourists are on a critical position at the hotel. Copyright © 2015 by IJSSER ISSN: 2149-5939
https://openalex.org/W4391645014	https://www.nature.com/articles/s41598-024-53214-w.pdf	English	null	A novel strategy towards efficient and reliable electric vehicle charging for the realisation of a true sustainable transportation landscape	Scientific reports	2,024	cc-by	13,569	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports A novel strategy towards efficient and reliable electric vehicle charging for the realisation of a true sustainable transportation landscape OPEN B. Anil Kumar 1, B. Jyothi 1, Arvind R. Singh 2, Mohit Bajaj 3,4,5,6, Rajkumar Singh Rathore 7 & Milkias Berhanu 8 This paper proposes an innovative approach for improving the charging efficiency of electric vehicles (EVs) by combining photovoltaic (PV) systems with AC–DC Power Factor Correction (PFC). The proposed approach employs bi-directional power flow management within the PFC system, allowing for enhanced resource utilization and EV battery capacity under a variety of environmental circumstances. A modified Lyapunov-based robust model reference adaptive controller (M-LRMRAC) is developed to provide real-time Maximum Power Point Tracking (MPPT) for the PV array. By quickly recording the MPP, this controller skilfully adjusts to shifting radiation and temperature dynamics. A noteworthy accomplishment is that the M-LRMRAC outperforms traditional Perturb and Observe (P&O) techniques by achieving quick MPP convergence (0.54 s). Additionally, the benefits of this integrated system go beyond effective MPPT. The method achieves operating at unity power factor and reduces total harmonic distortion, which results in improved power quality when charging EV Batteries (EVB). The entire solution provided by this multifaceted architecture improves the quality of electricity delivered to EV batteries while also increasing energy efficiency. This research helps to the evolution of sustainable and dependable EV charging infrastructure by solving difficulties and optimising performance. The combination of PV systems with AC–DC PFC, aided by the M-LRMRAC technology, presents a viable route for attaining efficient, clean, and high-quality EV charging, hence supporting the shift to a greener and more sustainable transportation landscape. List of symbols is Supply current vs Supply voltage F′(D) Connection between PV and D δIL Ripple current in the L γ Adaptation gain d(s) Small signal variation D θ1, θ2, θ3 Controlled parameters of plant am, km, bm Reference model parameters bp, ap, kp Plant parameters 1Department of Electrical and Electronics Engineering, Koneru Lakshmaiah Education Foundation, Vijayawada, India. 2Department of Electrical Engineering, School of Physics and Electronic Engineering, Hanjiang Normal University, Hubei, Shiyan 442000, People’s Republic of China. 3Department of Electrical Engineering, Graphic Era (Deemed to Be University), Dehradun 248002, India. 4Hourani Center for Applied Scientific Research, Al-Ahliyya Amman University, Amman, Jordan. 5Graphic Era Hill University, Dehradun 248002, India. 6Applied Science Research Center, Applied Science Private University, Amman 11937, Jordan. 7Cardiff School of Technologies, Cardiff Metropolitan University, Llandaff Campus, Western Avenue, Cardiff CF5 2YB, UK. 8Department of Electrical and Computer Engineering, Addis Ababa Science and Technology University, Adama, Ethiopia. www.nature.com/scientificreports/ The goal is to create a robust and reliable charging infrastructure that can support the growing demand for electric mobility while ensuring energy efficiency and stable power delivery to EVB18,19. Charging RV’s off-board without using solar panels faces key issues that could hinder EV popularity. Limited charging stations can make it hard to charge EVs without solar power, especially where charging infrastructure is lacking20,21. This also leads to concerns about running out of power on longer trips or in remote areas. Off-board charging relies on the grid, tying EVs to its capacity and energy sources22. If the grid uses fossil fuels, it reduces EVs’ environmental benefits due to carbon emissions. The growing number of EVs might strain the grid, needing costly upgrades and time. Another issue lies in charging costs. Off-board charging, particularly in regions where electricity prices are relatively high, might lead to increased operational expenses for EV owners, potentially impacting the overall cost-effectiveness of EV compared to traditional ICEV23,24. Lastly, the absence of solar PV panels means a missed opportunity to utilize renewable energy sources for charging EVs. Solar energy can be harnessed directly from the sun, providing a clean and sustainable power supply for EVs25. Without this renewable energy aspect, the full potential of EV’s in reducing greenhouse gas emissions and combating climate change might not be realized26. p g g g g g g Nevertheless, the frequent requirement of grid power for charging EVs presents several challenges that must be addressed27. The neighborhood distribution system may suffer as a result of the extensive use of EV. When many EV owners plug in their vehicles to charge simultaneously after returning home from work. The local power grid has to supply significantly more electricity as a result28,29. Technical concerns caused by this increase in demand may include fluctuations in frequency, harmonic disruption, and voltage regulation30,31. An unexpected and excessive peak load at the distribution network may result from the increasing EV usage and the regulation impact around their charging. Towards solve this problem, power utilities must upgrade the distribution grid to handle the increased demand for EV charging effectively32,33. g gf y Furthermore, the distribution grid is facing a growing presence of home PV systems. During periods of lower household consumption, such as noon time and the feeder receives the extra energy produced by HPV systems34. Leading to voltage rise and potential line overload. www.nature.com/scientificreports/ ensuring high efficiency to meet battery-side requirements like battery current and SOC3,4. Among the various DC/DC converter topologies investigated, researchers have explored different options such as DAB, DAB SRC5, QAB, and AQAB6 converters. While these converters offer galvanic isolation to achieve ZVS for all switches at the load side. The issues arise when multiple EVs are connected to a DAB converter. Balancing voltage on a dc link becomes challenging, leading to increased system size and complexity during the design process7. To tackle this problem, a QAB converter was suggested, comprising three bridges alongside one bridge on the MV and LV sides, offering an effective solution for multiple EV battery connections. However, relying solely on DC/DC converters presents other challenges, such as voltage flicker, increased THD, and low PF8,9. The varying current consumption of EV charging, particularly at initial connection and maximum charge, might result in voltage fluctuations. The electrical system may be momentarily affected by this sudden rise in current if there is a tran- sient voltage drop at the charging station. Factors like power system impedance and battery size determine the extent and duration of this voltage drop. Significant decreases can result in voltage flicker, which is the term for flickering lights or electronic faults. If EV charging stations have poor power factor, they may incur penalties and higher losses if they are not corrected10,11. Additionally, the absence of harmonic filtering may lead to voltage fluctuations and potential difficulties like transformer overheating. For the wider electrical grid and the charging infrastructure to continue operating efficiently, these issues must be resolved12. To mitigate these issues, a novel AC/DC converter has been introduced, which not only provides PFC but also achieves a high PF13. There have been studies on a number of AC/DC PFC converter configurations, including buck14, boost15, and buck-boost16. Among them, the Ferdowsi PFC converter stands out as a suitable option for EV charging applications due to its low semiconductor count and bidirectional power flow capability17. The Ferdowsi PFC converter is integrated buck boost converter, can handle variations in input voltage conditions, maintain a stable DC link voltage, and achieve high efficiency while minimizing THD. Considering all these parameters related to AC/DC PFC and DC/ DC converters are both, researchers go on designing off-board charging systems to efficiently charge EVB. A novel strategy towards efficient and reliable electric vehicle charging for the realisation of a true sustainable transportation landscape OPEN email: thebestbajaj@gmail.com; mil_ber2000@astu.edu.et \| https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 www.nature.com/scientificreports/ fs Switching frequency ibB Battery current ibchar Charge current of the battery ib Battery current iLp, iLn Inductor currents of PFC converter Isc Short circuit current km Positive gain Npe Number of parallel strings Nse Number of series strings Pmax Maximum power Ri, Cin Input resistance and input capacitor of so lar boost converter Vbchar Charge voltage of the battery vb Battery voltage Voc Open circuit voltage vpv, ipv PV array voltage and current vref Reference voltage AGMVC Adaptive generalized maximum versoria criterion ANFIS Adaptive neuro-fuzzy inference system AQAB Asymmetrical quadruple active bridge BES Battery energy storage BLDC Brushless DC CCM Continuous conduction mode D Duty cycle DAB Dual active bridge DAB SRC Dual active bridge series resonant converter DG Diesel generator DSP Digital signal processing ESS Energy storage system ESU Energy storage unit EV Electric vehicle EVB Electric vehicle battery FLC Fuzzy logic control ICEV Internal combustion electric vehicle INC Incremental conductance L Input inductor in boost converter LV, MV Low voltage, medium voltage M-LRMRAC Model Lyapunov predicated robust model reference adaptive controller MPP Maximum power point MRAC Model reference adaptive control MPPT Maximum power point tracking P&O Perturb and observe PCC Point of common coupling PEI Power electronic interface PEV Pug in electric vehicle PF Power factor PFC Power factor correction PHEV Pug in hybrid electric vehicle PID Proportional-integral-derivative PQ Power quality PRN Propulsion PV Photovoltaic PWM Pulse width modulation QAB Quadruple active bridge r(t) Input to the entire M-LRMRAC controller RBG Regenerative braking RV Road vehicles SMG Smart micro grid SOC State of charge TF Transfer function THD Total Harmonic distortion UVT Unit vector template Vdc Dc link voltage VSC Voltage source converter ZVS Zero voltage switching Researchers in the area of EV charging have concentrated on creating effective and efficient combine on and off-board charging1,2. Commonly, AC/DC and DC/DC converters are use ing. DC/DC converters which play a crucial role in voltage regulation, adapting renewabl Researchers in the area of EV charging have concentrated on creating effective and efficient charging systems that combine on and off-board charging1,2. Commonly, AC/DC and DC/DC converters are used for off-board charg- ing. DC/DC converters which play a crucial role in voltage regulation, adapting renewable energy sources, and https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Scientific Reports \| (2024) 14:3261 \| https://doi.org/10.1038/s41598-024-53214-w www.nature.com/scientificreports/ AC AC – DC + PFC Converter stage Boost converter is vs HV DC Bus LV DC Bus PV Panel Battery DC – DC Converter stage Figure 1. Off-board charging station with integrated PV. HV DC Bus HV DC Bus AC – DC + PFC Converter stage Boost converter PV Panel Figure 1. Off-board charging station with integrated PV. An integrated converter for EV’s that combines utility grid and solar PV charging uses a single converter for both sources, reducing component count, and employs an approach for PFC using inductor voltage detection without the need for a current sensor. The system operates seamlessly in all EV modes: charging, PRN, and RBG, functioning as a SEPIC converter for charging and as boost and buck converters for PRN and RBG modes, respectively46,47. Dual-source EV chargers combine grid power with solar electricity to overcome the drawbacks of solar PV-based chargers. Regardless of the solar variability or time of day, these hybrid systems guarantee dependability. In order to optimize energy use and save operating expenses, advanced energy management con- trols balance power sources, monitor solar generation, and integrate with the grid. This cost-effective and envi- ronmentally friendly method increases allure of electric cars by offering reliable, continuous charging services48. f In49 presents a multipurpose PEI for PEV, capable of utilizing dual charging sources encompass solar PV panels and the conventional grid supply for charging without additional components. Proposed PEI, derived from a conventional SEPIC converter, achieves MPPT during solar charging. It operates efficiently across all modes of transportation (charging, PRN, and RBG) with isolation in each mode, ensuring improved safety for batteries and vehicle users. The single converter design enhances charger compactness, making it an ideal solu- tion to power applications that charge on board batteries. In50 presents the creation of an EV charging station with solar energy. It places a strong emphasis on quiet, optimal transit using EVs and PHEVs compared to ICEVs. The study focuses on integrating renewable solar energy with the charging system. A charging circuit is included with PEV that maintains a near-unity PF through PFC and low current ripple through a PWM boost rectifier and DC/DC buck converter. By taking into account steady-state, transient, and configurable EV charging capacity responses relying upon battery SOC, a DSP con- troller board ensures optimal circuit operation. www.nature.com/scientificreports/ To address this issue, ESU are commonly used to mitigate the impacts of high HPV system integration. In recent years, the concept of utilizing EVB as an ESS addresses the intermittency of PV systems35. EVBs can function as ESS, allowing them to be charged with excess solar when necessary, pv power can be used to replace the grid. By absorbing power from widespread adoption of HPV in the distribution network are made possible by EVBs’ ability to reduce afternoon voltage surge concerns36,37. Addition- ally, the idea of using EVBs to support the high grid usage has been experienced also garnered increasing interest.f y g pp g g g p g g So the integration of PV into the off-board charging system includes converters for both ac and dc pro vides an adequate solution as shown in Fig. 1. Moreover, integrating PV into the off-board charging system enhances energy efficiency. Traditional charging methods rely solely on the electrical grid, which may involve energy losses during transmission and conversion processes. However, with PV panels directly generating electricity on-site, there are fewer energy losses, resulting in a more efficient charging process38,39. This efficiency not only benefits the environment but also contributes to cost savings and optimized resource utilization. Moreover, PV integration enhances the overall energy efficiency of the charging system40. Traditional electricity generation and distribution involve energy losses along the way, from power plants to charging stations. By tapping into solar energy directly at the charging location, these energy losses are minimized, resulting in a more efficient charging process41. This increased efficiency not only benefits the environment but also optimizes the use of avail- able resources, making EV charging more sustainable in the long run. Additionally, integrating PV into off-board charging can lead to cost savings for EV owners42. Solar energy is essentially free once the PV infrastructure is installed, making the cost of electricity for charging significantly lower compared to relying solely on grid power. While there may be an initial investment in setting up the PV panels and related equipment, the long-term savings on energy costs can make EV ownership more economically viable and appealing to a broader range of consumers43,44. Furthermore, in some regions, there may be incentives or government support programs that further reduce the financial burden of PV installation, making it even more attractive for EV users45. www.nature.com/scientificreports/ of a mechanical speed limiter, the frequency and voltage of the generator are controlled by the charging station ensures unity PF during nonlinear loading, and synchronizes PCC voltage with the grid/generator for cease- less charging. It also facilitates active/reactive power transfer and power exchange between EVs for enhanced efficiency. In both grid-connected and islanded environments, the Charging Station solves the problems asso- ciated with single-mode operation. In islanded mode, a storage battery provides steady power despite solar variability, while in grid mode, it guarantees solar PV use even in the absence of the grid. A DG set is integrated to ensure continuity, particularly in remote places, while EV charging harmonics impact its performance. To ensure efficient functioning, the CS uses a voltage source converter to deliver reactive current and harmonics, mitigating this. g g In54 discusses the growing utilization of EV’s worldwide and the need for self-sustainable charging stations to minimize fossil fuel consumption. It highlights the potential negative impact of fossil fuel-powered charging infrastructures on the distribution system and the environment, proposing PV energy as an efficient solution. To ensure effective grid planning and load management with a large number of EVs, centralized and decentralized control strategies should be employed, and EV load characteristics should be considered in substation planning and capacity design.h p y g The normal operation of smart houses in an SMG is improved using an intelligent strategy that uses a broad dragonfly algorithm and logical hierarchy approach55. The technique reduces the peak to average consumption of smart houses while simultaneously optimizing energy sources, which is accomplished by employing solar panels and PHEVs. A system that controls the bidirectional flow of power for a solar water pumping system that is connected to the grid. To run the water pump continuously regardless of weather, it uses a without current- phase indicators, BLDC motor-drive. The system enables the utility grid to receive any extra electricity that is generated. In order to reduce switching losses, a single-phase VSC with a UVT generation approach is employed for bidirectional power flow management. UPF and reduce THD of the grid is done by the PV array at MPP56. The recently installed PV-based pumping system with a BLDC motor drive enhances flexibility and efficiency, addressing shortcomings of previous systems with bidirectional power flow. www.nature.com/scientificreports/ This invention maximizes the use of solar energy by allowing power to move from the PV array to the utility grid when water pumping is not required. On the other hand, the system permits power to flow from the grid to the BLDC motor-pump when there is insufficient PV power or at night. By ensuring continuous functioning, its bidirectional capacity over- comes the drawbacks of unidirectional systems. It provides a more adaptable and durable solution that permits the best possible energy use under a range of operating conditions, hence enhancing the PV-based pumping system’s overall performance.h y p The synchronizing of EV charging and power allocation optimizing procedures is carried out by the two-stage system. Which involves the distribution of power based on their respective preferences, and the attainment of a balanced allocation is derived from game theory in relation to the Nash equilibrium. Moving into the subse- quent phase, the confirmed overall charging capacity is presented as a fused constraint, addressing the challenge of coordinating EV charging in a distributed manner, considering individual charging requirements showing improved battery SOC, smoother grid power profile with lower peak-to-average ratio, and peak power, confirm- ing its effectiveness57. In order to address power shortages at charging stations with PV and battery systems, a two-stage energy management technique is proposed. It intelligently distributes power between the grid, batteries, and solar PV in the initial stage. The second stage then makes sure that each EV receives an equitable share of the available electricity. This methodology takes into account the restricted capacity of the charging station, the constantly fluctuating demands of electric vehicles, and the sporadic nature of renewable energy sources such as solar power. By utilizing game theory, it maximizes decision-making and offers an adaptable and competitive framework to deal with power outages and improve charging operations’ overall efficiency. p g p g g pfi y Atmospheric conditions, dust, temperature, cloud cover, and geographic location are just a few of the variables that might affect how much solar energy can be harvested. Due to shifting azimuth angles, the amount of solar radiation varies during the day. Even under cloudy skies, electricity production from PV panels varies depend- ing even in the presence of constant irradiance and temperatures, generation is impacted by the panel’s resistive load. Due to day-to-day fluctuations in temperature and irradiance, determining the MPP and computing it at any given load becomes challenging. www.nature.com/scientificreports/ When it comes to connecting EVs to power sources, the CCS is essential. The vehicle’s connection to the grid, which includes solar PV or home power installations, is supported by the system. In order to provide effective communication for safe and efficient charging, the CCS functions as a smart connector, facilitating a complex “handshake” between the vehicle and the power source. In order for the EV to draw electricity and for intelligent communication to optimize the charging process and guarantee that safety rules are followed, a smooth connection must be established. y In51 Simple integration of PQ compensating characteristics into solar panels connected to a three-phase grid. It utilizes a three-phase VSC to convert generated DC power to AC. The control approach based on an AGMVC enables effective active power transfer and PQ compensation. With the help of the P&O based MPPT algorithm, the solar PV array is used effectively. The effectiveness of the AGMVC control technique among asymmetrical load conditions is demonstrated by the fact that the grid current maintains its balance and sinusoidal shape. In52 the incorporation of PV systems, traction batteries, and the AC grid is encompassed by the PEV battery charging, a centralized simultaneous multiport DC–DC converter is proposed. This converter allows both PV panels and the grid to deliver power to high-voltage batteries simultaneously or separately, making it more reli- able than conventional topologies. It uses a half-bridge CLLC converter with fewer switches for bidirectional power transfer between batteries and the AC grid. The unified controller, combined with an optimum MPPT algorithm, effectively regulates the converter’s operation. f In53 presents a charging station utilizing solar PV, BES, DG, and grid to provide continuous charging in various modes (islanded, connected to the grid, and connected to a DG set). This facility prioritizes solar PV and BES charging for EV batteries but intelligently switches either a DG set or the grid can be connected when necessary. For achieving optimal fuel efficiency, the DG set is operated at 80–85% of its capacity. In the absence Scientific Reports \| (2024) 14:3261 \| https://doi.org/10.1038/s41598-024-53214-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ great accuracy, ranging from 99.5 to 99.9%, and under a variety of radiation and temperature circumstances, an improved MPPT method based on FLC is achieved. Despite being easy to use and producing excellent results, classic MPPT techniques have undesirable oscil- lations close to the MPP, according to the scant literature review. Despite their effectiveness in MPPT, soft- computing techniques are characterized by intricacy, high costs, and significant computational demands. The conventional MRAC is also not totally appropriate for some plants, such as PV systems with boost converters, which exhibit second-order system characteristics, due to its primary design for first-order systems.i i A new M-LRMRAC is designed that extends the control from first order to second order and is implemented to reach MPPT in PV systems. The primary objective is to minimize system control complexity while effectively managing uncertainties and environmental disruptions. The uniqueness of this research lies in the development of an M-LRMRAC control law customized explicitly for second-order PV MPPT systems. Notable advantages of this M-LRMRAC technique include its simplicity, enhanced active adaptability, minimal Swings in close proxim- ity to the MPP, and rapid observation rate, particularly under dynamic weather conditions. y p p y y In addition, the M-LRMRAC controller has outstanding tracking ability, capturing the MPP in variable environmental variables like temperature and sun radiation changes with an average convergence time of 0.54 s. • A new M-LRMRAC controller is released for solar PV systems with improved MPPT.h The simplicity, quick convergence time, quick dynamic response, and low oscillations close to the MPP define this M-LRMRAC controller.h • The MRAC controller exhibits robustness against variations in temperature and solar radiation because of its adaptive nature. • The MRAC controller exhibits robustness against variations in temperature and solar radiation because of its adaptive nature. • For MPP tracking, the suggested controller performs better in terms of convergence time when compared to traditional algorithms like P&Oh For MPP tracking, the suggested controller performs better in terms of convergence time when compared to traditional algorithms like P&Oh to traditional algorithms like P&Oh • The suggested controller has a convergence speed that is ten times quicker than that of conventional P&O techniques, which is noteworthy. • The suggested controller has a convergence speed that is ten times quicker than that of conventional P&O techniques, which is noteworthy. www.nature.com/scientificreports/ The organization of the remaining portion of this article is as follows: “Boost converter model” section. Boost Converter Model, “Proposed MPPT Methodology” section. Proposed MPPT methodology, “System design” sec- tion. System design, “Procedure for M-LRMRAC” section. Procedure for M-LRMRAC, “Results and discussion” section. Results and Discussion. www.nature.com/scientificreports/ A significant MPPT topology for solar cells is the use of buck-boost, boost, and buck converters to solve this problem. Due to its benefits of reduced switching losses and inductivity, which reduce current ripples, the boost converter is especially ideal for PV applications. Furthermore, a stable current with reduced stress compared to other topologies during operation is upheld by this converter. For the purpose of performance enhancement, a control algorithm is necessitated by MPPT devices. Nevertheless, suboptimal performance in MPPT applications is brought about by the PID controller’s simple structure58. Under steady weather conditions the MPP is done by using the traditional approaches like INC59 and P&O60.hh y g pp The simplicity of common MPPT methods like INC and P&O makes them widely employed. There are drawbacks to these conventional methods, though. Although their simplicity of construction makes them less weather-adaptive, they are nonetheless effective at tracking MPP in conditions of consistent weather. Because they frequently show oscillations close to the MPP traditional MPPT algorithms—like INC and P&O—are less effective for solar power plants of a larger size. When trying to maximize solar panel power extraction under dynamic weather circumstances, where these traditional approaches might not be able to provide stable and accurate tracking, this disadvantage becomes very important. g g y p A novel approach combined to quickly get the MPP regarding fluctuating radiation and temperature, the modified MRAC using the Lyapunov and INC technique is designed. Simulations using PSIM demonstrate the robustness of the proposed control law, showcasing its superior MPP tracking capability compared to the INC-PI controller61. Another MPPT controller utilizing an ANFIS is presented, offering effective MPP tracking under varying environmental conditions, outperforming traditional methods like P&O and FLC. Furthermore, with Scientific Reports \| (2024) 14:3261 \| https://doi.org/10.1038/s41598-024-53214-w www.nature.com/scientificreports/ Boost converter modelh The boost converter, which plays a crucial role in contemporary Optimizing the system’s parts and reducing energy waste are necessary to achieve this is demonstrated in Fig. 2. Depending on specific needs, other convert- ers might potentially be used. In the boost converter approach depicted in Fig. 3, the duty cycle (d(t)) provided to the switch S is controlled as a consequence of MPPT controller, which also continually monitors the solar array voltage and current levels is modified as necessary. This modification is achieved using Eq. 1, which establishes the connection between both the array’s voltage and d(t). 6 4) 14:3261 \| https://doi.org/10.1038/s41598-024-53214-w Cin Co L + - MPPT Control Law(P&O) ^d(s) D vref ipv vpv S L o a d + - Figure 2. PV with Boost converter and MPPT control. D MPPT Control Law(P&O) Figure 2. PV with Boost converter and MPPT control. Scientific Reports \| (2024) 14:3261 \| https://doi.org/10.1038/s41598-024-53214-w www.nature.com/scientificreports/ ipv PWM Switch Cin BT1 vpv Ri Co L + - + - F’(D) d Figure 3. Small signal equivalent circuit of Boost converter. PWM Switch Figure 3. Small signal equivalent circuit of Boost converter. Figure 3. Small signal equivalent circuit of Boost converter. (1) vpv = ipvR0(1 −d)2 (1) ipv and vpv are commonly used notations to represent the PV array voltage and current. Every vpv, ipv and DC vpv, ipv components, which signify the load resistance R0. Equation (1) provides the foundation for the traditional MPPT method’s calculation of the steady-state duty cycle. Maximum transient response and suppression of oscillation with duty cycle across the connection are controlled by MPPT. Performance under a variety of various environmental situations is poor due to this lack of adaptation.h ipv and vpv are commonly used notations to represent the PV array voltage and current. Every vpv, ipv and DC vpv, ipv components, which signify the load resistance R0. Equation (1) provides the foundation for the traditional MPPT method’s calculation of the steady-state duty cycle. Maximum transient response and suppression of oscillation with duty cycle across the connection are controlled by MPPT. Performance under a variety of various environmental situations is poor due to this lack of adaptation.h The presentation and discussion of a detailed boost converter transient analysis are carried out. Toward improve every comprehensibility about transient response study, it is believed that a small-signal equivalent circuit can represent the system, as discussed in62. Boost converter modelh Figure 3 illustrates the linearized PV model which utilizes resis- tor Ri and small-signal representations of the vpv and ipv across its terminals to effectively model the solar array. Currently, the control signal (d(t)) actively regulates beyond the array voltage through the TF relationship at the supplied operating point. This TF represents dynamic of the system. A dynamic model showing a typical PV system battery load is shown in Fig. 3. Here, ignoring battery dynamics in the process manner in which fluctua- tions are the duty cycle of a system are brought about by variations in voltage across the array is revealed by the transfer function. Following correlation63 is obtained from this analysis of Fig. 3 and presented in Eqs. 2 and 3. (2) ˆvpv(s) Ri + sˆvpv(s)Cin = (F′(D)ˆd(s) −ˆvpv(s) sL (2) The derivative of F′(D) with respect to D is given in the following Eq. 3 The derivative of F′(D) with respect to D is given in the following Eq. 3 (3) ˆvpv(s) ˆds = F′(D) s2LCin + L Ri is + 1 ˆvpv(s) ˆds = F′(D) s2LCin + L Ri is + 1 (3) knows that F(d) in below Eq. 4 knows that F(d) in below Eq. 4 (4) F(D) = VPV = (1 −D)V (4) From Eq. 4 F′(D) presented in Eq. 5 q (5) ˆvpv(s) ˆds = −Vo LCin s2LCin + 1 RiCin is + 1 LCin (5) Proposed MPPT methodology Proposed MPPT methodology p gy In the framework of a second-order type PV MPPT system, the proposed study’s goal is to develop an M-LMRAC control rule that makes use of the Lyapunov stability theorem. Improving the efficiency of the MPPT algorithm for a second-order PV system is the primary goal of the proposed study. The main goals are to overcome the shortcomings of traditional methods and to increase overall efficiency, tracking speed, accuracy, and complexity in the face of changing environmental conditions. The second-order extension of the suggested adaptive MRAC architecture seeks to reduce system complexity and efficiently handle environmental and PV system uncertain- ties and disturbances. By developing an M-LMRAC control method using the Lyapunov stability theorem, the paper presents a novel strategy for improving MPPT in second-order PV systems. p p p gy p g y Figure 4 comprising of an initial MPPT control block succeeded by a subsequent M-LRMRAC module, show- casing a novel configuration represented consecutive two-tier hybrid strategy is encompassed by the proposed MPPT approach. In the first level, a vref for each MPPT is generated using the conventional P&O approach. Subsequently, this MPP voltage is updated continuously and compared to the varying vpv resulting from fluctua- tions in thermal conditions, electric demand, irradiation. The M-LRMRAC controller’s input is created by the difference between vpv and the adjusted vref and key challenge lies in determining suitable controller parameters for achieving effective performance. The suggested system parameters are refined throughout the adaptation process by exploiting the difference between the referring to both the controller and the system models. Using appropriate adaptive principles. Closed-loop stability is ensured, and the adaptive rules have the capability directed toward forecasting the undisclosed parameters of the controller. Using M-LRMRAC to serve as an Scientific Reports \| (2024) 14:3261 https://doi.org/10.1038/s41598-024-53214-w www.nature.com/scientificreports/ C Co L + - MPPT Control Law(P&O) M-LRMRAC Controller ^d(s) D vref ipv vpv S L o a d + - Figure 4. M-LRMRAC controller with Solar Boost converter. M-LRMRAC Controller vref Figure 4. M-LRMRAC controller with Solar Boost converter. initial signal for PWM duty cycle modification within the boost converter configuration, the Lyapunov stability theorem is implemented to actively maintain the PV panel at its MPP. initial signal for PWM duty cycle modification within the boost converter configuration, the Lyapunov stability theorem is implemented to actively maintain the PV panel at its MPP. y Using the formulation supplied in Eq. Proposed MPPT methodology 6, a tailored vref variation is determined for the controller, allowing the MPPT regulation law to effectively pinpoint the precise operational junction for best power extraction, where ∆v represents a small threshold voltage, and vpv. MPPT control block (P&O) ( ) Equation 6 depicts the connection that governs the variance of vref that corresponds with the P&O MPPT approach, ensuring optimal tracking of the MPP of the solar panel. (6) vref = vpv, dp dpv = 0, vpv, dp dpv < 0, vpv, dp dpv > 0 (6) Proposed M‑LRMRAC approach A l h For desired second order reference model along with plant, presented in the Eq. 9 Proposed M‑LRMRAC approach l h A responsive control mechanism is required, which can quickly adapt the duty cycle to be aligned with rapidly changing environmental factors, ensuring the optimal operation of the PV panel at its MPP, even when there are abrupt fluctuations in solar insolation, for instance shifting sunlight intensities or impacts caused by shading. A substantial preference for second-order dynamics is noted in the field of plant features, which includes a variety of systems including PV installations with boost converters. Despite their widespread adoption, traditional MRAC approaches fall short of offering sufficient tracking performance for second-order systems. ffi As a result, this study seeks to get around these limitations by developing a novel control law that is delib- erately designed to solve second-order system complexities. This breakthrough results in the development of a M-LRMRAC strategy that bridges the gap between first and second-order dynamics. Figure 5 depicts a visual representation of the suggested M-LRMRAC idea, with the signal r(t) playing a critical role. Equation 5’s transfer function precisely matches the plant model depicted in Fig. 5. p y p p g Here, the plant’s output and input are represented by yp(t) and up(t), respectively. (7) d2yp(t) dt2 = −ap dyp(t) dt −bp dyp(t) dt + kpup(t) (8) Gp(s) = yp(s) up(s) = kp s2 + aps + bp (7) d2yp(t) dt2 = −ap dyp(t) dt −bp dyp(t) dt + kpup(t) (7) (8) Gp(s) = yp(s) up(s) = kp s2 + aps + bp (8) For desired second order reference model along with plant, presented in the Eq. 9 For desired second order reference model along with plant, presented in the Eq. 9 https://doi.org/10.1038/s41598-024-53214-w https://doi.org/10.1038/s41598-024-53214-w https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ Figure 5. Design of M-LRMRAC controller. Figure 5. Design of M-LRMRAC controller. (11) up = θ1r −θ2yp −θ3 · yp = θT (11) Substitute Eq. 11 into the Eq. 7 will get the Eq. 12. Substitute Eq. 11 into the Eq. 7 will get the Eq. 12. (12) d2yp(t) dt2 = −(ap + kpθ3) dyp(t) dt −(bp + kpθ2)yp(t) + kpθ1r(t) (12) Compare the Eqs. 12 and 9 will get the controlled parameters of plant in terms θ1, θ2, θ3 Compare the Eqs. Number of panels p Number of panels is established by a total power rating of 4500 W, will use the specifications of the Risen-335-WH solar panel, which has a power rating of 335 W, a voltage at MPP of 37.65 V, and a current at MPP of 8.90 A. Considering standard irradiation and temperature conditions of 1000 W/m2 and 25 °C, will design a PV array to meet the required capacity. q p y Number of panels needed = Total required power/Power rating of each panel. 4500 q p y Number of panels needed = Total required power/Power rating of each panel. 4500 p q p Number of panels needed = 4500w 335w = 13.43 = 14 Number of panels needed = 4500w 335w = 13.43 = 14 335w But for suitable panels selection based on the series and parallel configuration will choose 15 panels. In which each row, 5 panels are connected in series. This will increase the voltage while keeping the current the same. Then, these three rows are connected in parallel. This will maintain the voltage at the level achieved by connecting 5 panels in series while increasing the total current by combining the currents of the three parallel rows. Design of boost converter Step 2: Consider the efficiency of the charging process. Assume a typical efficiency of around 80% for the entire charging system. Step 3: Calculate the power rating of the PV panels: Calculate the power rating of the PV panels:fi p g p Power Rating of PV Panels (W) = Total Charge Power/Charging Efficiency. Power Rating of PV Panels (W) = 3600W/0.8 ≈ 4500W. Proposed M‑LRMRAC approach l h 12 and 9 will get the controlled parameters of plant in terms θ1, θ2, θ3 (13) θ1 = km kp (14) θ2 = bm −bp kp (13) θ1 = km kp θ bm −bp (13) (14) θ2 = bm −bp kp (14) https://doi.org/10.1038/s41598-024-53214-w https://doi.org/10.1038/s41598-024-53214-w https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ (15) θ2 = am −ap kp (15) The eror from the plant controller and Vref will be the The eror from the plant controller and Vref will be the The eror from the plant controller and Vref will be the (16) e = yp −ym e = yp −ym (16) e = yp −ym (16) Double derivative of Eq. 16 with respect to t will give the Eq. 17 Double derivative of Eq. 16 with respect to t will give the Eq. 17 (17) d2e(t) dt2 = −(ap + kpθ3) dyp(t) dt −(bp + kpθ2)yp(t) + kpθ1r(t) + am dym(t) dt + bm dym(t) dt −kmr(t) Design of boost converter g As part of the design procedure, the sizing of the L in the boost converter is chosen in order to sustain CCM functioning under a broad range of weather scenarios along duty ratio D1. (18) D = Vdc −Vpv Vdc = 300 −188.25 300 = 0.3725 (18) Vpv is selected as 5 times of voltage at MPP. The input inductor estimated as (19) L = D1Vpv fswδIL = 0.3725 ∗188.25 50000 ∗0.2 ∗8.90 = 788 µH (19) Procedure for M‑LRMRACh System design y g Estimation of PV power rating p g In the research process, the PV array parameters, including the number of panels required, MPPT mechanism, and Boost converter specifications, will be designed based on the EVB specifications. The power rating of the PV array can be identified using three EVBs, each with an operational voltage and current of 48 V and 25 A, respectively. y Step 1: Determine total charge power required for the three batteries: Total Charge Power (W) = Total Voltage (V) x Charge Current (A). Total Voltage = 3 × Maximum Vb = 3 × 48 V = 144 V. Charge Current = 25 A. Determine total charge power required for the three batteries: Total Charge Power (W) = Total Voltage (V) x Charge Current (A). Total Voltage = 3 × Maximum Vb = 3 × 48 V = 144 V. Charge Current = 25 A. Determine total charge power required for the three batteries: Total Charge Power (W) = Total Voltage (V) x Charge Current (A) Total Voltage = 3 × Maximum Vb = 3 × 48 V = 144 V. Charge Current = 25 A. Total Charge Power (W) = 144 V × 25A = 3600 W. g Total Charge Power (W) = 144 V × 25A = 3600 W. Procedure for M‑LRMRACh The Lyapunov stability theorem is a fundamental concept in control theory used to analyze the stability of dynamical systems. When the framework is classified to exhibit asymptotic stability by the existence of a continu- ously differentiable real scalar function V(t) and its time derivative V(t) is consistently negative definite (V(t) 0) for all values of t larger than zero. Scientific Reports \| (2024) 14:3261 \| https://doi.org/10.1038/s41598-024-53214-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ Based on the idea of utilizing a Lyapunov function to assess the stability of a dynamical system, the Lyapunov stability theorem is presented in the study. An statement in mathematics that aids in evaluating the behavior of a system over time is called a Lyapunov function. A system must have a continuously decreasing derivative of a potential energy-like variable to be deemed “asymptotically stable,” which can be achieved by using a Lyapunov function. A system is said to be “asymptotically stable” if it naturally tends to approach a stable equilibrium point over time, which is identified by a minimum or zero value of the Lyapunov function.ii i y y p As for the Lyapunov function V(t) in this specific case, it is defined as follows steps: Step 1: i y y p As for the Lyapunov function V(t) in this specific case, it is defined as follows steps: Step-1: (20) V(t) = 0.5e2(t) Step-2: (20) V(t) = 0.5e2(t) (21) V·(t) = d dt 0.5e2(t) Step-2: Step-3: Step 4: (20) V(t) = 0.5e2(t) (21) V·(t) = d dt 0.5e2(t) (22) V·(t) = e(t) ∗e·(t) (20) V(t) = 0.5e2(t) Step-2: Step-2: (21) V·(t) = d dt 0.5e2(t) (21) Step-3: V·(t) = e(t) ∗e·(t) (22) Step-4: From Eq. 16 calculate e˙(t) Step-4: From Eq. 16 calculate e˙(t) (23) e·(t) = y· p(t) −y· m(t) e·(t) = y· p(t) −y· m(t) (23) Step-5: p Substitute Eqs. 23 in 22 will gives the (24) V·(t) = e(t) ∗y· p(t) −y·m(t) (24) Step-6: Step-6: Determine e(t)’s second order type derivative in relation to time “t” from Eq. 23 (25) d2e(t) dt2 = y··p(t) −y··m(t) d2e(t) dt2 = y··p(t) −y··m(t) (25) Step-7: From Eqs. 25 and 24 the Step-7: p From Eqs. 25 and 24 the (26 V·(t) = y· p(t) −y·m(t) ∗(−ap −kpθ3) dyp(t) dt −(bp + kpθ2)yp(t) + kpθ1r(t) + am dym(t) dt + bm dym(t) dt −kmr(t) h (26) Assuming kp γ > 0, let’s define the Lyapunov function V based on the given Eq. Procedure for M‑LRMRACh 26 will set to V’(t) = − γ. The Lyapunov function V is typically for any t > 0, the system is consistent because V(t) is negative definite (V(t) 0). If V(t) equals 0 for every t > 0 in addition system is equilibrium state and any errors or disturbances will converge towards zero over time. By carefully designing the parameters kp, γ, and the function V(t), the control system can be made robust and stable. The Lyapunov function V is an essential tool in control theory to prove stability properties of a system and design suitable controllers for desired performance. Based on Eq. 26 1. yp(t) −ym(t) (−ap −kpθ3) dyp(t) dt To make this term negative, present a yp(t) < ym(t) and both ap and to be positive. 1. yp(t) −ym(t) (−ap −kpθ3) dyp(t) dt To make this term negative, present a yp(t) < ym(t) and both ap and kpθ3 to be positive. 2. yp(t) −ym(t) (−bp −kpθ2)yp(t) To make this term negative, present a yp(t) < ym(t) and both bp and kpθ2 1. yp(t) −ym(t) (−ap −kpθ3) dyp(t) dt To make this term negative, present a yp(t) < ym(t) and both ap and kpθ3 to be positive. 2. yp(t) −ym(t) (−bp −kpθ2)yp(t) To make this term negative, present a yp(t) < ym(t) and both bp and kpθ2 to be positive. p 2. yp(t) −ym(t) (−bp −kpθ2)yp(t) To make this term negative, present a yp(t) < ym(t) and both bp and kpθ2 to be positive. 3. yp(t) −ym(t) kpθ1r(t) + am dym(t) dt + bm dym(t) dt −kmr(t) To make this term negative, present a yp(t) < ym(t) and the coefficients am, bm, km, kpθ1, to be such that the entire expression in the brackets becomes negative. 3. yp(t) −ym(t) kpθ1r(t) + am dym(t) dt + bm dym(t) dt −kmr(t) To make this term negative, present a yp(t) < ym(t) and the coefficients am, bm, km, kpθ1, to be such that the entire expression in the brackets becomes negative. The derivatives of the controller parameters with respect to time dθ1 dt , dθ2 dt , dθ3 dt can be found by setting the coef- ficients of each term to − γ. 1. For the term yp(t) −ym(t) (−ap −kpθ3) dyp(t) dt : −γ = − ap + kpθ3 . Therefore dθ3 dt = γ−ap kp Therefore dθ3 dt = γ−ap kp 2. Results and discussion Following the integration of solar PV into AC–DC PFC and DC–DC converter fed into an EVB, Fig. 1 displays a detailed Fig. 6.hih The remaining data parameters are presented in Table 1 and are based on the design specifications. T research focuses on solar PV arrays with M-LRMRAC (Fig. 7). y Two type of analysis are performed. yp y p 1.PV + AC–DC + PFC + DC–DC fed to EVB. 2.PV + PFC + DC–DC fed to EVB. Procedure for M‑LRMRACh For the term yp(t) −ym(t) (−bp −kpθ2)yp(t) : −γ = − bp + kpθ2 . 2. For the term yp(t) −ym(t) (−bp −kpθ2)yp(t) : −γ = − bp + kpθ2 . Therefore dθ2 dt = γ−bp kp 3. For the term, yp(t) −ym(t) kpθ1r(t) + am dym(t) dt + bm dym(t) dt −kmr(t) := −γ 3. For the term, yp(t) −ym(t) kpθ1r(t) + am dym(t) dt + bm dym(t) dt −kmr(t) := −γ https://doi.org/10.1038/s41598-024-53214-w https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Therefore dθ1 dt = −γ kp Th d d ll Therefore dt = kp The updated controlled parameters are defined in below and presented in Fig. 5: dt kp The updated controlled parameters are defined in below and presented in Fig. 5: (27) dθ1 dt = −γ kp (28) dθ2 dt = γ −bp kp (29) dθ3 dt = γ −ap kp (27) (28) (29) PV + AC–DC + PFC + DC–DC fed to EVBl Figure 8 shows the overall flowchart of PV + AC–DC + PFC + DC–DC fed to EV Battery. It is to be operated based on the vs, 220 V, and the AC–DC PFC Converter performance, demonstrating that the converter is functioning at UPF when the is peak is in phase with vs peak as depicted in Fig. 7. Figure 7c,d indicate converter’s operation in DCM due to transition, yet the inductor currents iLp and iLn remain continuous. This continuity results from the discharge of energy stored in inductors Lp and Ln, effectively transferred to output capacitor Cf.h g gy pf y f The inductor LP releases stored energy when the switch SP is open during the DCM transition, ensuring continuous current flow to the load through established inductor currents (iLp and iLn). The diode DP, which is forward-biased, stops current backflow. Concurrently, the capacitor CP maintains system stability by offering a steady voltage supply when the system is turned off. By coordinating their efforts, the load and output capacitor Co. will always receive power, and the converter will remain stable during the input voltage’s negative half-cycle. Additionally, the PV will be connected to the PFC converter’s DC connection and will be able to run MPPTs of 1000 w/m2 and PV panel outputs, as indicated in Figs. 9 and 10. Depending on how the panel circuit is con- figured, the ipv and vpv are reduced and increased until they reach the final steady state value shown in Table 1 as indicated in Fig. 10, can generate a maximum power of 4.5 kW. An essential part of EV charging stations that facilitates effective power transfer between the EVB and the grid is a bidirectional DC–DC converter. It functions as a connection point for various DC voltage sources and loads, enabling power to flow in both ways. f The inductor LP releases stored energy when the switch SP is open during the DCM transition, ensuring continuous current flow to the load through established inductor currents (iLp and iLn). The diode DP, which is forward-biased, stops current backflow. Concurrently, the capacitor CP maintains system stability by offering a steady voltage supply when the system is turned off. By coordinating their efforts, the load and output capacitor Co. will always receive power, and the converter will remain stable during the input voltage’s negative half-cycle. PV + AC–DC + PFC + DC–DC fed to EVBl Parameter Value Pmax 335w vpv at Pmax 37.65 V ipv at Pmax 8.90 A voc 45.90 V Isc 9.40 A vs 220 V fs 50 kHz vb 48 V ib 25A Cin 2.364 μF L 788 μH ap = 1 RiCin 100 × 103 rad/s am 8.17 × 103 (rad/s)2 bp = 1 LCin 5.368 × 108 (rad/s)2 bm 1.67 × 107 (rad/s)2 kp = vo LCin 257.664 × 108 (rad/s)2 km 5.75 × 108 V(rad/s)2 Nse, Npe 5 & 8 Ŵ 0.08 Table 1. Simulation results. Parameter Value Pmax 335w vpv at Pmax 37.65 V ipv at Pmax 8.90 A voc 45.90 V Isc 9.40 A vs 220 V fs 50 kHz vb 48 V ib 25A Cin 2.364 μF L 788 μH ap = 1 RiCin 100 × 103 rad/s am 8.17 × 103 (rad/s)2 bp = 1 LCin 5.368 × 108 (rad/s)2 bm 1.67 × 107 (rad/s)2 kp = vo LCin 257.664 × 108 (rad/s)2 km 5.75 × 108 V(rad/s)2 Nse, Npe 5 & 8 Ŵ 0.08 Table 1. Simulation results. Parameter Value Pmax 335w vpv at Pmax 37.65 V ipv at Pmax 8.90 A voc 45.90 V Isc 9.40 A vs 220 V fs 50 kHz vb 48 V ib 25A Cin 2.364 μF L 788 μH ap = 1 RiCin 100 × 103 rad/s am 8.17 × 103 (rad/s)2 bp = 1 LCin 5.368 × 108 (rad/s)2 bm 1.67 × 107 (rad/s)2 kp = vo LCin 257.664 × 108 (rad/s)2 km 5.75 × 108 V(rad/s)2 Nse, Npe 5 & 8 Ŵ 0.08 Table 1. Simulation results. Figure 7. Performance of PFC: (a,b) vs and is. (c,d) Inductor currents iLp, iLn. Figure 7. Performance of PFC: (a,b) vs and is. (c,d) Inductor currents iLp, iLn. For a number of applications in the EV ecosystem and the charging infrastructure, this bidirectional func- tionality is crucial. Circulating currents develop when a DC–DC converter operates outside of its nominal volt- age conversion ratio. As a result of applying a ZVS for DC–DC converter to mitigate circulating currents, the trapezoidal current seen in Fig. 11. Figure 12a,b depict total system fed to the 48 V/100 Ah Battery. Figure 12a shows that the Vbchar is 51.93 V, suggesting that the vb will be raised to 51.93 V while charging. PV + AC–DC + PFC + DC–DC fed to EVBl Additionally, the PV will be connected to the PFC converter’s DC connection and will be able to run MPPTs of 1000 w/m2 and PV panel outputs, as indicated in Figs. 9 and 10. Depending on how the panel circuit is con- figured, the ipv and vpv are reduced and increased until they reach the final steady state value shown in Table 1 as indicated in Fig. 10, can generate a maximum power of 4.5 kW. An essential part of EV charging stations that facilitates effective power transfer between the EVB and the grid is a bidirectional DC–DC converter. It functions as a connection point for various DC voltage sources and loads, enabling power to flow in both ways. AC AC – DC + PFC Converter stage DC – DC Converter + M-LRMRAC technique T/F iLLV iLMV is vs HV DC Bus LV DC Bus DC-DC Converter PV Panel Battery Figure 6. Detailed description of PV with PFC fed to EVB. LV DC Bus HV DC Bus AC – DC + PFC Converter stage PV Panel Figure 6. Detailed description of PV with PFC fed to EVB. Figure 6. Detailed description of PV with PFC fed to EVB. https://doi.org/10.1038/s41598-024-53214-w https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| scientificreports/ For a number of applications in the EV ecosystem and the charging infrastructure, this bidirectional func- tionality is crucial. Circulating currents develop when a DC–DC converter operates outside of its nominal volt- i i A l f l i ZVS f DC DC i i i l i h Table 1. Simulation results. Parameter Value Pmax 335w vpv at Pmax 37.65 V ipv at Pmax 8.90 A voc 45.90 V Isc 9.40 A vs 220 V fs 50 kHz vb 48 V ib 25A Cin 2.364 μF L 788 μH ap = 1 RiCin 100 × 103 rad/s am 8.17 × 103 (rad/s)2 bp = 1 LCin 5.368 × 108 (rad/s)2 bm 1.67 × 107 (rad/s)2 kp = vo LCin 257.664 × 108 (rad/s)2 km 5.75 × 108 V(rad/s)2 Nse, Npe 5 & 8 Ŵ 0.08 Figure 7. Performance of PFC: (a,b) vs and is. (c,d) Inductor currents iLp, iLn. www.nature.com/scientificreports/ Table 1. Simulation results. PV + AC–DC + PFC + DC–DC fed to EVBl EVB is capable of being charged at a maximum of 25.06 A, in accordance with an Ibchar value of -25.06A, verifying the expected inward current flow through this phase as indicated by the negative sign before the current measurement. https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ PV + DC–DC fed to EVB The EVB is charged by the combination of PV and DC–DC converter contribute 1.12 kW for charging. This process involves monitoring vpv, ipv and Ppv as depicted in Fig. 13a–c. Meanwhile, the EVB’s operating parameters, vbchar and ibchar, are maintained at 51.86 V and − 32.88A, respectively, indicating a maximum charging current of 32.88A. The negative sign denotes that current entered EVB, aligning with expectations illustrated in Fig. 14a,b. Figures 15 and 16 shows the variation in irradaince and temperature Figure 17 illustrates four alternative Start Vdc, Vdccap, SOC, EVB Vdccap < Vdc Ferdowsi PFC converter LV DC Bus 25% < SOC < 90% EVB Present EVB Charging Mode SOC < 60% Constant Voltage Mode SOC > 80% EVB Charged Constant Current Mode No Yes Yes No No No Yes Yes Yes n=NMV/NLV ilLV < iMV QAB DC-DC Converter No Yes Solar PV panel Define the Nse and Npe Vload = Nse Vpv Iload = Npe* Ipv Yes No Yes HV DC Bus 25% < SOC < 90% EVB Present EVB Charging Mode SOC < 60% Constant Voltage Mode SOC > 80% EVB Charged Constant Current Mode No No No Yes Yes Yes 25% < SOC < 90% EVB Present EVB Charging Mode SOC < 60% Constant Voltage Mode SOC > 80% EVB Charged Constant Current Mode No No No Yes Yes Yes No Yes AC-DC PFC + PV + DC-DC + EVB PV + DC-DC + EVB Figure 8. Flowchart of PV + AC/DC PFC + DC − DC + EVB. Vdccap < Vdc n=NMV/NL No ilLV < iMV Yes 25% < SOC < 90% Constant Current Mode SOC < 60% Constant Current Mode SOC < 60% SOC < 60% Constant Voltage Mode SOC > 80% SOC > 80% SOC > 80% EVB Charged EVB Charged EVB Charged Figure 8. Flowchart of PV + AC/DC PFC + DC − DC + EVB. PV + DC–DC fed to EVBh PV + DC–DC fed to EVB The EVB is charged by the combination of PV and DC–DC converter contribute 1.12 kW for charging. This process involves monitoring vpv, ipv and Ppv as depicted in Fig. 13a–c. Meanwhile, the EVB’s operating parameters, vbchar and ibchar, are maintained at 51.86 V and − 32.88A, respectively, indicating a maximum charging current of 32.88A. The negative sign denotes that current entered EVB, aligning with expectations illustrated in Fig. 14a,b. Figures 15 and 16 shows the variation in irradaince and temperature. Figure 17 illustrates four alternative states in which the intensity varies with 400/m2, 600/m2, 800/m2, and 1000/m2. The temperature varies with 25 degrees and the PV outputs are vpv, ipv, and ppv. 25 °C, 35 °C, 45 °C, 55 °C. All of these were fed to three EVB. Regardless of variances, each battery displays ibchar of -32.88A and vbchar of 51.86 V. The negative sign before the current measurement means that the current is flowing into the battery. PV DC DC fed to EVB The EVB is charged by the combination of PV and DC–DC converter contribute 1.12 kW for charging. This process involves monitoring vpv, ipv and Ppv as depicted in Fig. 13a–c. Meanwhile, the EVB’s operating parameters, vbchar and ibchar, are maintained at 51.86 V and − 32.88A, respectively, indicating a maximum charging current of 32.88A. The negative sign denotes that current entered EVB, aligning with expectations illustrated in Fig. 14a,b. Figures 15 and 16 shows the variation in irradaince and temperature. Figure 17 illustrates four alternative states in which the intensity varies with 400/m2, 600/m2, 800/m2, and 1000/m2. The temperature varies with 25 degrees and the PV outputs are vpv, ipv, and ppv. 25 °C, 35 °C, 45 °C, 55 °C. All of these were fed to three EVB. Regardless of variances, each battery displays ibchar of -32.88A and vbchar of 51.86 V. The negative sign before the current measurement means that the current is flowing into the battery. https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ Utilizing the M-LRMRAC technique for second-order solar PV systems yields remarkably MPP attainment across varying irradiance and temperature conditions, often within fractions of a second. The convergence time for these specific parameters is detailed in Table 2. PV + DC–DC fed to EVBh T bl 2 l th t f i t t MPP t 37 65 V d 8 9A i ldi 335W th M LRMRAC th d t Figure 9. MPPT at 1000 w/m2. Figure 10. PV panel outputs. Figure 9. MPPT at 1000 w/m2. Figure 9. MPPT at 1000 w/m2. Figure 10. PV panel outputs. Utilizing the M-LRMRAC technique for second-order solar PV systems yields remarkably MPP attainment across varying irradiance and temperature conditions, often within fractions of a second. The convergence time for these specific parameters is detailed in Table 2. Table 2 reveals that for a consistent MPP at 37.65 V and 8.9A, yielding 335W, the M-LRMRAC method out- performs the P&O technique in achieving rapid MPP. For instance, under specific conditions such as 1000 w/ m2, irradiation and 25 °C temperature, the M-LRMRAC technique attains MPP within just 0.54 s, while the P&O method takes 10.1 s. A detailed comparison for the proposed system along with existing topologies present in Table3. Utilizing the M-LRMRAC technique for second-order solar PV systems yields remarkably MPP attainment across varying irradiance and temperature conditions, often within fractions of a second. The convergence time for these specific parameters is detailed in Table 2. Table 2 reveals that for a consistent MPP at 37.65 V and 8.9A, yielding 335W, the M-LRMRAC method out- performs the P&O technique in achieving rapid MPP. For instance, under specific conditions such as 1000 w/ m2, irradiation and 25 °C temperature, the M-LRMRAC technique attains MPP within just 0.54 s, while the P&O method takes 10.1 s. A detailed comparison for the proposed system along with existing topologies present in Table3. Utilizing the M-LRMRAC technique for second-order solar PV systems yields remarkably MPP attainment across varying irradiance and temperature conditions, often within fractions of a second. The convergence time for these specific parameters is detailed in Table 2. Table 2 reveals that for a consistent MPP at 37.65 V and 8.9A, yielding 335W, the M-LRMRAC method out- Table 2 reveals that for a consistent MPP at 37.65 V and 8.9A, yielding 335W, the M-LRMRAC method out- performs the P&O technique in achieving rapid MPP. For instance, under specific conditions such as 1000 w/ m2, irradiation and 25 °C temperature, the M-LRMRAC technique attains MPP within just 0.54 s, while the P&O method takes 10.1 s. PV + DC–DC fed to EVBh A detailed comparison for the proposed system along with existing topologies present in Table3. Scientific Reports \| (2024) 14:3261 \| https://doi.org/10.1038/s41598-024-53214-w entificreports/ Figure 11. DC–DC converter triangular currents. Figure 12. Battery outputs (a) vbchar (b) ibchar. www.nature.com/scientificreports/ Figure 11. DC–DC converter triangular currents. Figure 11. DC–DC converter triangular currents. Figure 11. DC–DC converter triangular currents. Figure 12. Battery outputs (a) vbchar (b) ibchar. Figure 12. Battery outputs (a) vbchar (b) ibchar. Conclusion ll h d Finally, this study presents a novel strategy for integrating photovoltaic systems with AC–DC Power Factor Cor- rection for efficient and reliable electric vehicle charging. The PFC system’s bi-directional power flow regulation optimizes resource utilization and increases EV battery capacity under variable environmental circumstances. The Modified Lyapunov-Based Robust Model Reference Adaptive Controller (M-LRMRAC) implementation for real-time Maximum Power Point Tracking (MPPT) improves MPP convergence speed, exceeding traditional methods such as Perturb and Observe (P&O). Furthermore, the benefits of the suggested approach go beyond efficient MPPT. The EV charging process shows a significant improvement in power quality with Unity Power Factor (UPF) operating and reduced Total Harmonic Distortion (THD). This holistic strategy is in line with the goals of efficient energy use and sustainable transportation networks. PV system integration with AC–DC PFC, combined with the M-LRMRAC method, represents an appealing possibility for expanding clean and efficient EV charging technology. This study contributes to the realization of a cleaner and more sustainable transportation landscape, so assisting the global shift towards lower carbon emissions and better energy efficiency. https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| ientificreports/ Since a result of this study’s solid foundation for effective EV charging various options for future research Figure 13. PV Outputs (a) vpv (b) ipv (c) Ppv. Figure 14. Battery outputs (a) vbchar (b) ibchar. www.nature.com/scientificreports/ Figure 13. PV Outputs (a) vpv (b) ipv (c) Ppv. Figure 14. Battery outputs (a) vbchar (b) ibchar. Figure 14. Battery outputs (a) vbchar (b) ibchar. Since a result of this study’s solid foundation for effective EV charging, various options for future research arise. Additional studies into advanced control techniques, such as AI-based controllers, could improve MPPT precision and system efficiency as a whole. Explore strategies for smoothly integrating the EV charging infra- structure with the grid, taking into account load management, smart grid technology, and bidirectional energy flow. Investigate the use of energy storage devices when combined with the proposed system to handle energy fluctuations, improve charging flexibility, and improve grid interaction. Real-world testing and validation should be carried out to evaluate the efficacy and efficacy of the proposed integrated EV charging system in practical cir- cumstances. Conduct thorough economic evaluations to determine the economic value of the suggested method in contrast to traditional EV charging alternatives. Table 2. Convergence time for different techniques with M-LRMRAC and P&O. Conclusion ll h d Look into the incorporation of Vehicle-to-Grid (V2G) features, https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ which let EVs send energy back to the grid during times of high demand. To calculate the decrease in carbon emissions brought on by the integrated PV-based EV charging strategy, do an environmental impact assessment. By exploring these topics, future research can help to improve and enhance the integration of renewable energy sources, cutting-edge control techniques, and grid interconnections within the context of EV charging, eventually leading to the development of more sustainable and effective transportation systems. Figure 15. Irradiance variation. Figure 16. Temperature Variation. Figure 15. Irradiance variation. Figure 16. Temperature Variation. which let EVs send energy back to the grid during times of high demand. To calculate the decrease in carbon emissions brought on by the integrated PV-based EV charging strategy, do an environmental impact assessment. By exploring these topics, future research can help to improve and enhance the integration of renewable energy sources, cutting-edge control techniques, and grid interconnections within the context of EV charging, eventually leading to the development of more sustainable and effective transportation systems. https://doi.org/10.1038/s41598-024-53214-w Scientific Reports \| (2024) 14:3261 \| www.nature.com/scientificreports/ Figure 17. PV outputs with Variation of irradiance and temperature. Figure 17. PV outputs with Variation of irradiance and temperature. Table 2. Convergence time for different techniques with M-LR Irradiance (W/m2) Temperature (°C) M-LRMRAC (s) P&O (s) 1000 25 0.54 10.1 1000 30 0.54 10.2 1000 35 0.54 10.3 1000 40 0.54 10.4 1000 45 0.54 10.5 800 25 0.47 11.9 800 30 0.47 11.8 800 35 0.47 11.7 800 40 0.47 11.6 800 45 0.47 11.5 600 25 0.39 13.9 600 30 0.39 13.8 600 35 0.39 13.7 600 40 0.39 13.6 600 45 0.39 13.5 500 25 0.29 14.9 500 30 0.29 14.8 500 35 0.29 14.7 500 40 0.29 14.6 500 45 0.29 14.5 400 25 0.09 15.9 400 30 0.09 15.8 400 35 0.09 15.7 400 40 0.09 15.6 400 45 0.09 15.5 Table 2. Convergence time for different techniques with M-LRMRAC and P&O. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 3. Comparison of different topologies with the proposed system. References 12 50 53 Proposed System Integration EV integrated converter EV charging station with solar assistance at level 2 DG, grid-based CS, solar PV, and battery storage MPPT PV system with PFC and DC–DC for EV charging Power Sources Utility grid and solar PV Solar PV Solar PV, Battery, Diesel Genera- tor, Grid Utility grid and solar PV Converter Types Isolated SEPIC for charging, Boost, Buck Buck Converter for charging SEPIC, Boost, Buck Boost Converter for MPPT with PFC and DC–DC for EV charging MPPT Technique P & O P & O P & O M-LRMRAC Tracking Speed (MPP Capture Time) Not specified Not specified Not specified 0.54 s MPPT under dynamic condition of PV Oscillation present at MPP Oscillation present at MPP Oscillation present at MPP Oscillation is absent at MPP Table 3. Comparison of different topologies with the proposed system. Table 3. Comparison of different topologies with the proposed system. Data availability Th d d d/ y The datasets used and/or analysed during the current study available from the corresponding author on reason- able request. Received: 26 December 2023; Accepted: 30 January 2024 References Limited sensing and deep data mining: A new exploration of developing city-wide parking guidance systems. IEEE Intell. Transp. Syst. Mag. 14(1), 198–215. https://doi.org/10.1109/MITS.2020.2970185 (2022).fi g g yst. Mag. 14(1), 198–215. https://doi.org/10.1109/MITS.2020.2970 p y g g 14. Xu, J., Guo, K. & Sun, P. Z. H. Driving performance under violations of traffic rules: Novice vs. experienced drivers. IEEE T Intell. Veh. 7(4), 908–917. https://doi.org/10.1109/TIV.2022.3200592 (2022). p g 5. Wang, H., Wu, X., Zheng, X. & Yuan, X. Model predictive current control of nine-phase open-end winding PMSMs with an online virtual vector synthesis strategy. IEEE Trans. Ind. Electron. 70(3), 2199–2208. https://doi.org/10.1109/TIE.2022.3174241 (2023). y gy p g 16. 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https://openalex.org/W2024321534	https://sapientia.ualg.pt/bitstream/10400.1/11819/1/fbi073.pdf	English	null	Correct diagnosis of early zoeal stages of Athanas nitescens (Leach, 1814) (Decapoda, Caridea, Alpheidae) using laboratory-raised larvae	Journal of plankton research	2,005	cc-by	3,461	Correct diagnosis of early zoeal stages of Athanas nitescens (Leach, 1814) (Decapoda, Caridea, Alpheidae) using laboratory-raised larvae Correct diagnosis of early zoeal stages of Athanas nitescens (Leach, 1814) (Decapoda, Caridea, Alpheidae) using laboratory-raised larvae Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 CA´ TIA BARTILOTTI1, RICARDO CALADO2 AND ANTONINA DOS SANTOS1 1INSTITUTO NACIONAL DE INVESTIGAC¸ A˜ O AGRA´ RIA E PESCAS, IPIMAR, AVENIDA DE BRASI´LIA, S/N 1449-006 LISBOA, PORTUGAL AND 2CCMAR, UNIVERSIDADE DO ALGARVE, CAMPUS GAMBELAS, 8005-139 FARO, PORTUGAL CORRESPONDING AUTHOR: cbartilotti@ipimar pt CA´ TIA BARTILOTTI1, RICARDO CALADO2 AND ANTONINA DOS SANTOS1 1INSTITUTO NACIONAL DE INVESTIGAC¸ A˜ O AGRA´ RIA E PESCAS, IPIMAR, AVENIDA DE BRASI´LIA, S/N 1449-006 LISBOA, PORTUGAL AND 2CCMAR, UNIVERSIDADE DO ALGARVE, CAMPUS GAMBELAS, 8005-139 FARO, PORTUGAL CORRESPONDING AUTHOR: cbartilotti@ipimar.pt CA´ TIA BARTILOTTI1, RICARDO CALADO2 AND ANTONINA DOS SANTOS1 1INSTITUTO NACIONAL DE INVESTIGAC¸ A˜ O AGRA´ RIA E PESCAS, IPIMAR, AVENIDA DE BRASI´LIA, S/N 1449-006 LISBOA, PORTUGAL AND 2CCMAR, UNIVERSIDADE DO ALGARVE, CAMPUS GAMBELAS, 8005-139 FARO, PORTUGAL Received April 27, 2005; accepted in principle August 30, 2005; accepted for publication September 14, 2005; published online September 27, 2005 Communicating editor: K.J. Flynn The morphology of the ﬁrst two larval stages of Athanas nitescens (Leach, 1814), reared under laboratory conditions, is redescribed. The present data are compared with previous works, since a clariﬁcation of the morphological characters of the ﬁrst two larval stages of A. nitescens is needed, in order to avoid misidentiﬁcation of these stages in the future. doi:10.1093/plankt/fbi073, available online at www.plankt.oxfordjournals.org The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org. SHORT COMMUNICATION Correct diagnosis of early zoeal stages of Athanas nitescens (Leach, 1814) (Decapoda, Caridea, Alpheidae) using laboratory-raised larvae CA´ TIA BARTILOTTI1, RICARDO CALADO2 AND ANTONINA DOS SANTOS1 1INSTITUTO NACIONAL DE INVESTIGAC¸ A˜ O AGRA´ RIA E PESCAS, IPIMAR, AVENIDA DE BRASI´LIA, S/N 1449-006 LISBOA, PORTUGAL AND 2CCMAR, UNIVERSIDADE DO ALGARVE, CAMPUS GAMBELAS, 8005-139 FARO, PORTUGAL *CORRESPONDING AUTHOR: cbartilotti@ipimar.pt Received April 27, 2005; accepted in principle August 30, 2005; accepted for publication September 14, 2005; published online September 27, 2005 Communicating editor: K.J. Flynn The morphology of the ﬁrst two larval stages of Athanas nitescens (Leach, 1814), reared under laboratory conditions, is redescribed. The present data are compared with previous works, since a clariﬁcation of the morphological characters of the ﬁrst two larval stages of A. nitescens is needed, in order to avoid misidentiﬁcation of these stages in the future. INTRODUCTION Athanas nitescens (Leach, 1814) is a widely distributed species, occurring from southern Scandinavia to Congo, Madeira, Canaries and Cape Verde Islands and the Mediterranean Sea (d’Udekem d’Acoz, 1999). Although several authors have pointed to the existence of morphological variations between Atlantic and Medi- terranean specimens (e.g. Nouvel, 1941; Holthuis, 1951; Holthuis and Gottlieb, 1958), a study analysing morpho- logical and genetic differences is still missing (d’Udekem d’Acoz, 1999). S l l l t f A it (L h 1814) h A. nitescens stating that the larval stage ﬁgured by Sars as the ﬁrst therefore corresponds to the third one (Webb, 1921). Unfortunately, Webb provided no illustrations of these early larval stages (Webb, 1921). Later, Lebour hatched the larvae of A. nitescens in the laboratory and noticed that the newly hatched larvae had some unex- pected larval characters, namely a short rostrum and stalked eyes, and that the next larval stage was consid- ered as a ‘typical third stage’ (Lebour, 1932). Taking into account Webb’s (Webb, 1921) previous work, Lebour considered that the ‘normal second stage had been ki d’ (L b 1932) B id A it l l t di JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 11 j PAGES 1189–1194 j 2005 JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 11 j PAGES 1189–1194 j 2005 JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 11 j PAGES 1189–1194 j 2005 A. nitescens hatched under laboratory conditions and to compare them with previous works, since the clariﬁca- tion of the morphological characters of these larval stages will avoid misidentiﬁcation of these stages in the future. A. nitescens hatched under laboratory conditions and to compare them with previous works, since the clariﬁca- tion of the morphological characters of these larval stages will avoid misidentiﬁcation of these stages in the future. Maxilla (Fig. 1F): Coxal endite unilobed, with three setae; basial endite unilobed, with three setae; endopod unsegmented with four marginal setae and minute spines sparsely distributed; exopod with four marginal plumose setae. First maxilliped (Fig. 1G): Coxa and basis without setae; endopod ﬁve segmented with one apical seta on distal segment; exopod three segmented with 1,1,4 plumose distal setae. Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 RESULTS AND DISCUSSION Five A. nitescens ovigerous females were collected dur- ing September 2001 using baited traps at Cape Raso (38420 N; 09290 W), 30 km west of Lisbon, Portugal. The females were kept in the laboratory individually in 2000 mL beakers provided with aeration, at 20C and in darkness until hatching. Thirty larvae from each female were reared individually in small plastic containers (20 mL each) and were fed with the microalgae Nannochloropsis minuta, supplied at a ﬁnal density of 50 106 cellL–1. Ten larvae, from each female, at the ﬁrst and second zoeal stage were ran- domly sampled, ﬁxed with 4% formalin and preserved in 70% ethanol. Second maxilliped (Fig. 1H): Basis without setae; endopod four segmented with 0,0,1,2+1 setae; exopod two segmented, with 1,4 distal plumose setae. Third maxilliped (Fig. 1I): Basis without setae; endo- pod four segmented, with 0,0,2,3 setae; exopod three segmented, with 1,1,4 plumose terminal setae. First pereiopod (Fig. 1J): Biramous bud. Second to fourth pereiopods (Fig. 1A): Absent. Second to fourth pereiopods (Fig. 1A): Absent. Fifth pereiopod (Fig. 1K): Uniramous long bud reaching the ﬁrst maxilliped. Abdomen (Fig. 1A): Six abdominal somites without any spines; the sixth longer and fused with telson; anal spine absent. Drawings and measurements were made with the aid of a camera lucida on a binocular Wild M8. Setal observations and drawings were made using a Zeiss microscope with camera lucida. Setal counts and other morphological features are described according to Clark et al. (Clark et al., 1998). The spent females and larval stages have been deposited in the Instituto Nacional de Investigac¸a˜o Agra´ria e Pescas (IPIMAR) in Lisbon, Por- tugal (number IPIMAR/A/An/11. 2001). Pleopods (Fig. 1A): Absent. Pleopods (Fig. 1A): Absent. Uropods (Fig. 1A): Absent. Uropods (Fig. 1A): Absent. Telson (Fig. 1L): Narrow, with very small median cleft and 7 + 7 processes posteriorly, being the innermost pair very small. Telson (Fig. 1L): Narrow, with very small median cleft and 7 + 7 processes posteriorly, being the innermost pair very small. Zoea II Measurements: TL = 1.87–1.94 mm; CL = 0.55–0.60 mm. Carapace (Fig. 2A): Unchanged. The general morphological features of A. nitescens ﬁrst and second larval stage are as follows: Antennule (Fig. 2B): Peduncle two segmented, with one small plumose seta on inner margin and two plu- mose setae distally; second segment with two distal long setae on ventral side, and a lobe with four small setae on dorsal side; otherwise unchanged. INTRODUCTION A. nitescens stating that the larval stage ﬁgured by Sars as the ﬁrst therefore corresponds to the third one (Webb, 1921). Unfortunately, Webb provided no illustrations of these early larval stages (Webb, 1921). Later, Lebour hatched the larvae of A. nitescens in the laboratory and noticed that the newly hatched larvae had some unex- pected larval characters, namely a short rostrum and stalked eyes, and that the next larval stage was consid- ered as a ‘typical third stage’ (Lebour, 1932). Taking into account Webb’s (Webb, 1921) previous work, Lebour considered that the ‘normal second stage had been skipped’ (Lebour, 1932). Besides A. nitescens larval studies, and although the genus Athanas is spread over the world, only the early larval stages of four other species are known: Athanas djiboutensis by Gurney (Gurney, 1927), Athanas dimorphus by Gurney and Bhuti et al. (Gurney, 1927; Bhuti et al., 1977), and Athanas japonicus and Athanas parvus by Yang (Yang, 2003a,b). Athanas nitescens (Leach, 1814) is a widely distributed species, occurring from southern Scandinavia to Congo, Madeira, Canaries and Cape Verde Islands and the Mediterranean Sea (d’Udekem d’Acoz, 1999). Although several authors have pointed to the existence of morphological variations between Atlantic and Medi- terranean specimens (e.g. Nouvel, 1941; Holthuis, 1951; Holthuis and Gottlieb, 1958), a study analysing morpho- logical and genetic differences is still missing (d’Udekem d’Acoz, 1999). Several larval stages of A. nitescens (Leach, 1814) have been ﬁrst described in some detail, from plankton sam- ples, by Sars (Sars, 1906). Sars noted that the ﬁrst larval stage of this species presented morphological characters generally attributed to a more advanced caridean larval stage (e.g. telson separated from the sixth abdominal somite and the presence of uropods) (Sars, 1906). In 1921, Webb described, from plankton samples, what she considered to be the ﬁrst two zoeal stages of The purposes of the present work are to present a standardized description of the two ﬁrst zoeal stages of JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 11 j PAGES 1189–1194 j 2005 Zoea I Measurements: TL = 1.73–1.82 mm; CL = 0.55–0.58 mm. Carapace (Fig. 1A): Smooth, with anterior region broad, displaying a spine at anterior angle; pointed ros- trum, not reaching the end of the antennule peduncle; eyes large and stalked. Antenna (Fig. 2C): Protopod without setae; exopod with one distal segment, with one plumose setae on outer side and 10 plumose setae on inner side, plus a simple small seta on apex; otherwise unchanged. Antennule (Fig. 1B): Peduncle two segmented, with 2,2 distal setae; exopod with two thick aesthetascs and two small setae terminally; endopod smaller, with one long plumose terminal seta. Mandibles (Fig. 2D): Incisor process smaller than molar process; palp absent; right mandible with a strong tooth in median part. Antenna (Fig. 1C): Protopod with a small spine on the inner posterior side; endopod with one apical seta; exopod with three distal segments, with nine plumose setae on inner side and two plumose setae on outer side, plus a simple small seta on apex. Maxillule (Fig. 2E): Coxal endite with ﬁve setae; otherwise unchanged. Maxilla (Fig. 2F): Coxal endite unilobed, with two setae; basial endite unilobed, with three setae; unseg- mented bilobed endopod with six marginal setae arranged as ﬁgured; exopod with ﬁve marginal plumose setae. Mandibles (Fig. 1D): Incisor process smaller than molar process; palp absent. Maxillule (Fig. 1E): Coxal endite with four setae; basial endite with four setae; endopod unsegmented with two distal setae; exopod absent. First maxilliped (Fig. 2G): Basis without setae; endo- pod four segmented with one sub-apical seta and one 1190 C. BARTILOTTI ETAL. j EARLY ZOEAL STAGES OF ATHANAS NITESCENS irst zoeal stage of Athanas nitescens (Leach, 1814). A, lateral view; B, antennule; C, antenna; D, mandibles; E, maxillule; F, maxilla; G, ﬁrst H, second maxilliped; I, third maxilliped; J, ﬁrst pereiopod; K, ﬁfth pereiopod; L, telson. Scale bars: A–D and G–L, 100 mm; E–F, 10 mm. Downloaded from https://academic.oup.com/plankt/article abstract/27/11/1189/1576264 by B On Consortium Portugal user on 04 Jun Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 cle-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 Fig. 1. First zoeal stage of Athanas nitescens (Leach, 1814). A, lateral view; B, antennule; C, antenna; D, mandibles; E, maxillule; F, maxilla; G, ﬁrst maxilliped; H, second maxilliped; I, third maxilliped; J, ﬁrst pereiopod; K, ﬁfth pereiopod; L, telson. C. BARTILOTTI ETAL. j EARLY ZOEAL STAGES OF ATHANAS NITESCENS A. nitescens described by Sars (Sars, 1906) corresponds to the second zoeal stage of the present work. We agree with Sars (Sars, 1906) when he suggests that he may have skipped the ﬁrst stage (saying that he has not witnessed its escape from the ova). Lebour (Lebour, 1932) considered Sars’s (Sars, 1906) ﬁrst larva as the third due to its resemblance with the stage she consid- ered as the third, believing that she skipped the typical second stage in her study. displayed are not commonly found in the ﬁrst zoeal stage of this genus. The ﬁrst zoea of A. nitescens already pre- sents a short rostrum and stalked eyes rather than the usual sessile eyes of other Athanas species ﬁrst larval stage (Gurney, 1927, 1938; Bhuti et al., 1977; Yang, 2003a,b). Another uncommon feature of the ﬁrst zoeal stage is the antennular peduncle segmented when in all other Atha- nas larvae it is still unsegmented. The second larval stage of this species also presents some more advanced mor- phological characters, namely the telson already sepa- rated from the sixth abdominal somite and the presence of uropods, which are only found in the third zoeal stage of other Athanas species. Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 The thought that the larvae described by Webb (Webb, 1921) actually belongs to the species A. nitescens, and Lebour’s (Lebour, 1932) assumption on the validity of Webb’s laboratory study on A. nitescens larvae, could have led researchers working with plankton samples to consider the ﬁrst and second zoeal stages of this species as the second and third stages, respectively. This mis- identiﬁcation of larval stages is probably the reason of the absence of the ﬁrst larval stage of this species from plankton samples in European waters (e.g. see Barnich, 1996). Although the present larvae were not reared until metamorphosis to megalopa, it is reasonable to assume that the larval development of A. nitescens could pass through eight stages instead of nine zoeal stages as gen- erally considered (e.g. Gonza´lez-Gordillo et al., 2001). Therefore Sars (Sars, 1906) Fig. 6, Plate 1 probably represents stage 3, Fig. 7 represents stage 7, and Fig. 5 is probably illustrating the eighth and last larval stage changing to a young form. These uncommon characters were also pointed out by Lebour’s description of the ﬁrst zoeal stages of A. nitescens (Lebour, 1932). ACKNOWLEDGEMENTS The authors thank Fundac¸a˜o para Cieˆncia e Tecnologia (scholarship SFRH/BD/16695/2004). The authors thank Fundac¸a˜o para Cieˆncia e Tecnologia (scholarship SFRH/BD/16695/2004). C. BARTILOTTI ETAL. j EARLY ZOEAL STAGES OF ATHANAS NITESCENS Her description agrees with the present one, even in the presence of stalked eyes, only differing in the shape of the telson (ﬁgured as more triangular). Nevertheless, as previously mentioned, Lebour (Lebour, 1932) considered that her laboratory results did not correspond to the correct larval series of this species in the plankton. According to Lebour (Lebour, 1932), this might be due to laboratory artefacts, while in Sars description (Sars, 1906) it could be a possible abbrevia- tion in the development of A. nitescens in the Christiania Fjord where his specimens were collected. These conclu- sions were made based on Webb’s (Webb, 1921) work. Webb’s (Webb, 1921) larval description refers to the two earliest larval stages that should appear before the ﬁrst larval stage ﬁgured by Sars (Webb, 1921). Since Webb was referring to a ‘normal’ caridean ﬁrst larval stage (Webb, 1921), it was assumed by Lebour that the eyes described were sessile and not stalked (Lebour, 1932). Besides this feature, we also noticed that the antennule protopod described by Webb (Webb, 1921) is a simple unjointed process, which in the present description is two segmented. In our larvae, the telson is narrow and presents a very small median cleft, while the one described by Webb (Webb, 1921) has the shape of a ﬂattened triangular swimming plate. The mandible palp is absent in our description and all maxillipeds present four long plumose setae on the exopod, rather than two or three setae. All these differences lead us to conclude that the larvae described by Webb (Webb, 1921) are not A. nitescens larvae but probably another caridean larvae common in Plymouth area. Webb’s (Webb, 1921) larvae were collected from the plankton and the actual parental species of those larvae remains unknown, since no larvae were reared to the postlarval stage. Zoea I Scale bars: A–D and G–L, 100 mm; E–F, 10 mm. apical seta on distal segment; exopod three segmented with 1,1,4 plumose distal setae. exopod three segmented, with 1,1,4 plumose terminal setae. Second maxilliped (Fig. 2H): Basis with two simple setae; exopod three segmented, with 1,1,4 distal plumose setae; otherwise unchanged. First pereiopod (Fig. 2J): Basis with one simple seta; endopod four segmented, with 0,0,2,2 (one subterminal and one terminal) setae; exopod two segmented, with 1,4 plumose terminal setae. Third maxilliped (Fig. 2I): Basis with one seta; endopod four segmented, with 0,0,2,3 setae; Second to fourth pereiopods (Fig. 2A): Absent. 1191 JOURNAL OF PLANKTON RESEARCH j VOLUME 27 j NUMBER 11 j PAGES 1189–1194 j 2005 2. Second zoeal stage of Athanas nitescens (Leach, 1814). A, lateral view; B, antennule; C, antenna; D, mandibles; E, maxillule; F, maxilla; rst maxilliped; H, second maxilliped; I, third maxilliped; J, ﬁrst pereiopod; K, ﬁfth pereiopod; L, telson and uropods. Scale bars: A–L, mm. Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 J Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 kt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 Fig. 2. Second zoeal stage of Athanas nitescens (Leach, 1814). A, lateral view; B, antennule; C, antenna; D, mandibles; E, maxillule; F, maxilla; G, ﬁrst maxilliped; H, second maxilliped; I, third maxilliped; J, ﬁrst pereiopod; K, ﬁfth pereiopod; L, telson and uropods. Scale bars: A–L, 100 mm. Telson (Fig. 2L): More rectangular, with an almost imperceptible median cleft. Fifth pereiopod (Fig. 2K): Five segmented, with the last segment shaped as a long and slender stylet, displaying a serrated extremity (as ﬁgured). Telson (Fig. 2L): More rectangular, with an almost imperceptible median cleft. Athanas nitescens ﬁrst larval stage presents the typical form of the genus, namely rounded eyes, telson rather narrow, slightly indented, and body double bent with dorsal connection between the carapace and the abdo- men making a 90 angle. However, some of the features Pleopods (Fig. 2A): Absent. Uropods (Fig. 2L): Biramous; exopods well developed not reaching the end of telson, with six plumose setae; endopod small without any setae. 1192 REFERENCES Barnich, R. (1996) The larvae of the Crustacea Decapoda (Excl. Brachyura) in the plankton of the French Mediterranean coast (Identiﬁcation Keys and Systematic Review). PhD Dissertation. Cuvillier Verlag, Go¨ttingen, Germany, 189 pp. Bhuti, G. S., Shenoy, S. and Sankolli, K. N. (1977) Laboratory reared alpheid larvae of the genera Automate, Athanas and Synalpheus (Crus- tacea Decapoda, Alpheidae). In Proceedings of the Symposium on Warm Water Zooplankton. Spec. Natl. Inst. Oceanogr., Goa, India, pp. 588–600. Clark, P. 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(1906) Postembryonal development of Athanas nitescens, Leach. Arch. Math. Nat., 27, 1–29. d’Udekem d’Acoz, C. (1999) Inventaire et distribution des crustace´s de´capodes de l’Atlantique nord-oriental, de la Me´diterrane´e et des eaux continentales adjacentes au nord de 25N. Collection Patrimoines Naturels (M.N.H.N./S.P.N.), 40, 1–383. Holthuis, L. B. (1951) The caridean Crustacea of tropical West Africa. Atlantide Report, 2, 7–187. Holthuis, L. B. and Gottlieb, E. (1958) An annotated list of the Decapod Crustacea of the Mediterranean coast of Israel, with an appendix listing the Decapoda of the eastern Mediterranean. Bull. Res. Counc. Isr., 7B, 1–126. Webb, G. (1921) The larvae of the Decapoda Macrura and Anomura of Plymouth. J. Mar. Biol. Ass. U. K., 12, 385–425. Downloaded from https://academic.oup.com/plankt/article-abstract/27/11/1189/1576264 by B-On Consortium Portugal user on 04 June 2019 Yang, H. J. 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https://openalex.org/W2937376639	https://bibliotecadigital.ipb.pt/bitstream/10198/20075/1/142.pdf	English	null	The male and female complete mitochondrial genomes of the threatened freshwater pearl mussel <i>Margaritifera margaritifera</i> (Linnaeus, 1758) (Bivalvia: Margaritiferidae)	Mitochondrial DNA. Part B. Resources	2,019	cc-by	3,681	Mitochondrial DNA Part B Resources ISSN: (Print) 2380-2359 (Online) Journal homepage: https://www.tandfonline.com/loi/tmdn20 Mitochondrial DNA Part B CONTACT Andre Gomes-dos-Santos andrepousa64@gmail.com CIIMAR/CIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros de Leix~oes. Av. General Norton De Matos s/n, 4450-208 Matosinhos, Portugal 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The male and female complete mitochondrial genomes of the threatened freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) (Bivalvia: Margaritiferidae) Andre Gomes-dos-Santosa,n , Elsa Froufea , Rafaela Amarob, Paz Ondinab, Sophie Bretonc, Davide Guerrac, David C. Aldridged, Ivan N. Bolotove , Ilya V. Vikhreve , Han Ming Ganf, Duarte V. Gonc¸alvesa, Arthur E. Bogang , Ronaldo Sousah , Donald Stewarti , Amılcar Teixeiraj, Simone Varandask , David Zanattal and Manuel Lopes-Limaa,m aCIIMAR/CIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Matosinhos, Portugal; bDepartment of Zoology, Genetics, and Physical Anthropology, Faculty of Veterinary Science, University of Santiago de Compostela, Lugo, Spain; cDepartement de Sciences Biologiques, Universite de Montreal, Montreal, Canada; dDepartment of Zoology, University of Cambridge, Cambridge, United Kingdom; eFederal Center for Integrated Arctic Research of the Russian Academy of Sciences; fDeakin Genomics Centre School of Life and Environmental Sciences, Deakin University, Geelong, Australia; gResearch Laboratory, North Carolina Museum of Natural Sciences, Raleigh, NC, USA; hCBMA – Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Braga, Portugal; iDepartment of Biology, Acadia University, Wolfville, Canada; jCentro de Investigac¸~ao de Montanha (CIMO), Instituto Politecnico de Braganc¸a, Braganc¸a, Portugal; kCITAB-UTAD – Centre for Research and Technology of Agro-Environment and Biological Sciences, University of Tras-os-Montes and Alto Douro, Vila Real, Portugal; lBiology Department, Institute for Great Lakes Research, Central Michigan University, Mount Pleasant, MI, USA; mCIBIO – Centro de Investigac¸~ao em Biodiversidade e Recursos Geneticos, InBio Laboratorio Associado, Universidade do Porto, Agrario de Vair~ao, Portugal; nDepartment of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre 1021/1055, Porto, Portugal The male and female complete mitochondrial genomes of the threatened freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) (Bivalvia: Margaritiferidae) André Gomes-dos-Santos, Elsa Froufe, Rafaela Amaro, Paz Ondina, Sophie Breton, Davide Guerra, David C. Aldridge, Ivan N. Bolotov, Ilya V. Vikhrev, Han Ming Gan, Duarte V. Gonçalves, Arthur E. Bogan, Ronaldo Sousa, Donald Stewart, Amílcar Teixeira, Simone Varandas, David Zanatta & Manuel Lopes- Lima To cite this article: André Gomes-dos-Santos, Elsa Froufe, Rafaela Amaro, Paz Ondina, Sophie Breton, Davide Guerra, David C. Aldridge, Ivan N. Bolotov, Ilya V. Vikhrev, Han Ming Gan, Duarte V. Gonçalves, Arthur E. Bogan, Ronaldo Sousa, Donald Stewart, Amílcar Teixeira, Simone Varandas, David Zanatta & Manuel Lopes-Lima (2019) The male and female complete mitochondrial genomes of the threatened freshwater pearl mussel Margaritiferamargaritifera (Linnaeus, 1758) (Bivalvia: Margaritiferidae), Mitochondrial DNA Part B, 4:1, 1417-1420, DOI: 10.1080/23802359.2019.1598794 p g © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 08 Apr 2019. Submit your article to this journal Article views: 316 View related articles View Crossmark data Citing articles: 1 View citing articles MITOCHONDRIAL DNA PART B 2019, VOL. 4, NO. 1, 1417–1420 https://doi.org/10.1080/23802359.2019.1598794 ABSTRACT ARTICLE HISTORY Received 25 January 2019 Accepted 13 March 2019 ABSTRACT The complete mitogenomes of one (M-)ale (North America), one Hermaphroditic (Europe), and two (F-)emale (North America and Europe) individuals of the freshwater pearl mussel Margaritifera margariti- fera were sequenced. The M-type and F-type (Female and Hermaphroditic) mitogenomes have 17,421 and 16,122 nucleotides, respectively. All with the same content: 13 protein-coding genes, 22 transfer RNA, two ribosomal RNA genes, and one sex-related ORF. The M-type is highly divergent (37.6% uncor- rected p-distance) from the F-type mitogenomes. North American and European F-type mitogenomes exhibit low genetic divergence (68 nt substitutions), and the Female and Hermaphroditic European mitogenomes are almost identical, and matching sex-related ORFs. KEYWORDS Doubly uniparental inheritance; mitoge- nome; Unionida gonad tissue (Hoeh et al. 1996; Amaro et al. 2017). Furthermore, many hermaphroditic species of freshwater mussels seem to have lost DUI and do not possess the M- type mitochondrial genome in their gonad tissues (Breton et al. 2011). he Margaritiferidae (Bivalvia: Unionida), comprising 16 extant pecies, represents the most threatened freshwater mussel mily (Lopes-Lima et al. 2018). Within this family, the fresh- ater pearl mussel Margaritifera margaritifera (Linnaeus, 758) is one of the most threatened species; it is subject to umerous conservation projects and is listed as Endangered obally (Geist 2010; Moorkens et al. 2018). Margaritifera mar- aritifera is a long lived species (reaching over 100 years) hat generally inhabits cool oligotrophic running waters hroughout freshwater systems of northwest Europe and ortheast North America (Geist 2010; Lopes-Lima, Sousa, t al. 2017). As noted for other margaritiferid bivalves, M. argaritifera shows an unusual mitochondrial inheritance rocess called doubly uniparental inheritance (DUI). Under UI, both males and females inherit F-type mitochondrial NA from their mothers, while males also inherit M-type itochondrial DNA from their fathers, which predominates in gonad tissue (Hoeh et al. 1996; Amaro et al. 2017). Furthermore, many hermaphroditic species of freshwater mussels seem to have lost DUI and do not possess the M- type mitochondrial genome in their gonad tissues (Breton et al. 2011). The M-type and F-type mitochondrial lineages show high levels of divergence within species of Unionida freshwater mussels and even distinct gene order arrangements (Fonseca et al. 2016; Froufe et al. 2016; Guerra et al. 2017). The Margaritiferidae also exhibit a unique M-type and F-type gene order (Lopes-Lima, Fonseca, et al. 2017). ARTICLE HISTORY Received 25 January 2019 Accepted 13 March 2019 ABSTRACT Figure 1. Margaritiferidae and Unionidae Bayesian phylogenetic tree of Male and Female mitogenomes sequences based on concatenated nucleotide sequences of 13 mitochondrial protein-coding genes and the two rRNA genes. GenBank accession numbers are behind species names, numbers at the nodes indicate the per- centage posterior probabilities and bootstrap support values. The above the branches indicate posterior probabilities and bootstrap support values > 95%. et al. 2017). The larger size of the M-type genomes is expected given the larger cox2 gene and the presence of M-specific coding regions (Breton et al. 2009). Both haplo- types have the same gene content: 13 protein-coding genes (PCGs), 22 transfer RNA (trn) and two ribosomal RNA (rrn) genes. ORFs specific to each type of mtDNA, F-orf in the F mitogenome and M-orfs in the M, are also present. Regarding the gene orientation, again, both have the same genes (four PCGs, 20 tRNAs, and two rRNAs) encoded on the heavy strand and the remaining (nine PCGs and two tRNAs) encoded on the complementary strand. The exception is the sex related ORFs, with the M-orf on the complementary strand and the F-orf on the heavy strand, located at different positions. A nucleotide alignment of the mitochondrial genomes shows that the M-type mitogenome is highly diver- gent (37.6% uncorrected p-distance) from the F-type mitoge- nomes. The F-type mitogenomes from North America and Europe exhibit a low genetic divergence (68 nt substitutions ¼ 0.04% uncorrected p-distance), with the European mitoge- nomes of the female and hermaphroditic individuals being almost identical with only 5 nt substitutions. This pattern may reflect a recent (Pleistocene) dispersal event of freshwater pearl mussels from Europe to North America or slow mtDNA substitution rates in this species (Lopes-Lima et al. 2018; Zanatta et al. 2018). The F-orfs of the European hermaphro- ditic and female individuals are identical. Secondarily herm- aphroditic species generally contain a distinct and longer F- like ORF (Breton et al. 2011). Therefore, these results seem to indicate that hermaphroditic individuals of typically dioecious species may maintain their F-type ORFs unchanged. and Europe; (2) determine and compare the gene order and content of those mitogenomes; and (3) produce phylogenetic analyses using all available F-types and M-type mitogenomes of the Margaritiferidae family. Four complete mitogenomes of M. ABSTRACT Available phylogenetic studies within the family are based on only a few markers still lacking a more robust multi-marker approach (Lopes-Lima et al. 2018). Given this background, the aims of this study are to (1) obtain the whole mitogenomes of male, female, and herm- aphroditic specimens of M. margaritifera from North America ONTACT A d G d S t d 64@ il CIIMAR/CIMAR I t di i li C t f M i d E i t l R h U i it The Margaritiferidae (Bivalvia: Unionida), comprising 16 extant species, represents the most threatened freshwater mussel family (Lopes-Lima et al. 2018). Within this family, the fresh- water pearl mussel Margaritifera margaritifera (Linnaeus, 1758) is one of the most threatened species; it is subject to numerous conservation projects and is listed as Endangered globally (Geist 2010; Moorkens et al. 2018). Margaritifera mar- garitifera is a long lived species (reaching over 100 years) that generally inhabits cool oligotrophic running waters throughout freshwater systems of northwest Europe and northeast North America (Geist 2010; Lopes-Lima, Sousa, et al. 2017). As noted for other margaritiferid bivalves, M. margaritifera shows an unusual mitochondrial inheritance process called doubly uniparental inheritance (DUI). Under DUI, both males and females inherit F-type mitochondrial DNA from their mothers, while males also inherit M-type mitochondrial DNA from their fathers, which predominates in The M-type and F-type mitochondrial lineages show high levels of divergence within species of Unionida freshwater mussels and even distinct gene order arrangements (Fonseca et al. 2016; Froufe et al. 2016; Guerra et al. 2017). The Margaritiferidae also exhibit a unique M-type and F-type gene order (Lopes-Lima, Fonseca, et al. 2017). Available phylogenetic studies within the family are based on only a few markers still lacking a more robust multi-marker approach (Lopes-Lima et al. 2018). Given this background, the aims of this study are to (1) obtain the whole mitogenomes of male, female, and herm- aphroditic specimens of M. margaritifera from North America 1418 A. GOMES-DOS-SANTOS ET AL. 1418 A. GOMES-DOS-SANTOS ET AL. Figure 1. Margaritiferidae and Unionidae Bayesian phylogenetic tree of Male and Female mitogenomes sequences based on concatenated nucleotide sequences of 13 mitochondrial protein-coding genes and the two rRNA genes. GenBank accession numbers are behind species names, numbers at the nodes indicate the per- centage posterior probabilities and bootstrap support values. The above the branches indicate posterior probabilities and bootstrap support values > 95%. References Reproductive cycle and strategy of Anodonta anatina (L., 1758): Notes on hermaphroditism. J Exp Zool A Ecol Genet Physiol. 319:378–390. Guerra D, Plazzi F, Stewart DT, Bogan AE, Hoeh WR, Breton S. 2017. Evolution of sex-dependent mtDNA transmission in freshwater mus- sels (Bivalvia: Unionida). Sci Rep. 7:1551. ( ) p Hinzmann M, Lopes -lima M, Teixeira A, Varandas S, Sousa R, Lopes A, Froufe E, Machado J. 2013. Reproductive cycle and strategy of Anodonta anatina (L., 1758): Notes on hermaphroditism. J Exp Zool A Ecol Genet Physiol. 319:378–390. ORCID Andre Gomes-dos-Santos http://orcid.org/0000-0001-9973-486 Elsa Froufe http://orcid.org/0000-0003-0262-0791 Ivan N. Bolotov http://orcid.org/0000-0002-3878-4192 Ilya V. Vikhrev http://orcid.org/0000-0002-8612-7736 Arthur E. Bogan http://orcid.org/0000-0003-4042-7706 Ronaldo Sousa http://orcid.org/0000-0002-5961-5515 Donald Stewart http://orcid.org/0000-0002-4402-1757 Simone Varandas http://orcid.org/0000-0001-5038-6085 Manuel Lopes-Lima http://orcid.org/0000-0002-2761-7962 The best obtained phylogenetic BI and ML trees revealed an identical topology (Figure 1). Both the F and M clades are div- ided into the two Unionida families, Margaritiferidae, and Unionidae. Maximum support values were obtained for all nodes with two exceptions for the relationships of Pseudunio marocanus both in the female and in the male clades (Figure 1). The phylogenies are consistent with the systematic divisions of the Margaritiferidae in four genera (Margaritifera, Cumberlandia, Pseudunio, and Gibbosula) and two subfamilies (Margaritiferinae (Margaritifera þ Cumberlandia þ Pseudunio) and Gibbosulinae (Gibbosula)) (Lopes-Lima et al. 2018). The newly sequenced M. margaritifera genomes cluster inside the Margaritifera genus in the F-type clade, being the M-type mitogenome sequence the first available for this genus, following the most recent system- atics for the family (Lopes-Lima et al. 2018). MITOCHONDRIAL DNA PART B 1419 Each chain started with a ran- domly generated tree and ran for 1 106 generations with a sampling frequency of 1 tree for every 100 generations. The resultant trees, after discarding the first 25% as burn-in, were combined in a 50% majority rule consensus tree. The final trees were rooted at the split between Male and Female hap- lotypes (based on previous studies, e.g. Huang et al. 2013). Funding This work was supported by Portuguese Foundation for Science and Technology (FCT) [grant number SFRH/BD/115728/2016 (MLL), grant number SFRH/BD/137935/2018 (AGS)]; Russian Foundation for Basic Research [grant number 18-34-20033 (IVV)]; Dawson Fellowship at St. Catharine’s College, Cambridge (DCA); Life Margal Ulla [number LIFE09 NAT/ES/000514 (RA and PO)]; COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by Portuguese Foundation for Science and Technology (FCT) through national funds [UID/Multi/04423/ 2019] under project ConBiomics: the missing approach for the Conservation of freshwater Bivalves Project, and [project number NORTE- 01-0145-FEDER-030286]; Federal Agency for Scientific Organizations under Grants [grant number 0409-2015-0143 (INB and IVV)] MITOCHONDRIAL DNA PART B 1419 single gene alignments the following GUIDANCE parameters were used: score algorithm: GUIDANCE; bootstraps replicates: 100; Sequence cut-off score: 0.0 (no sequences removed); Column cut-off score: below 0.8. The final concatenated data set included the 13 mitochondrial PCG and the 2 rrn genes for each mitogenome reaching a total length of 13,505 nt. Phylogenetic relationships were estimated by Bayesian infer- ence using MrBayes v. 3.2.6 (Ronquist et al. 2012) and Maximum Likelihood using RAxML v. 8.2.10 (Stamatakis 2014) HPC Black Box with 100 rapid bootstrap replicates and 20 ML searches at the San Diego Supercomputer Center through the CIPRES Science Gateway (https://www.phylo.org). The final alignment was partitioned in 11 subsets according to the best scheme determined using PartitionFinder v.2.1.1 (Lanfear et al. 2016). For the ML a unique GTR model was applied for each partition with corrections for gamma distri- bution. For the BI, the GTR þ G, GTR þ IþG, HKY þ G, HKY þ IþG models were used. Each chain started with a ran- domly generated tree and ran for 1 106 generations with a sampling frequency of 1 tree for every 100 generations. The resultant trees, after discarding the first 25% as burn-in, were combined in a 50% majority rule consensus tree. The final trees were rooted at the split between Male and Female hap- lotypes (based on previous studies, e.g. Huang et al. 2013). single gene alignments the following GUIDANCE parameters were used: score algorithm: GUIDANCE; bootstraps replicates: 100; Sequence cut-off score: 0.0 (no sequences removed); Column cut-off score: below 0.8. The final concatenated data set included the 13 mitochondrial PCG and the 2 rrn genes for each mitogenome reaching a total length of 13,505 nt. Phylogenetic relationships were estimated by Bayesian infer- ence using MrBayes v. 3.2.6 (Ronquist et al. 2012) and Maximum Likelihood using RAxML v. 8.2.10 (Stamatakis 2014) HPC Black Box with 100 rapid bootstrap replicates and 20 ML searches at the San Diego Supercomputer Center through the CIPRES Science Gateway (https://www.phylo.org). The final alignment was partitioned in 11 subsets according to the best scheme determined using PartitionFinder v.2.1.1 (Lanfear et al. 2016). For the ML a unique GTR model was applied for each partition with corrections for gamma distri- bution. For the BI, the GTR þ G, GTR þ IþG, HKY þ G, HKY þ IþG models were used. ABSTRACT margaritifera were sequenced: one M-type and one F-type from a North American male specimen (River Annapolis near Auburn, Canada: 45.014999, -64.856344) and two F-type from European specimens, one from a female (River Ulla near Barazon, Galicia, Spain: approximate coordinates 42.846676, -8.025244) and another from a hermaphrodite (River Tuela near Vinhais, northeast Portugal: approximate coordinates 41.862414, -6.931596). DNA samples are stored at the CIIMAR Institute Unionoid DNA and Tissue Databank (Voucher num- bers P2, MM63, 155G, and 165G). Sex was determined for all specimens under a microscope following Hinzmann et al. (2013). DNA was sheared to 500 bp using an M220 Covaris Ultrasonicator (Covaris, Woburn, MA, USA) and processed with the NEBultra Illumina library preparation kit (NEB, Ipswich, MA, USA). Sequencing was performed on the MiSeq (Illumina, San Diego, CA, USA) located at Monash University Malaysia using a run configuration of 2 250 bp. Mitogenomes were assembled from the paired-end reads and annotated using an established pipeline (Gan et al. 2014). The four mitogenomes have been deposited in the GenBank database under the accession numbers (MK421959 and MK421956; M-type and F-type, respectively for the North American specimens), and (MK421957 and MK421958; for the Spanish and Portuguese F-type European specimens). Sequence divergence (uncorrected p-distance) was assessed using MEGA7 software (Kumar et al. 2016). The length of both mitogenome types (M-type: 17,421 nt; and F-type: 16,122 nt) of M. margaritifera sequenced in this study is within the expected range for each gender-specific haplo- types within Margaritiferidae (Guerra et al. 2017; Lopes-Lima For the phylogenies, additional mitogenome sequences (M-type and F-type) available from all Margaritiferidae and three Unionidae species were downloaded from GenBank. Each gene sequence was aligned using GUIDANCE v. 1.5 (Penn et al. 2010) with the MAFFT v. 7.304 multiple sequence alignment algorithm (Katoh and Standley 2013). To build References Amaro R, Bouza C, Pardo BG, Castro J, San Miguel E, Villalba A, Lois S, Outeiro A, Ondina P. 2017. Identification of novel gender-associated mitochondrial haplotypes in Margaritifera margaritifera (Linnaeus, 1758). Zool J Linn Soc. 179:738–750. Breton S, Beaupre HD, Stewart DT, Piontkivska H, Karmakar M, Bogan AE, Blier PU, Hoeh WR. 2009. Comparative mitochondrial genomics of freshwater mussels (Bivalvia: Unionoida) with doubly uniparental inheritance of mtDNA: gender-specific open reading frames and puta- tive origins of replication. Genetics. 183:1575–1589. Breton S, Stewart DT, Shepardson S, Trdan RJ, Bogan AE, Chapman EG, Ruminas AJ, Piontkivska H, Hoeh WR. 2011. Novel protein genes in animal mtDNA: a new sex determination system in freshwater mussels (Bivalvia: Unionoida)? Mol Biol Evol. 28:1645–1659. Fonseca MM, Lopes-Lima M, Eackles MS, King TL, Froufe E. 2016. The female and male mitochondrial genomes of Unio delphinus and the phylogeny of freshwater mussels (Bivalvia: Unionida). Mitochondrial DNA B. 1:954–957. The present results confirm the usefulness of the mitoge- nomes gene arrangements as diagnostic character for the Margaritiferidae and provide additional confirmation for the sys- tematics of the family as recently proposed by Lopes-Lima et al. (2018). These results also highlight the low intraspecific genetic divergence of M. margaritifera even between specimens from the edges of distribution. Furthermore, the current study pro- vides novel information about mtDNA structure and sequence of hermaphroditic individuals of typical dioecious species pro- viding opportunities for further studies on the sex determin- ation mechanism and mtDNA evolution of freshwater bivalves. Froufe E, Gan HM, Lee YP, Carneiro J, Varandas S, Teixeira A, Zieritz A, Sousa R, Lopes-Lima M. 2016. The male and female complete mito- chondrial genome sequences of the Endangered freshwater mussel Potomida littoralis (Cuvier, 1798) (Bivalvia: Unionidae). Mitochondrial DNA A. 27:3571–3572. Gan HM, Schultz MB, Austin CM. 2014. Integrated shotgun sequencing and bioinformatics pipeline allows ultra-fast mitogenome recovery and confirms substantial gene rearrangements in Australian fresh- water crayfishes. BMC Evol Biol. 14:19. Geist J. 2010. Strategies for the conservation of endangered freshwater pearl mussels (Margaritifera margaritifera L.): a synthesis of conserva- tion genetics and ecology. Hydrobiologia. 644:69–88. tion genetics and ecology. Hydrobiologia. 644:69–88. Guerra D, Plazzi F, Stewart DT, Bogan AE, Hoeh WR, Breton S. 2017. Evolution of sex-dependent mtDNA transmission in freshwater mus- sels (Bivalvia: Unionida). Sci Rep. 7:1551. Hinzmann M, Lopes -lima M, Teixeira A, Varandas S, Sousa R, Lopes A, Froufe E, Machado J. 2013. Disclosure statement The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper. A. GOMES-DOS-SANTOS ET AL. 1420 freshwater mussels in Europe: state of the art and future challenges. Biol Rev. 92:572–607. Hoeh WR, Stewart DT, Sutherland GW, Zouros E. 1996. Multiple origins of gender-associated mitochondrial DNA lineages in bivalves (Mollusca: Bivalvia). Evol. 50:2276–2286. Lopes-Lima M, Fonseca MM, Aldridge DC, Bogan AE, Gan HM, Ghamizi M, Sousa R, Teixeira A, Varandas S, Zanatta D, et al. 2017. The first Margaritiferidae male (M-type) mitogenome: mitochondrial gene order as a potential character for determining higher-order phylogeny within Unionida (Bivalvia). J Molluscan Stud. 83:249–252. Huang XC, Rong J, Liu Y, Zhang MH, Wan Y, Ouyang S, Zhou CH, Wu XP. 2013. The complete maternally and paternally inherited mitochondrial genomes of the endangered freshwater mussel Solenaia carinatus (Bivalvia: Unionidae) and implications for Unionidae taxonomy. PLoS One. 8:e84352 Moorkens E, Cordeiro J, Seddon MB, von Proschwitz T, Woolnough D. 2018. Margaritifera margaritifera (errata version published in 2018). The IUCN Red List of Threatened Species 2018: e.T12799A128686456. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment soft- ware version 7: improvements in performance and usability. Mol Biol Evol. 30:772–780. Penn O, Privman E, Ashkenazy H, Landan G, Graur D, Pupko T. 2010. GUIDANCE: a web server for assessing alignment confidence scores. Nucleic Acids Res. 38:W23–W28. Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Mol Biol Evol. 33: 1870–1874. Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Hna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 61:539–542. Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B. 2016. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol Biol Evol. 34:772–773. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 30: 1312–1313. Lopes-Lima M, Bolotov IN, Do VT, Aldridge DC, Fonseca MM, Gan HM, Gofarov MY, Kondakov AV, Prie V, Sousa R, et al. 2018. Expansion and systematics redefinition of the most threatened freshwater mussel family, the Margaritiferidae. Mol Phylogenetics Evol. 127:98–118. Zanatta DT, Stoeckle BC, Inoue K, Paquet A, Martel AL, Kuehn R, Geist J. 2018. Disclosure statement High genetic diversity and low differentiation in North American Margaritifera margaritifera (Bivalvia: Unionida: Margaritiferidae). Biol J Linn Soc. 123:850–863. Lopes-Lima M, Sousa R, Geist J, Aldridge DC, Araujo R, Bergengren J, Bespalaya Y, Bodis E, Burlakova L, et al. 2017. Conservation status of
https://openalex.org/W4205451696	https://cadmus.eui.eu/bitstream/1814/74741/1/Manufacturing_Statelessness_2022.pdf	English	null	Manufacturing Statelessness	American journal of international law	2,022	cc-by	37,145	* Associate professor of law, University of Minnesota Law School, United States; Professor of Public International Law and Co-Director of the Academy of European Law, European University Institute in Italy. For illuminating comments and discussions, I am grateful to Jessica Clarke, Martijn Hesselink, Sarah Nouwen, Maarten Vink, and participants in workshops at Northwestern Pritzker School of Law, European University Institute, O.P. Jindal Global University, Hebrew University, and Edinburgh Law School. 1 ABSTRACT Having recently emerged from its unenviable status as the runt of international law, the phenomenon of statelessness nonetheless eludes traditional international legal instruments. Confronted with questions of nationality that typically fall within the domain of sovereignty, international and regional human rights bodies struggle to rein in the increasingly creative measures that states adopt to obscure the production and persistence of statelessness. This Article uncovers and dissects the different ways in which states manufacture statelessness not through explicitly discriminatory laws and unequal treatment, but through manipulating ostensibly neutral criteria for nationality. The Article identiﬁes three such criteria that are not traditionally considered “suspect” categories for the grant or denial of nationality: time, territory, and administrative practice. It also suggests doctrinal, policy, and strategic tools for identifying and responding to the types of statelessness that are not a collateral consequence of state failure or incompetence, but the outcome of state intentionality. Copyright © The Author(s), 2022. Published by Cambridge University Press for The American Society of International Law doi:10.1017/ajil.2022.2 Copyright © The Author(s), 2022. Published by Cambridge University Press for The American Society of International Law doi:10.1017/ajil.2022.2 1 B. TRAVEN, THE DEATH SHIP (1934). B. TRAVEN, THE DEATH SHIP (1934). INTRODUCTION The time is the 1920s, after the “Great War of Freedom.” Gerard Gales, an American sea- man and the ﬁctional protagonist of B. Traven’s sensational novel The Death Ship, is stranded in Antwerp, penniless and paperless. To be paperless is to ofﬁcially not exist. Unable to prove either citizenship or alien-ness, Gales is churned and spat out by one faceless bureaucracy after another, shunted between prisons, consulates, and police stations as he is repeatedly arrested and deported. Without a seaman’s pass or any other form of identiﬁcation, and with no means of securing legal residence or employment in any state, Gales eventually ﬁnds refuge on the only entity that will accept him, no questions asked––the “death ship” Yorikke––an arms smuggling vessel manned by a crew consisting almost entirely of stateless, homeless, or undoc- umented individuals like Gales, trapped in horriﬁcally brutal slave-like conditions on a living hell ship that is bound for destruction.1 Described as an arresting allegory of the “horrible things that can happen to a man in the cockeyed postwar world of Europe . . . a world 237 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW 238 Vol. 116:2 gone mad,”2 the narrative is no less applicable to the cockeyed world of today where a reported 3.9 million people are currently stateless.3 Unlike the statelessness on the Death Ship, which unfolds against the backdrop of war- time and post-war nationalism and scatters the dispossessed in far ﬂung territories––or even on the no-man’s land of the high seas––statelessness today is not merely, or even mostly, a product of state conﬂict.4 Nor does it involve displacement beyond the territory of the state.5 And, most importantly, statelessness is not a random act of misfortune that could have just as easily have befallen some other hapless victim. Nevertheless, the long shadow of the post-war discourse on statelessness continues to inform international legal responses to the plight of the stateless. This Article argues that the post-war topography of statelessness has been reshaped in signiﬁcant ways by statelessness as statecraft.6 This terrain calls for sophisticated litigation and policy tools to successfully navigate and challenge statelessness that is not a collateral consequence of state failure or incompetence, but the outcome of state intentionality. 2 See John Anthony West, Traven’s “Death Ship” –– Authentic, Hypnotic, and Maybe Alchemical, N.Y. TIMES (Nov. 10, 1985) (quoting Bruce Catton). 3 5 See UNHCR, HANDBOOK ON PROTECTION OF STATELESS PERSONS UNDER THE 1954 CONVENTION RELATING TO THE STATUS OF STATELESS PERSONS, para. 1 (2014) (“Most stateless persons, however, have never crossed borders and ﬁnd themselves in their ‘own country.’”). 6 4 See, e.g., UNHCR, Ending Statelessness, at https://www.unhcr.org/ending-statelessness.html (identifying var- ious causes for statelessness, including gaps in nationality laws, movement of people outside the country of their birth, state creation and border changes, and citizenship loss or deprivation). As I will argue, these only scratch the surface of the processes through which states manufacture statelessness. 5 6 There is sophisticated scholarship focusing on speciﬁc countries and the ways in which they have deliberately sought to exclude individuals who would be entitled to citizenship status, or indeed may already possess that status and be subsequently stripped of it. This Article draws from this scholarship in the various examples it puts forward to illustrate its thesis. However, most of this scholarship takes the form of in-depth historical or rich ethnographic accounts and it does not typically zoom out from individual case studies to speak to the broader phenomenon of statelessness as manufactured by impartial, neutral criteria that are considered universal prerequisites for citizen- ship. The bulk of this scholarship is also not written by legal scholars, leaving the international legal dimensions of the phenomena somewhat unexplored. For a notable recent exception, see Michelle Foster & Jade Roberts, Manufacturing Foreigners: The Law and Politics of Transforming Citizens into Migrants, in RESEARCH HANDBOOK ON THE LAW AND POLITICS OF MIGRATION 218 (Catherine Dauvergne ed., 2021) (analyzing the practice of states turning minority populations into foreigners, thus forcibly rendering them stateless, through case studies of the Dominican Republic and India). INTRODUCTION Drawing on a wide range of both familiar and more obscure country-speciﬁc cases of state- lessness, this Article uncovers and highlights how states deliberately produce statelessness not through explicitly discriminatory laws or unequal treatment, but through tampering with facially impartial criteria for access to citizenship. The Article identiﬁes three such criteria that are not traditionally considered as “suspect” classiﬁcations for the grant or denial of polit- ical membership: time, territory, and administrative practice. For example, instead of enact- ing citizenship laws that exclude individuals on the basis of ethnic origin, states achieve the same result by “zero option” rules that award citizenship based on physical presence on the territory of the state on a certain date. Members of marginalized groups who cannot furnish documentary proof of this residence may be excluded from citizenship altogether, or may be subjected to protracted administrative procedures whereby the acquisition of nationality is 2 See John Anthony West, Traven’s “Death Ship” –– Authentic, Hypnotic, and Maybe Alchemical, N.Y. TIMES (Nov. 10, 1985) (quoting Bruce Catton). 3 3 Lily Chen, Petra Nahmias & Sebastian Steinmueller, UNHCR Statistical Reporting on Statelessness, UNHCR Statistics Technical Series 2019/1 (Oct. 2019), at https://www.unhcr.org/statistics/unhcrstats/5d9e182e7/unhcr- statistical-reporting-statelessness.html. 4 See, e.g., UNHCR, Ending Statelessness, at https://www.unhcr.org/ending-statelessness.html (identifying var- ious causes for statelessness, including gaps in nationality laws, movement of people outside the country of their birth, state creation and border changes, and citizenship loss or deprivation). As I will argue, these only scratch the surface of the processes through which states manufacture statelessness. 5 See UNHCR, HANDBOOK ON PROTECTION OF STATELESS PERSONS UNDER THE 1954 CONVENTION RELATING TO THE STATUS OF STATELESS PERSONS, para. 1 (2014) (“Most stateless persons, however, have never crossed borders and ﬁnd themselves in their ‘own country.’”). 6 6 There is sophisticated scholarship focusing on speciﬁc countries and the ways in which they have deliberately sought to exclude individuals who would be entitled to citizenship status, or indeed may already possess that status and be subsequently stripped of it. This Article draws from this scholarship in the various examples it puts forward to illustrate its thesis. However, most of this scholarship takes the form of in-depth historical or rich ethnographic accounts and it does not typically zoom out from individual case studies to speak to the broader phenomenon of statelessness as manufactured by impartial, neutral criteria that are considered universal prerequisites for citizen- ship. 3 Lily Chen, Petra Nahmias & Sebastian Steinmueller, UNHCR Statistical Reporting on Statelessness, UNHCR Statistics Technical Series 2019/1 (Oct. 2019), at https://www.unhcr.org/statistics/unhcrstats/5d9e182e7/unhcr- statistical-reporting-statelessness.html. g g p g g ( y g ious causes for statelessness, including gaps in nationality laws, movement of people outside the country of their birth, state creation and border changes, and citizenship loss or deprivation). As I will argue, these only scratch the surface of the processes through which states manufacture statelessness. 5 See UNHCR, HANDBOOK ON PROTECTION OF STATELESS PERSONS UNDER THE 1954 CONVENTION RELATING TO THE STATUS OF STATELESS PERSONS, para. 1 (2014) (“Most stateless persons, however, have never crossed borders and ﬁnd themselves in their ‘own country.’”). 6 There is sophisticated scholarship focusing on speciﬁc countries and the ways in which they have deliberately sought to exclude individuals who would be entitled to citizenship status, or indeed may already possess that status and be subsequently stripped of it. This Article draws from this scholarship in the various examples it puts forward to illustrate its thesis. However, most of this scholarship takes the form of in-depth historical or rich ethnographic accounts and it does not typically zoom out from individual case studies to speak to the broader phenomenon of statelessness as manufactured by impartial, neutral criteria that are considered universal prerequisites for citizen- ship. The bulk of this scholarship is also not written by legal scholars, leaving the international legal dimensions of the phenomena somewhat unexplored. For a notable recent exception, see Michelle Foster & Jade Roberts, Manufacturing Foreigners: The Law and Politics of Transforming Citizens into Migrants, in RESEARCH HANDBOOK ON THE LAW AND POLITICS OF MIGRATION 218 (Catherine Dauvergne ed., 2021) (analyzing the practice of states turning minority populations into foreigners, thus forcibly rendering them stateless, through case studies of the 2 See John Anthony West, Traven’s “Death Ship” –– Authentic, Hypnotic, and Maybe Alchemical, N.Y. TIMES (Nov. 10, 1985) (quoting Bruce Catton). 3 Lily Chen, Petra Nahmias & Sebastian Steinmueller, UNHCR Statistical Reporting on Statelessness, UNHCR Statistics Technical Series 2019/1 (Oct. 2019), at https://www.unhcr.org/statistics/unhcrstats/5d9e182e7/unhcr- statistical-reporting-statelessness.html. 4 See, e.g., UNHCR, Ending Statelessness, at https://www.unhcr.org/ending-statelessness.html (identifying var- ious causes for statelessness, including gaps in nationality laws, movement of people outside the country of their birth, state creation and border changes, and citizenship loss or deprivation). As I will argue, these only scratch the surface of the processes through which states manufacture statelessness. 5 See UNHCR, HANDBOOK ON PROTECTION OF STATELESS PERSONS UNDER THE 1954 CONVENTION RELATING TO THE STATUS OF STATELESS PERSONS, para. 1 (2014) (“Most stateless persons, however, have never crossed borders and ﬁnd themselves in their ‘own country.’”). 6 There is sophisticated scholarship focusing on speciﬁc countries and the ways in which they have deliberately sought to exclude individuals who would be entitled to citizenship status, or indeed may already possess that status and be subsequently stripped of it. This Article draws from this scholarship in the various examples it puts forward to illustrate its thesis. However, most of this scholarship takes the form of in-depth historical or rich ethnographic accounts and it does not typically zoom out from individual case studies to speak to the broader phenomenon of statelessness as manufactured by impartial, neutral criteria that are considered universal prerequisites for citizen- ship. The bulk of this scholarship is also not written by legal scholars, leaving the international legal dimensions of the phenomena somewhat unexplored. For a notable recent exception, see Michelle Foster & Jade Roberts, Manufacturing Foreigners: The Law and Politics of Transforming Citizens into Migrants, in RESEARCH HANDBOOK ON THE LAW AND POLITICS OF MIGRATION 218 (Catherine Dauvergne ed., 2021) (analyzing the practice of states turning minority populations into foreigners, thus forcibly rendering them stateless, through case studies of the Dominican Republic and India). INTRODUCTION The bulk of this scholarship is also not written by legal scholars, leaving the international legal dimensions of the phenomena somewhat unexplored. For a notable recent exception, see Michelle Foster & Jade Roberts, Manufacturing Foreigners: The Law and Politics of Transforming Citizens into Migrants, in RESEARCH HANDBOOK ON THE LAW AND POLITICS OF MIGRATION 218 (Catherine Dauvergne ed., 2021) (analyzing the practice of states turning minority populations into foreigners, thus forcibly rendering them stateless, through case studies of the Dominican Republic and India). https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 239 MANUFACTURING STATELESSNESS contingent on the vagaries of bureaucratic decision making. This Article aims to draw the attention of international legal actors to the ways in which states practice discrimination in disguise to manufacture statelessness under the shadow of the law and to counter efforts at its legitimation. g In doing so, this Article tackles head-on the question posed by the ambitious 2014 #IBelong Campaign on statelessness by the United Nations High Commissioner for Refugees (UNHCR): what would it take to end statelessness?7 The UNHCR has called state- lessness an “easily resolvable and preventable issue”8 and has drawn a ten-point action plan to identify and resolve existing situations of statelessness and prevent future ones from occur- ring.9 However, as this Article shows, these measures are both too little, and too late. Focused as they are on either inadvertent or overtly discriminatory causes of statelessness, and on incentivizing states to adopt formal, technical solutions to statelessness, these policy prescriptions fail to recognize covert state-manufactured statelessness. Even worse, state responses to some of these international recommendations might render effective statelessness all the more invisible, in the process exacerbating rather than eliminating statelessness. p g g The Article instead capitalizes on the diffused and infrequent challenges that international and regional actors––including courts, human rights bodies, and civil society groups––are beginning to mount in response to the state’s production of statelessness to propose a bolder and more sustained international legal response. Much like the shape-shifting nature of the strategies deployed by the state to choreograph statelessness, this new legal roadmap must be sufﬁciently crafty and agile to match, and even surpass, states’ creativity. 7 UNHCR, Global Action Plan to End Statelessness, 2014–2024, at https://www.unhcr.org/ibelong/global- action-plan-2014-2024. 8 UN News, Ending Statelessness “A Matter of Political Will,” Says UN Refugee Agency Chief (Nov. 11, 2020), at https://news.un.org/en/story/2020/11/1077392. 9 Global Action Plan to End Statelessness, supra note 7. INTRODUCTION The Article thus seizes on the potential of indirect discrimination as a doctrinal tool to prevent states from circumventing the prohibition of direct discrimination against categories of individuals who have a sufﬁciently close connection to the state by targeting ostensibly “neutral” policies and practices that amount to systemic discrimination in the production of statelessness. This tool, however, can only be developed and sharpened if advocates make use of it. Strategic litigation should be part of a broad-based advocacy strategy that brings statelessness claims before different international and regional fora––including those that may not issue binding rulings––with a view to both developing international law norms and empowering domestic human rights actors. The Article also speaks to the policy dimension of combatting stateless- ness by urging international development and human rights agencies such as the UNHCR to be more targeted in advocating for legal identity documentation and registration as stateless- ness prevention and reduction measures. Recognizing that these measures can do more harm than good, the Article urges these agencies to scrutinize the legal framework and political con- ditions within which modern identity management systems are implemented to avoid per- petuating or expanding existing forms of exclusion. Rather than focusing on the utopian prospect championed by the UNHCR to end statelessness in a decade, the Article instead adopts a pragmatic approach that incrementally develops forward-looking ideas, principles, and tools that can be used by actors in plural and diverse national settings to steadily chip away at the circumstances in which statelessness can be produced. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW 240 Vol. 116:2 This Article proceeds in three Parts. Part I situates the discourse on statelessness within broader debates relating to international law’s preoccupation with the stability of the interna- tional legal order, the importance of nationality, and the inﬂuence of international human rights law on issues concerning citizenship. Part II highlights that in addition to cases of manifest ille- gality, statelessness is manufactured in more subtle, less visible ways through creative techniques that circumvent international law standards by tampering with ostensibly neutral criteria relevant to nationality. It uncovers three such criteria that masquerade as unbiased markers for citizenship acquisition and the concomitant exclusion from its beneﬁts: time,space, and documents. INTRODUCTION Part III argues that the halting efforts that international actors are beginning to take to arrest statelessness must be transformed into a bolder legal roadmap with doctrinal, litigation, and policy tools that are equal to the state’s creative capacity to manufacture statelessness. A brief conclusion follows. I. STATELESSNESS CREEP AND THE LAW This Part unpacks the instability of the international legal discourse on statelessness in order to set the stage for accurately diagnosing and responding to international law’s blind spots on statelessness. It traces how the emergence of the nation-state as the predominant form of political organization transformed statelessness from an ambivalent status that could be both empowering and disempowering to one of absolute vulnerability. However, international recognition of this vulnerability was overshadowed by the plight of refugee pop- ulations during the political upheaval and mass displacements of the mid-twentieth century. As a matter of international legal regulation, statelessness was thus suspended between being viewed as a problem of reconciling state interests through avoiding conﬂicts in nationality laws, or as subsumed within the more capacious framework of refugee law.10 It took contin- ued political turmoil in the form of state dissolutions, state successions, armed conﬂicts, and boundary disputes, for the international legal community to begin to recognize the stateless as a distinct category in need of international––and not merely domestic––protection. 10 See MIRA L. SIEGELBERG, STATELESSNESS: A MODERN HISTORY (2020) (for a comprehensive analysis of the historical evolution of the status of statelessness). 11 Id. at 14–15. 12 John Torpey, The Great War and the Birth of the Modern Passport System, in DOCUMENTING INDIVIDUAL IDENTITY: THE DEVELOPMENT OF STATE PRACTICES IN THE MODERN WORLD 256, 269–70 (Jane Caplan & John Torpey eds., 2001). 11 Id. at 14–15. A. The Regulation of People Without Countries The emergence of statelessness in the consciousness of the international community is strongly linked to the rise of protectionism and regulation of human mobility in the wake of World War I. The war signaled an end to the exceptional movement of labor and capital fueled by the nineteenth century spirit of internationalism, technological innovation, and lib- eral capitalism.11 The advent of welfare states in combination with nationalist ideologies resulted in states drawing ever sharper boundaries between those who were their nation- als––and thus entitled to the social beneﬁts stemming from political membership––and non-nationals. Central to this boundary drawing was the question of identiﬁcation, resulting in the proliferation of documentation such as passports and identity cards to serve bureau- cratic needs of migration control and internal administration.12 As Torpey argues, this https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 241 MANUFACTURING STATELESSNESS extraordinary capacity to regulate human mobility through documentary practices was not merely evidence of the state’s expanding authority, but constitutive of its identity as a state.13 The signiﬁcance of nationality as a form of identiﬁcation, and what its absence represented, must be seen against this evolving interstate order. The end of World War I saw a rise in the number of claims before domestic courts from individuals seeking to be declared stateless in order to avoid penalties associated with their designation as enemy aliens during the war. While these claims had previously been treated with ambivalence and even suspicion due to concerns of national security, courts now began to accord them greater sympathy, spurring efforts toward the recognition of statelessness as a legally valid category.14 The stateless person nonetheless continued to be treated as a “res nullius,”15 or a “caput lupinum”16 whose presence threatened the stability of the post-war international order. As the numbers of stateless per- sons grew, the question of whether and how their status could be secured on the international plane gained increasing urgency.17 Mass denationalization of Russians labeled as enemies of the Bolshevik Revolution coupled with refugee ﬂows from the collapse of the Hapsburg empire served as a catalyst for the emergence of the League of Nations as a potential forum for securing the rights of the dispossessed.18 The League was none too keen to assume this mantle, which threatened to destabilize its carefully constructed boundaries between issues that were within the League’s “international” mandate, and those that implicated state sovereignty. p 22 Manley O. Hudson, The First Conference for the Codiﬁcation of International Law, 24 AJIL 447, 450 (1930). 23 Id.; Richard W. Flournoy, Nationality Convention, Protocols and Recommendations Adopted by the First Conference on the Codiﬁcation of International Law, 24 AJIL 467, 481–83 (1930). 13 Id. at 270. 14 SIEGELBERG, supra note 10, at 45–46. 15 See GEORG SCHWARZENBERGER, INTERNATIONAL LAW 171 (1949). 16 HERSCH LAUTERPACHT, AN INTERNATIONAL BILL OF THE RIGHTS OF MAN 126 (1945). 17 See 1 LASSA OPPENHEIM, INTERNATIONAL LAW 668 (1955) (stating that, with the loss of the link of nationality, the stateless had no means of claiming protection under international law). 18 SIEGELBERG, supra note 10, at 65–67. 19 Id. at 68–69. 20 179 LNTS 89, Apr. 13, 1930. 21 179 LNTS 115, Apr. 12, 1930. 22 Manley O. Hudson, The First Conference for the Codiﬁcation of International Law, 24 AJIL 447, 450 (1930). 23 Id.; Richard W. Flournoy, Nationality Convention, Protocols and Recommendations Adopted by the First Conference on the Codiﬁcation of International Law, 24 AJIL 467, 481–83 (1930). 13 Id. at 270. 15 See GEORG SCHWARZENBERGER, INTERNATIONAL LAW 171 (1949). 14 SIEGELBERG, supra note 10, at 45–46. 16 HERSCH LAUTERPACHT, AN INTERNATIONAL BILL OF THE RIGHTS OF MAN 126 (1945). A. The Regulation of People Without Countries It thus pressed for a technical, legal sol- ution to the status of those without nationality, painting the problem as one of conﬂict of nationality laws that could be overcome by harmonization of the diversity of conditions for naturalization and denaturalization in national legal systems.19 The outcome of these efforts was the 1930 Hague Conference for the Codiﬁcation of International Law, which adopted a Convention on Certain Questions Relating to the Conﬂict of Nationality Laws20 and a Protocol Relating to a Certain Case of Statelessness.21 Even though the codiﬁcation of nationality laws was motivated by the object to “reduce the number of cases of, if not to abolish, statelessness and multiple nationality,”22 the Convention and Protocol were regarded by commentators as having achieved fairly little in this respect.23 The provisions on reduction of statelessness were in the nature of “negative” obligations on states to prevent the loss of nationality rather than positive obligations to grant nationality. Moreover, the instruments mainly targeted statelessness at birth rather than subsequent 21 179 LNTS 115, Apr. 12, 1930. 20 179 LNTS 89, Apr. 13, 1930. 19 Id. at 68–69. 18 SIEGELBERG, supra note 10, at 65–67. 19 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 Vol. 116:2 242 loss of nationality.24 Aware of these limitations, the Conference sought to convey a sense of urgency to states and to the League to persist in efforts to minimize the problem of stateless- ness, a call that the League failed to heed.25 Enmeshed as they were in concerns relating to the constitution of the nation-state and the political division of authority between the domestic and international legal orders, these inter- national efforts to address the problem of those without nationality viewed the dispossessed though the lens of state interests.26 The stateless person was a ﬂotsam who posed a problem of order management and risked exacerbating friction between states. By creating a class of peo- ple who were “‘gatecrashers’ in the backyards of others,” statelessness posed challenges for states who were their unwilling recipients and thus prevented from controlling the boundaries of their membership.27 Seen from this angle, statelessness was “an unfortunate consequence of public international law’s errors, rather than a phenomenon with its own weight.”28 While the inter-war period saw increased international attention to statelessness as a problem in its own right, as the subsequent section shows, the plight of stateless persons came to be over- shadowed by the more pressing need to respond to another category of dispossessed persons: refugees. Id. 26 Peter J. Spiro, A New International Law of Citizenship, 105 AJIL 694, 709 (2011). 27 Matthew J. Gibney, Statelessness and Citizenship in Ethical and Political Perspective, in NATIONALITY AND STATELESSNESS UNDER INTERNATIONAL LAW 44, 49 (Alice Edwards & Laura van Waas eds., 2014). 28 Will Hanley, Statelessness: An Invisible Theme in the History of International Law, 25 EUR. J. INT’L L. 321, 322 (2014). 29 Carol A. Batchelor, Stateless Persons: Some Gaps in International Protection, 7 INT’L J. REFUGEE L. 232, 239 (1995), citing Guy Goodwin-Gill, The Rights of Refugees and Stateless Persons: Problems of Stateless Persons and the Need for International Measures of Protection, in HUMAN RIGHTS PERSPECTIVE AND CHALLENGES (IN 1990 AND BEYOND) 378, 389–90 (K. P. Saksena ed., 1994). 30 Arthur K. Kuhn, International Measures for the Relief of Stateless Persons, 30 AJIL 495, 499 (1936). 31 See SIEGELBERG, supra note 10, at 152–54. See generally MICHELLE FOSTER & HÉLÈNE LAMBERT, S ( ) 24 Paul Weis, The United Nations Convention on the Reduction of Statelessness, 1961, 11 INT’L & COMP. L. Q. 1073, 1074 (1962). 25 30 Arthur K. Kuhn, International Measures for the Relief of Stateless Persons, 30 AJIL 495, 499 (1936). 31 29 Carol A. Batchelor, Stateless Persons: Some Gaps in International Protection, 7 INT’L J. REFUGEE L. 232, 239 (1995), citing Guy Goodwin-Gill, The Rights of Refugees and Stateless Persons: Problems of Stateless Persons and the Need for International Measures of Protection, in HUMAN RIGHTS PERSPECTIVE AND CHALLENGES (IN 1990 AND BEYOND) 378, 389–90 (K. P. Saksena ed., 1994). 31 See SIEGELBERG, supra note 10, at 152–54. See generally MICHELLE FOSTER & HÉLÈNE LAMBE INTERNATIONAL REFUGEE LAW AND THE PROTECTION OF STATELESS PERSONS 23 (2019). Peter J. Spiro, A New International Law of Citizenship, 105 AJIL 694, 709 (2011). 27 Matthew J. Gibney, Statelessness and Citizenship in Ethical and Political Perspective, in NATIONALITY AND STATELESSNESS UNDER INTERNATIONAL LAW 44, 49 (Alice Edwards & Laura van Waas eds., 2014). 28 Will Hanley, Statelessness: An Invisible Theme in the History of International Law, 25 EUR. J. INT’L L. 321, 322 (2014). 29 Carol A. Batchelor, Stateless Persons: Some Gaps in International Protection, 7 INT’L J. REFUGEE L. 232, 239 (1995) i i G G d i Gill Th Ri h f R f d S l P P bl f S l P d h 30 Arthur K. Kuhn, International Measures for the Relief of Stateless Persons, 30 AJIL 495, 499 (1936). 31 See SIEGELBERG, supra note 10, at 152–54. See generally MICHELLE FOSTER & HÉLÈNE LAMBERT, INTERNATIONAL REFUGEE LAW AND THE PROTECTION OF STATELESS PERSONS 23 (2019) B. Statelessness as the Stepchild to Refugee Status According to Article 1 of the Convention, a stateless person is one “who is not considered as a national by any State under the operation of its law.”36 Commentators have been critical of this deﬁnition, arguing that what it gains in brevity, it loses in treating nationality as a purely technical problem and limiting its scope to de jure stateless persons, excluding situa- tions where an individual is formally a national of a state but this nationality is not effective.37 As the drafting history of the Convention makes clear, however, this was not a question of oversight, but a deliberate attempt to avoid overlaps between the 1951 Refugee Convention and the 1954 Statelessness Con ention 38 The emphasis on de jure statelessness and the dis This background is important for understanding the deﬁnition of statelessness that was eventually adopted in the 1954 Convention Relating to the Status of Stateless Persons. According to Article 1 of the Convention, a stateless person is one “who is not considered as a national by any State under the operation of its law.”36 Commentators have been critical This background is important for understanding the deﬁnition of statelessness that was eventually adopted in the 1954 Convention Relating to the Status of Stateless Persons. According to Article 1 of the Convention, a stateless person is one “who is not considered as a national by any State under the operation of its law.”36 Commentators have been critical of this deﬁnition, arguing that what it gains in brevity, it loses in treating nationality as a purely technical problem and limiting its scope to de jure stateless persons, excluding situa- tions where an individual is formally a national of a state but this nationality is not effective.37 As the drafting history of the Convention makes clear, however, this was not a question of oversight, but a deliberate attempt to avoid overlaps between the 1951 Refugee Convention and the 1954 Statelessness Convention.38 The emphasis on de jure statelessness, and the dis- tinction between de jure statelessness and refugees, was retained in the 1961 Convention on the Reduction of Statelessness. 32 UN Economic and Social Council (UNESC), Res. 116 D (VI), UN Doc. E/777 (Mar. 1–2, 1948). 33 UN Ad Hoc Committee on Refugees and Stateless Persons, A Study of Statelessness, at 4, 10, UN Doc. E/1112 (1949); FOSTER AND LAMBERT, supra note 31, at 29. 34 UNESC, Res. 248(IX) (Aug. 6, 1949). 35 FOSTER & LAMBERT, supra note 31, at 36–39. 36 Convention Relating to the Status of Stateless Persons, Art. 1, Sept. 28, 1954, 360 UNTS 117 (hereinafter 1954 Stateless Convention). 37 Batchelor, supra note 29, at 232. 38 Id. at 247–48. 39 Convention on the Reduction of Statelessness, Art. 1, Aug. 30, 1961, 989 UNTS 175. 40 See Laura van Waas, The UN Statelessness Conventions, in NATIONALITY AND STATELESSNESS UNDER INTERNATIONAL LAW, supra note 27, at 78. 41 See Batchelor, supra note 29, at 246–49. 39 Convention on the Reduction of Statelessness, Art. 1, Aug. 30, 1961, 989 UNTS 175. 40 See Laura van Waas, The UN Statelessness Conventions, in NATIONALITY AND STATELESSNESS UNDER INTERNATIONAL LAW, supra note 27, at 78. 41 See Batchelor, supra note 29, at 246–49. 37 Batchelor, supra note 29, at 232. 41 See Batchelor, supra note 29, at 246–49. B. Statelessness as the Stepchild to Refugee Status The oft-repeated statement that initially, refugees and stateless persons “walked hand in hand,” and that the stateless came to be treated as a subset of the refugee population only after World War II,29 is only partially accurate. Indeed, the inter-war period saw several attempts to distinguish between the status and needs of the two categories of dispossessed as exempliﬁed by a 1936 editorial comment in the American Journal of International Law that distinguished between stateless persons as individuals who had “lost the diplomatic pro- tection of their national State without acquiring any other national protection” and refugees who had found asylum in other countries.30 These nuances became difﬁcult to sustain in the face of national laws such as those enacted by Nazi Germany stripping Jews of their citizenship rights, and subsequently of their nation- ality.31 By the end of World War II, the growing number and scale of the problem of persons who could no longer claim the protection of any nation gave fresh impetus to international efforts to address their status. In 1948, the Economic and Social Council (ECOSOC) charged https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 243 MANUFACTURING STATELESSNESS the UN secretary-general with “undertak[ing] a study of the existing situation in regard to the protection of stateless persons” and “submit[ting] recommendations to the Council as to the desirability of concluding a further convention on this subject.”32 The Study reinforced the notion that the categories of refugees and stateless persons overlapped in large part, focusing as it did primarily on displaced stateless persons rather than those in situ.33 This Study and related Memoranda formed the basis for the deliberations of the Ad Hoc Committee on Statelessness and Related Problems constituted by the ECOSOC.34 Though the Ad Hoc Committee opted to distinguish between the two categories of persons by proposing a Draft Convention relating to the Status of Refugees and an attached Protocol Concerning Stateless Persons, only the former was eventually adopted by the United Nations Conference of Plenipotentiaries on the Status of Refugees and Stateless Persons in the form of the Refugee Convention. The Protocol Concerning Stateless Persons was referred for further study.35 y This background is important for understanding the deﬁnition of statelessness that was eventually adopted in the 1954 Convention Relating to the Status of Stateless Persons. 38 Id. at 247–48. B. Statelessness as the Stepchild to Refugee Status The 1961 Convention, initially motivated by the objective of eliminating statelessness, instead dialed this back to the more modest aim of reducing the number of stateless at birth, later in life, and in the course of state succession.39 Notwithstanding this cautious and pragmatic approach, the Statelessness Conventions were slow to gain traction, struggling to attract signatories and largely ignored in academic scholarship for the ﬁrst four decades of their existence, earning them the moniker of “orphan conventions.”40 One of the reasons for this international obscurity was the ambiguity sur- rounding which agency, if any, was responsible for their promotion and enforcement.41 And even though the 1974 Resolution of the General Assembly designated the UNHCR as https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW 244 Vol. 116:2 the agency to which individuals who claimed the protection of the 1961 Convention could apply for assistance, the UNHCR did little to pursue this mandate until the 1990s.42 Thus, beginning in the mid-twentieth century, the issue of statelessness came to be con- signed to obscurity and was addressed only as an afterthought to the more urgent problem of refugees. This posture has only recently changed, in large part due to the UNHCR’s expanded mandate to actively promote accession to the Statelessness Conventions and provide technical assistance to states in drafting and implementing nationality legislation.43 The UNHCR has achieved considerable success in its advocacy efforts for the former goal, registering a steady uptick in state accessions to the Conventions.44 In addition, it has made important headway in providing technical guidance and advice to states on statelessness by developing guidance on important doctrinal issues, including the deﬁnition of stateless persons, stateless determi- nation procedures, the status of stateless persons in the domestic system, and prevention of child statelessness.45 It has also worked with a variety of actors on standard setting on nation- ality issues, played a seminal role in collecting data on and raising awareness of the problems faced by stateless persons, helped states to develop and reform their nationality legislation, and sought to promote birth registration and acquisition of identity documentations.46 Even though the UNHCR’s recent work demonstrates an awareness of the limitations of a framework oriented to formal, technical solutions for addressing statelessness, it nonetheless exhibits a reluctance to test the boundaries of sovereignty over nationality issues. 45 UNHCR, Guidelines on Statelessness No. 1: The Deﬁnition of “Stateless Person” in Article 1(1) of the 1954 Convention Relating to the Status of Stateless Persons, HCR/GS/12/01 (Feb. 20, 2012); UNHCR, Guidelines on Statelessness No. 2: Procedures for Determining Whether an Individual Is a Stateless Person, HCR/GS/12/02 (Apr. 5, 2012); UNHCR, Guidelines on Statelessness No. 3: The Status of Stateless Persons at the National Level, HCR/GS/12/03 (July 17, 2012); UNHCR, Guidelines on Statelessness No. 4: Ensuring Every Child’s Right to Acquire a Nationality Through Articles 1–4 of the 1961 Convention on the Reduction of Statelessness, HCR/GS/12/04 (Dec. 21, 2012). 42 GA Res. 3274(XXIX) (Dec. 10, 1974); GA Res. 31/36 (Nov. 30, 1976). See van Waas, supra note 40, at 78. 43 GA Res. 50/152 (Feb. 9, 1996). 44 See the status at https://treaties.un.org/pages/Home.aspx?clang¼_en. As of early 2021, the 1954 Convention has ninety-ﬁve parties and twenty-three signatories, whereas the 1961 Convention has seventy-six parties and ﬁve signatories. 45 UNHCR, Guidelines on Statelessness No. 1: The Deﬁnition of “Stateless Person” in Article 1(1) of the 1954 Convention Relating to the Status of Stateless Persons, HCR/GS/12/01 (Feb. 20, 2012); UNHCR, Guidelines on Statelessness No. 2: Procedures for Determining Whether an Individual Is a Stateless Person, HCR/GS/12/02 (Apr. 5, 2012); UNHCR, Guidelines on Statelessness No. 3: The Status of Stateless Persons at the National Level, HCR/GS/12/03 (July 17, 2012); UNHCR, Guidelines on Statelessness No. 4: Ensuring Every Child’s Right to Acquire a Nationality Through Articles 1–4 of the 1961 Convention on the Reduction of Statelessness, HCR/GS/12/04 (Dec. 21, 2012). 46 See Mark Manly, UNHCR’s Mandate and Activities to Address Statelessness, in NATIONALITY AND STATELESSNESS UNDER INTERNATIONAL LAW, supra note 27, at 88, 97–113. 47 UNHCR HANDBOOK, supra note 5, para. 1. 48 Id., para. 7, n. 4. 46 See Mark Manly, UNHCR’s Mandate and Activities to Address Statelessness, in NATIONALITY AND STATELESSN UNDER INTERNATIONAL LAW, supra note 27, at 88, 97–113. 4 42 GA Res. 3274(XXIX) (Dec. 10, 1974); GA Res. 31/36 (Nov. 30, 1976). See van Waas, supra note 40, at 78. 43 GA Res. 50/152 (Feb. 9, 1996). 44 See the status at https://treaties.un.org/pages/Home.aspx?clang¼_en. As of early 2021, the 1954 Convention has ninety-ﬁve parties and twenty-three signatories, whereas the 1961 Convention has seventy-six parties and ﬁve signatories. 45 p 47 UNHCR HANDBOOK, supra note 5, para. 1. 48 Id., para. 7, n. 4. 42 GA Res. 3274(XXIX) (Dec. 10, 1974); GA Res. 31/36 (Nov. 30, 1976). See van Waas, supra note 40, at 78. 43 GA Res. 50/152 (Feb. 9, 1996). 44 S h h // i / /H ? l A f l 2021 h 1954 C i 44 See the status at https://treaties.un.org/pages/Home.aspx?clang¼_en. As of early 2021, the 1954 Conven has ninety-ﬁve parties and twenty-three signatories, whereas the 1961 Convention has seventy-six parties and signatories. 45 43 GA Res. 50/152 (Feb. 9, 1996). 44 C. The Human Rights Turn in Statelessness C. The Human Rights Turn in Statelessness While state sovereignty over nationality matters continues to pose a challenge for the inter- national regulation of statelessness, in the past few decades, two developments in interna- tional law jurisprudence and scholarship have made incursions into this sovereign space: the ﬁrst seeks to close the gap between rights accorded to nationals and non-nationals while the second emphasizes the limits set by international law to state control over nationality. The distinction between the treatment of nationals and non-nationals when it comes to human rights has been questioned by scholars who proclaim the universality of human rights. Decoupling the possession of rights from citizenship status, they argue that one’s status as a human being is the only requirement for entitlement to human rights, even though the implementation of these rights requires governmental action at the national level.49 This view ﬁnds some recognition in both international human rights instruments as well as the Statelessness Conventions, which minimize the importance of nationality.50 While these pro- visions go some way in addressing the rights deprivations experienced by stateless persons, they are nonetheless not a substitute for the right to nationality. The second, more ambitious, development in international law is the shift from conceiving of nationality in terms of state capacity to control the boundaries of its membership to an individual’s right to citizenship.51 This reconceptualization, which Peter J. Spiro labels the “new international law of citizenship,” is the result of the convergence of legal trends seen across a range of treaty instruments, international tribunals, international and regional orga- nizations, and state practice. In the context of statelessness, the most signiﬁcant developments relate to greater scrutiny and, in some cases, overruling, of state measures relating to legal and administrative restrictions on acquisition of nationality, in particular in situations of state suc- cession, and the prohibition of discrimination in access to citizenship.52 There is, however, signiﬁcant diversity on how the different human rights “regimes” choose to frame the issue of statelessness, which is typically contingent on the narrow focus of the regime. And the regimes themselves remain underdeveloped in important respects and leave much to state discretion. 50 Id. at 249–50. For example, Article 4 of the 1954 Stateless Convention enjoins states to grant lawfully res- ident stateless persons rights that are equivalent to nationals in certain respects such as religion, access to elemen- tary education, and public relief. 51 52 See id. at 721–22. 51 See Spiro, supra note 26, at 695–96. 49 See David Weissbrodt & Clay Collins, The Human Rights of Stateless Persons, 28 HUM. RTS. Q. 245, 248–49 (2006). 50 Id. at 249–50. For example, Article 4 of the 1954 Stateless Convention enjoins states to grant lawfully res- ident stateless persons rights that are equivalent to nationals in certain respects such as religion, access to elemen- tary education, and public relief. 51 See Spiro, supra note 26, at 695–96. 52 See id. at 721–22. 53 James G. Devaney, What Happens Next? The Law of State Succession, 1, 19 (GCILS Working Paper, No. 6, Nov. 2020). 49 See David Weissbrodt & Clay Collins, The Human Rights of Stateless Persons, 28 HUM. RTS. Q. 245, 248 (2006). 50 d 4 l l 4 f h 4 l l f ll B. Statelessness as the Stepchild to Refugee Status Thus, for instance, while the 2014 UNHCR Handbook on Protection of Stateless Persons recognizes that statelessness does not only occur in the context of migration but also in situ, it treats the latter as “often the result of problems in the framing and implementation of nationality laws.”47 Similarly, in elaborating on the meaning of when an individual is “not considered as a national by any State under the operation of its law” for the purposes of Article 1 of the 1954 Convention, the Handbook endorses an understanding of de facto statelessness as referring to individuals outside the country of their nationality who cannot avail themselves of its diplomatic protection notwithstanding that “some participants [in the UNHRC’s 2010 expert consultation] were of the view that a person’s nationality could be ineffective inside as well as outside of his or her country of nationality.”48 Moreover, as this Article goes on to discuss in Part III, some of the UNHCR’s proposed actions to eliminate statelessness, https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 245 MANUFACTURING STATELESSNESS such as identity registration and documentation drives, may unwittingly exacerbate rather than reduce the incidence of statelessness. such as identity registration and documentation drives, may unwittingly exacerbate rather than reduce the incidence of statelessness. 49 See David Weissbrodt & Clay Collins, The Human Rights of Stateless Persons, 28 HUM. RTS. Q. 245, 248–49 (2006). 50 Id. at 249–50. For example, Article 4 of the 1954 Stateless Convention enjoins states to grant lawfully res- ident stateless persons rights that are equivalent to nationals in certain respects such as religion, access to elemen- tary education, and public relief. 51 See Spiro supra note 26 at 695–96 54 See Andreas Zimmermann, State Succession and the Nationality of Natural Persons – Facts and Possible Codiﬁcation, in LA SUCCESSION D’ÉTATS: LA CODIFICATION À L’ÉPROUVE DES FACTS/STATE SUCCESSION: CODIFICATIONS TESTED AGAINST THE FACTS 612, 659–61 (Pierre Michel Eisemann & Martti Koskenniemi eds., 2000) (surveying state practice and evidence of customary international law on the question of conferral of nation- ality and obligation to avoid statelessness in the context of state succession). 55 See Jeffrey L. Blackman, State Successions and Statelessness: The Emerging Right to an Effective Nationality Under International Law, 19 MICH. J. INT’L L. 1141, 1164–94 (1998) (for an excellent overview of these developments). 56 56 European Convention on Nationality, Art. 18(2), Nov. 6, 1997, 37 ILM 44; Council of Europe Convention on the Avoidance of Statelessness in relation to State Succession, Arts. 5–6, May 19, 2006, ETS No. 200, Explanatory Report, para. 19. 7 International Law Commission, Draft Articles on Nationality of Natural Persons in Relation to the cession of States, UN Doc. A/54/10 (Apr. 3, 1999). 58 Ineta Ziemele, State Succession and Issues of Nationality and Statelessness, in NATIONALITY AND STATELESSNESS UNDER INTERNATIONAL LAW, supra note 27, at 217, 229. 61 International Convention on the Elimination of All Forms of Racial Discrimination, Mar. 7, 1966, UNTS 195. 60 Michelle Foster & Timnah Rachel Baker, Racial Discrimination in Nationality Laws: A Blindspot of International Law?, 11 COLUM. J. RACE & L. 83, 90–91 (2021). 61 See Andreas Zimmermann, State Succession and the Nationality of Natural Persons – Facts and Possible Codiﬁcation, in LA SUCCESSION D’ÉTATS: LA CODIFICATION À L’ÉPROUVE DES FACTS/STATE SUCCESSION: CODIFICATIONS TESTED AGAINST THE FACTS 612, 659–61 (Pierre Michel Eisemann & Martti Koskenniemi eds., 2000) (surveying state practice and evidence of customary international law on the question of conferral of nation- ality and obligation to avoid statelessness in the context of state succession). 55 See Jeffrey L. Blackman, State Successions and Statelessness: The Emerging Right to an Effective Nationality Under International Law, 19 MICH. J. INT’L L. 1141, 1164–94 (1998) (for an excellent overview of these developments). 56 European Convention on Nationality, Art. 18(2), Nov. 6, 1997, 37 ILM 44; Council of Europe Convention on the Avoidance of Statelessness in relation to State Succession, Arts. 5–6, May 19, 2006, ETS No. 200, Explanatory Report, para. 19. 57 International Law Commission, Draft Articles on Nationality of Natural Persons in Relation to the Succession of States, UN Doc. A/54/10 (Apr. 3, 1999). 58 Ineta Ziemele, State Succession and Issues of Nationality and Statelessness, in NATIONALITY AND STATELESSNESS UNDER INTERNATIONAL LAW, supra note 27, at 217, 229. 59 ILC Draft Articles, supra note 57, Art. 4. 60 Michelle Foster & Timnah Rachel Baker, Racial Discrimination in Nationality Laws: A Blindspot of International Law?, 11 COLUM. J. RACE & L. 83, 90–91 (2021). 61 International Convention on the Elimination of All Forms of Racial Discrimination, Mar. 7, 1966, 660 UNTS 195. C. The Human Rights Turn in Statelessness For example, while there have been signiﬁcant developments in the international law of state succession that seek to avoid statelessness, there is lack of clarity on the exact content of state obligations in this respect, leading one commentator to remark “[i]f the law of state succession in respect of treaties is the most well-developed area of the law of state suc- cession, the nationality of persons is a strong contender for being the least well-developed.”53 3 James G. Devaney, What Happens Next? The Law of State Succession, 1, 19 (GCILS Working Paper, No. 6, v. 2020). https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 59 ILC Draft Articles, supra note 57, Art. 4. THE AMERICAN JOURNAL OF INTERNATIONAL LAW It is worth noting that––not- withstanding discrimination having been recognized as both a cause and a consequence of statelessness––unlike gender discrimination and statelessness of children, statelessness on account of racial discrimination is not at the forefront of international initiatives such as the UNHCR’s #IBelong campaign aiming to eliminate statelessness by 2024.60 This is in part due to the wording of the 1966 International Convention on the Elimination of All Forms of Racial Discrimination.61 Though Article 5(iii)(d) of the Convention explicitly pro- hibits discrimination with respect to a right to nationality, its scope is limited by operation of Article 1(2), which excludes from the Convention’s ambit distinctions between nationals and non-nationals and Article 1(3), which afﬁrms that “[n]othing in this Convention may be interpreted as affecting in any way the legal provisions of States Parties concerning nationality, https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 247 MANUFACTURING STATELESSNESS citizenship or naturalization, provided that such provisions do not discriminate against any particular nationality.” The net effect is that while states may not discriminate internally between citizens on the basis of race, they are nonetheless not constrained from having racial criteria for acquisition of nationality in the ﬁrst place.62 Nor are they prevented from enacting laws that target multiple nationalities rather than one particular nationality.63 g p p y The monitoring body for the Convention—the Committee on the Elimination of Racial Discrimination—has also historically been reluctant to interfere with naturalization laws.64 In more recent years, however, it has adopted a more aggressive stance as exempliﬁed in its 2004 General Recommendation Thirty, which enjoins states to ensure that “particular groups of non-citizens are not discriminated against with regard to access to citizenship or naturalization, and to pay due attention to possible barriers to naturalization that may exist for long-term or permanent residents.” States are reminded that “deprivation of citizen- ship on the basis of race, colour, descent, or national or ethnic origin is a breach of States parties’ obligations to ensure non-discriminatory enjoyment of the right to nationality.”65 However, notwithstanding this statement of principles, in practice, the Committee has not always been consistent in its willingness to call out and condemn discriminatory laws on nationality.66 Surveying the Committee’s observations on individual country reports over a period of thirty years, Foster and Baker conclude that, with few exceptions, the Committee generally uses “soft” language to evaluate discriminatory laws when it comes to state denial of nationality, reserving the stronger language of breach for nationality deprivations and with- drawals.67 There is also no overarching analysis of the relationship between Article 5(iii)(d) and Article 1(3), leaving it open to states to rely on the latter as excluding the Convention’s application to acquisition of nationality.68 In contrast to the relatively weak international legal framework, some of the most signiﬁ- cant legal efforts in cabining state discretion over nationality—and in the process, addressing statelessness as a matter of international concern—can be seen at the regional level. THE AMERICAN JOURNAL OF INTERNATIONAL LAW THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 246 There is no multilateral treaty or clear customary international law imposing binding obliga- tions on states with respect to conferral of nationality and avoidance of statelessness in the context of state succession.54 However, there have been important regional initiatives to cod- ify and develop the international law on this issue, in particular in Europe, in the form of the 1997 European Convention on Nationality and the 2006 Council of Europe Convention on the Avoidance of Statelessness in relation to State Succession.55 These instruments nonethe- less give states ample discretion as to the procedure for granting nationality.56 A parallel international effort is the International Law Commission’s (ILC) 1999 Articles on Nationality of Natural Persons in relation to the Succession of States.57 The Articles pro- vide progressive principles for the attribution of nationality on various grounds, whereby, among others, those with “habitual residence” in the state affected by state succession acquire the nationality of the successor state.58 However, the Articles ultimately refrain from impos- ing concrete obligations on states, only urging states to take “all reasonable measures” to avoid persons becoming stateless as a result of state succession.59 And despite this cautious approach, the Articles have not led to the conclusion of a binding multilateral instrument. pp g Another area in which the international law framework governing statelessness is progres- sively gathering pace, but nonetheless contains some ambiguity and loopholes, relates to non-discrimination in the context of nationality acquisition. 69 See, e.g., The Girls Yean v. Dominican Republic, Preliminary Objections, Merits, Reparations and Costs, Judgment, Inter-Am. Ct. H.R. (ser. C) No. 130 (Sept. 8, 2005); Expelled Dominicans and Haitians v. Dominican Republic, Preliminary Objections, Merits, Reparations and Costs, Judgment, Inter-Am. Ct. H.R. (ser. C) No. 282 (Aug. 28, 2014); Anudo v. Tanzania, Decision, African Court on Human and Peoples’ Rights [2108] AFCHPR 5 (Mar. 22, 2018); Penessis v. Tanzania, Decision, African Court on Human and Peoples’ Rights [2109] AFCHPR 8 (Nov. 28, 2019). 63 Foster & Baker, supra note 60, at 87. 64 62 See Spiro, supra note 26, at 716. 63 Foster & Baker, supra note 60, at 87. 64 See Drew Mahalic & Joan Gambee Mahalic, The Limitation Provisions of the International Convention on the Elimination of All Forms of Racial Discrimination, 9 HUM. RTS. Q. 74, 79, 82 (1987). 65 Comm. on Elimination Racial Discrimination, General Recommendation Thirty, on Discrimination Against Non-citizens, paras. 13–14, UN Doc. HRI/GEN/1/Rev.7/Add.1 (May 4, 2005). See also PATRICK THORNBERRY, THE INTERNATIONAL CONVENTION ON THE ELIMINATION OF ALL FORMS OF RACIAL DISCRIMINATION: A COMMENTARY 146 (2016). 66 Foster & Baker, supra note 60, at 116. 67 Id. at 118–123. 68 Id. at 124–26. 69 See, e.g., The Girls Yean v. Dominican Republic, Preliminary Objections, Merits, Reparations and Costs, Judgment, Inter-Am. Ct. H.R. (ser. C) No. 130 (Sept. 8, 2005); Expelled Dominicans and Haitians v. Dominican Republic, Preliminary Objections, Merits, Reparations and Costs, Judgment, Inter-Am. Ct. H.R. (ser. C) No. 282 (Aug. 28, 2014); Anudo v. Tanzania, Decision, African Court on Human and Peoples’ Rights [2108] AFCHPR 5 (Mar. 22, 2018); Penessis v. Tanzania, Decision, African Court on Human and Peoples’ Rights [2109] AFCHPR 8 (Nov. 28, 2019). 67 Id. at 118–123. 64 See Drew Mahalic & Joan Gambee Mahalic, The Limitation Provisions of the International Convention on the Elimination of All Forms of Racial Discrimination, 9 HUM. RTS. Q. 74, 79, 82 (1987). 68 Id. at 124–26. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 65 Comm. on Elimination Racial Discrimination, General Recommendation Thirty, on Discrimination Against Non-citizens, paras. 13–14, UN Doc. HRI/GEN/1/Rev.7/Add.1 (May 4, 2005). See also PATRICK THORNBERRY, THE INTERNATIONAL CONVENTION ON THE ELIMINATION OF ALL FORMS OF RACIAL DISCRIMINATION: A COMMENTARY 146 (2016). 66 62 See Spiro, supra note 26, at 716. 63 66 Foster & Baker, supra note 60, at 116. p 64 See Drew Mahalic & Joan Gambee Mahalic, The Limitation Provisions of the International Convention on the Elimination of All Forms of Racial Discrimination, 9 HUM. RTS. Q. 74, 79, 82 (1987). 65 Comm. on Elimination Racial Discrimination, General Recommendation Thirty, on Discrimination Against Non-citizens, paras. 13–14, UN Doc. HRI/GEN/1/Rev.7/Add.1 (May 4, 2005). See also PATRICK THORNBERRY, THE INTERNATIONAL CONVENTION ON THE ELIMINATION OF ALL FORMS OF RACIAL DISCRIMINATION: A COMMENTARY 146 (2016). 66 F t & B k p t 60 t 116 70 BRONWEN MANBY, CITIZENSHIP IN AFRICA: THE LAW OF BELONGING 193 (2018). 71 BRONWEN MANBY, NATIONALITY, MIGRATION AND STATELESSNESS IN WEST AFRICA: A STUDY FOR UNHCR AND IOM 21 (2015), available at https://www.refworld.org/docid/55b886154.html. 72 See UNHCR, Background Note on Gender Equality, Nationality Laws and Statelessness 2018 (Mar. 8, 2018), available at https://www.refworld.org/docid/5aa10fd94.html. 73 See generally Thomas McGee, “Rainbow Statelessness”––Between Sexual Citizenship and Legal Theory: Exploring the Statelessness-LGBTQ+ Nexus, 2 STATELESSNESS & CITIZENSHIP REV. 64, 80–84 (2020). THE AMERICAN JOURNAL OF INTERNATIONAL LAW The Inter- American Court of Human of Rights (IACtHR) and the African Court on Human and Peoples’ Rights (AfCHPR) have elected to take a progressively bold stance in this respect, with important pronouncements of general international law principles governing state obli- gations in respect of statelessness.69 These judgments, however, are highly fact-speciﬁc and 67 Id. at 118–123. 6 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 248 primarily focus on state laws and practices relating to deprivation of nationality rather than laws that limit the acquisition of nationality in the ﬁrst place. Further, as the Article discusses in Part II, these examples of successful supranational litigation represent only a fraction of the strategies that states use to manufacture statelessness in the shadow of the law. Nonetheless, as Part II will show, this emerging regional jurisprudence and other forms of international legal mobilization provides a useful lens for analyzing the ways in which supranational and inter- national institutions––both formal and informal––are beginning to challenge state discretion over questions of nationality and the statelessness that can follow in its wake. International law’s uneasy relationship with and response to the notion of statelessness sits at the conﬂuence of shifting conceptions of state sovereignty, political membership, and the boundaries between the international and domestic legal orders. Wary of trespassing into the sovereign space, international laws, institutions, and actors have variously sought to frame statelessness as a technical, legal problem that can be addressed through avoiding conﬂicts in nationality laws, as the handmaiden to refugee law, or as a highly fact-speciﬁc evaluation that is directed toward preventing loss of nationality, once acquired, rather than dictating who gets to be a national in the ﬁrst place. The next Part shows this framework is at best only partially capable of, and at worst counterproductive for, resolving cases of statelessness that are not a result of incompetence, oversight, or error, but ﬂow from the intentional exercise of state power. II. STRATEGIC STATELESSNESS The increasingly emboldened stance that international and regional bodies are willing to take to circumscribe states’ ability to set the terms of their political membership is largely pre- mised on the belief that statelessness is a product of manifest illegality. To be sure, there are certainly instances, both in the distant past, and also more recently, where states have enacted nationality laws that are blatantly discriminatory or involve mass denationalization. These include well-known cases such as the disenfranchisement and effective loss of nationality of German Jews during the Nazi era to less familiar situations that are closer in time, such as the Sierra Leonean and Liberian nationality laws that restrict citizenship by birth or descent to particular racial groups.70 This overt racial discrimination can result in statelessness for those who do not have access to another nationality.71 y Additionally, a substantial number of countries retain nationality laws that explicitly dis- criminate on grounds of gender, for example, by preventing women from passing on their nationality to their children. A child who is unable to acquire the nationality of the other parent risks being rendered stateless.72 Children of same-sex couples may likewise ﬁnd them- selves at risk of statelessness due to discriminatory laws and practices concerning the recog- nition of LGBTIQþ partnerships and legal parentage rights.73 In the European context, for https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 MANUFACTURING STATELESSNESS 249 example, advocates have highlighted the problems in civil registration and recognition of legal parenthood in the country of nationality of one or both parents in a same-sex couple, leading to difﬁculties in acquisition of identity documents and nationality by the child, an issue that is the subject of recent litigation before the Court of Justice of the European Union.74 Such transparently discriminatory laws are, however, relatively uncommon and represent one extreme of the spectrum of measures through which states may produce statelessness. Statelessness may also be manufactured in more subtle, less visible ways—increasingly, states rely on more creative techniques to circumvent international law standards, including norms on non-discrimination, by tampering with “neutral” criteria that are relevant to nationality and its absence. This Part uncovers three such criteria that masquerade as unbiased markers for citizenship acquisition and the concomitant exclusion from its beneﬁts: time, space, and documents. 74 See V.M.A. v. Stolichna Obsthina, Rayon “Pancharevo,” Case No. C-490/20, Grand Chamber Judgment (Dec. 14, 2021); Patricia Cabral, Protecting the Right to a Nationality for Children of Same-Sex Couples in the EU – A Key Issue Before the CJEU in V.M.A. v. Stolichna Obsthina (C-490/20), EUR. NETWORK STATELESSNESS BLOG (Feb. 3, 2021), at https://www.statelessness.eu/updates/blog/protecting-right-nationality-children-same-sex-couples- eu-key-issue-cjeu-vma-v. 75 For instance, while the situation of the Palestinians has been described as one of the “most widely known situations of statelessness in the world,” the Article does not use this as a case study since it cannot easily be sep- arated from the larger question of Palestinian statehood, which is beyond the scope of the Article. The situation is equally complex from the point of view of international regulation, where the UN Reliefs and Works Agency (UNRWA) established with a mandate to assist “Palestinian Refugees” may also include stateless Palestinians in certain cases. For a detailed analysis, see INSTITUTE ON STATELESSNESS AND INCLUSION, THE WORLD’S STATELESS 127–132 (2014). Similarly, the Article does not address issues of statelessness that are inextricably linked to the contested international legal status of the putative country of nationality, as is the case for the UN-listed non-self-governing territory of Western Sahara. As Manby notes, the absence of UN recognition of Morocco as the administering power coupled with the limited international recognition of the Sahrawi Arab Democratic Republic as an independent state has resulted in a “nationality vacuum” for the Sahrawis. Nevertheless, some states consider persons of Sahrawi origin who cannot be attributed with Moroccan nationality to be stateless. See Bronwen Manby, Nationality and Statelessness Among Persons of Western Saharan Origin, 34 J. IMMIGRATION, ASYLUM & NATIONALITY L. 9, 25–29 (2020). 6 See Carol Batchelor, Assessment of the #IBelong Campaign Mid-point and the High-Level Segment on elessness, 1 STATELESSNESS & CITIZENSHIP REV. 307–08 (2019). II. STRATEGIC STATELESSNESS Drawing on concrete country-speciﬁc examples, it illustrates how statelessness as statecraft involves the use and misuse of legal ﬁctions that exploit the disjunction between the physical or factual reality of these elements, their subjective experience, and their legal interpretation. It is worth stressing that these illustrative cases, which encompass familiar as well as more obscure situations of statelessness, are intended to highlight the ubiquity of the phenomenon across different regions of the world and do not in any way exhaust the universe of state man- ufactured statelessness.75 Indeed, as various international actors as well as the scholarly liter- ature on statelessness note, the UNHCR’s statistical count of 3.9 million stateless individuals around the world is likely to be a vast underestimate given that many statelessness people live in the shadows and most states do not have designated statelessness determination proce- dures.76 It is also for this reason that I include cases of both de jure and de facto statelessness: the shape-shifting character of the techniques that are used to produce statelessness means that there is often a deliberate blurring of the different forms of statelessness in state practice that make it all the more difﬁcult for domestic as well as international institutions, processes, and actors to respond to statelessness as statecraft. States may also toggle between, or simul- taneously adopt, different strategies to choreograph and sustain statelessness. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 250 Vol. 116:2 While international legal efforts to counter this strategic production of statelessness have been diffused and infrequent, the examples in this Part have also been chosen to capture instances where international legal actors are beginning to mount challenges that have been partially successful in asserting limits to the state’s prerogative to engage in statelessness production. These halting efforts, as Part III will go on to discuss, must be transformed into a bolder and more comprehensive international legal response to statelessness creep. A. Statelessness and Time Governance scholars have remarked on the nation-state “as an unexceptional container of time, as of space. States govern through temporal devices and rationalities, including censuses and other surveys, qualiﬁcation periods for everything from citizenship to bus passes.”77 Marking, measuring, and assigning political value to durational time—the ostensibly neutral and universal time of clocks and calendars—is intrinsic to the structuring and distribution of political power in liberal democracies.78 This political time is used to “carve sovereign bound- aries around citizenries, as a means for measuring qualitative attributes of citizens, and for political exchange value through which amounts of time are demanded of individuals in exchange for rights.”79 The state’s capacity to suspend, contract, expand, and accelerate dura- tional time gives it the superpower to create and erase political membership not through the messy business of moving populations or physical boundaries, but through manipulating its counting of the abstract unit of time. 81 See Rainer Obliger, Ius Sanguinis, in 1 IMMIGRATION AND ASYLUM: FROM 1900 TO THE PRESENT 342, 342–46 (Matthew J. Gibney & Randall Hansen eds., 2005). 82 NOORA LORI, OFFSHORE CITIZENS: PERMANENT TEMPORARY STATUS IN THE GULF 94 (2019). 77 Melanie Grifﬁths, Ali Rogers & Bridget Anderson, Migration, Time, and Temporalities: Review and Prospects (COMPAS Research Resources Paper, Mar. 2013). 78 ELIZABETH F. COHEN, THE POLITICAL VALUE OF TIME: CITIZENSHIP, DURATION, AND DEMOCRATIC JUSTICE 1–4 (2018). 79 80 Id. at 5. 79 Id. at 25. 77 Melanie Grifﬁths, Ali Rogers & Bridget Anderson, Migration, Time, and Temporalities: Review and Prospects (COMPAS Research Resources Paper, Mar. 2013). 78 ELIZABETH F. COHEN, THE POLITICAL VALUE OF TIME: CITIZENSHIP, DURATION, AND DEMOCRATIC JUSTICE 1–4 (2018). 79 Id. at 25. 80 Id. at 5. 81 See Rainer Obliger, Ius Sanguinis, in 1 IMMIGRATION AND ASYLUM: FROM 1900 TO THE PRESENT 342, 342–46 (Matthew J. Gibney & Randall Hansen eds., 2005). 82 NOORA LORI, OFFSHORE CITIZENS: PERMANENT TEMPORARY STATUS IN THE GULF 94 (2019). 1. Freezing Time State formation is an act inscribed not only in space but also in time: the physical bound- aries of a state at the time of its founding often also simultaneously dictate who will be con- sidered its citizens by demanding certain relationships between the individual and their physical location at a particular moment in time. In the words of Elizabeth Cohen, the tem- poral boundaries of state formation highlight how an individual’s rights in relation to the state are not merely a function of who and where they are, but equally, when they happen to be there.80 The widespread temporal device of “zero option rules” acts as a sorting mechanism for differentiating between members of the polity and foreign others by specifying a cut off point for residence or physical presence on the territory that entitles one to citizenship.81 While facially neutral, by demarcating and freezing a population that is labeled as “native” on a certain date or dates, temporal deadlines can be used strategically by states to shape the ethnic, racial, religious, and linguistic composition of their population and create a class of quasi-citizens and stateless persons.82 Like time itself, this act of boundary drawing https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 MANUFACTURING STATELESSNESS 251 is both backward and forward looking: temporal boundaries can harken back to a primordial vision of an original, true population to which the state seeks to return itself. They can signal a deliberate and violent break from the past where the population of the future is no longer shaped in the image of the past. 83 See COHEN, supra note 78, at 43. 84 Nida M. Gelazis, The European Union and the Statelessness Problem in the Baltic States, 6 EUR. J. MIGRATION & L. 225, 228–34 (2004). 85 Priit Järve & Vadim Poleshchuk, Country Report: Estonia, EUDO CITIZENSHIP OBSERVATORY 1 (2013), available at https://cadmus.eui.eu/bitstream/handle/1814/19611/RSCAS_EUDO_CIT_2013_6.pdf?sequence¼3&is Allowed¼y. 86 See Kristine Krūma, Country Report on Citizenship Law: Latvia, EUDO CITIZENSHIP OBSERVATORY 6–7 (2015), available at http://hdl.handle.net/1814/34481. 87 Gelazis, supra note 84, at 234–36. 88 Vello Pettai & Klara Hallik, Understanding Processes of Ethnic Control: Segmentation, Dependency and Co-opta- tion in Post-communist Estonia, 8 NATIONS AND NATIONALISM 505, 510–11 (2002). 89 Järve & Poleshchuk, supra note 85, at 13. 90 See Dimitry Kochenov, EU Inﬂuence on the Citizenship Policies of the Candidate Countries: The Case of the Roma Exclusion in the Czech Republic, 3 J. CONTEMP. EUR. RES. 124, 139 (2007). 0 See Dimitry Kochenov, EU Inﬂuence on the Citizenship Policies of the Candidate Countries: The Case of the ma Exclusion in the Czech Republic, 3 J. CONTEMP. EUR. RES. 124, 139 (2007). 83 See COHEN, supra note 78, at 43. 8 Vello Pettai & Klara Hallik, Understanding Processes of Ethnic Control: Segmentation, Dependency and Co-opta- in Post-communist Estonia, 8 NATIONS AND NATIONALISM 505, 510–11 (2002). 6 See Kristine Krūma, Country Report on Citizenship Law: Latvia, EUDO CITIZENSHIP OBSERVATORY 6–7 15), available at http://hdl.handle.net/1814/34481. 1. Freezing Time 116:2 of entry into the EU and NATO.91 International pressure to resolve the situation of non-cit- izens in the two countries has since ceased—arguably because this would involve international actors admitting their own failures and oversights92—and the issue has been relegated to domestic politics, where this class of non-citizens has gradually acquired greater rights and privileges that nonetheless stop short of full citizenship.93 As Estonian and Latvian cases show, zero-options rules that are argued to be part of the decolonization process present a particularly challenging case for addressing the statelessness that follows. From Asia to Africa to Latin America, newly independent states have inherited artiﬁcial borders that were put in place by colonial powers. These state boundaries were imposed in disregard of pre-existing geopolitical realities, arbitrarily grouping together peo- ples from diverse communities within a single territorial state and simultaneously dividing political, cultural, religious, and ethnic polities into separate territorial units.94 The legacy of these colonial boundaries lives on in the ways in which nationality is conceived and trans- mitted in post-colonial states, putting certain groups at risk of statelessness. In the UAE, for instance, the 1971 law of citizenship has sought to limit UAE citizenship to persons who can trace their ancestry to the 1925 Arab tribes documented by the British census, requiring non- Arab minorities such as Persians, East Africans, and South Asians to undergo naturalization and resulting in a limbo population that is not counted as citizens even if their families have been settled in the country for generations.95 y g The invocation of temporal deadlines to resolve contestations over which groups are enti- tled to membership in newly formed and modernizing nations has been particularly fraught in the African context. Notwithstanding the egregious colonial drawing of the political map in Africa, African states elected to preserve the borders in place at the time of their independence through the Organization of African Unity’s 1964 Cairo Declaration on Border Disputes Among African States, afﬁrming Member States’ commitment to “respect the borders exist- ing on their achievement of national independence.”96 This decision has been criticized in various quarters as indefensibly ahistorical and a catalyst for future conﬂicts. 1. Freezing Time And they can be used to set the terms of a newborn, imagined community that knows no past, but only the future.83 One such instance of temporal deadlines being used to ethnically engineer populations can be seen in the aftermath of the breakup of the Soviet Union, where states such as Latvia and Estonia granted automatic citizenship to individuals who were citizens or permanent resi- dents of these states and their descendants prior to the period of Soviet occupation, rendering stateless thousands of ethnic Russians and other Russian-speaking minorities resident on their territories.84 In Estonia, these “individuals with undeﬁned citizenship” constituting a third of the country’s population could then either seek naturalization as Estonian citizens, opt for the citizenship of another country (including Russia), or simply continue to retain the status of “individuals with undeﬁned citizenship.”85 In Latvia, the 1994 citizenship law enacted after signiﬁcant domestic political wrangling, provided for a “window system” for the naturaliza- tion of “non-citizens” with staggered time periods during which different categories of resi- dents could apply for citizenship.86 Both states argued that their nationality laws were a decolonization measure intended to rectify discrimination against ethnic Estonians and Latvians during the period of Soviet occupation.87 Rather than an ethno-nationalist political project, the rhetoric was thus one of a rightful assertion of sovereignty and independence fol- lowing decades of illegal occupation.88 Demanding naturalization requirements, especially those related to language proﬁciency, meant that the percentage of non-citizens remained fairly signiﬁcant in both countries until the naturalization policies were softened under pressure from international and regional actors such as the Organization for Security and Co-operation in Europe, the Council of Europe, and the European Commission, in particular through policy recommendations championed by the Commission during the pre-accession process to the EU.89 However, as scholars have argued, the Commission largely refrained from questioning the ethnocentric state model adopted by both countries and focused narrowly on the mechanisms and proce- dures for naturalization instead of challenging the discriminatory grounds for citizenship acquisition.90 Political elites in Estonia as well as Latvia were thus spared from having to undertake major reforms in their citizenship policies in order to achieve their policy goals https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW 252 Vol. 91 James Hughes, “Exit” in Deeply Divided Societies: Regimes of Discrimination in Estonia and Latvia and the Potential for Russophone Migration, 43 J. COMMON MARKET STUD. 739, 759 (2005). 92 Id at 756 7 See Frontier Dispute (Burk. Faso v. Niger), 2013 ICJ Rep. 134, paras. 16–19 (Apr. 16) (sep. op., Yusuf, J.). 6 Organization of African Unity, Border Disputes Among African States, AHG/res. 16(I), Assembly of Heads tate and Government Meeting in Its First Ordinary Session in Cairo, UAR, 17–21 July (1964). p p 94 On the continuing legacy of artiﬁcial borders, in particular in Africa and the Middle East, see TIM MARSHALL, PRISONERS OF GEOGRAPHY: TEN MAPS THAT EXPLAIN EVERYTHING ABOUT THE WORLD (2015). 95 LORI supra note 82 at 94 91 James Hughes, “Exit” in Deeply Divided Societies: Regimes of Discrimination in Estonia and Latvia and the Potential for Russophone Migration, 43 J. COMMON MARKET STUD. 739, 759 (2005). 92 Id. at 756. 93 See Järve & Poleshchuk, supra note 85, at 1; Krūma, supra note 86, at 10–11. 94 On the continuing legacy of artiﬁcial borders, in particular in Africa and the Middle East, see TIM MARSHALL, PRISONERS OF GEOGRAPHY: TEN MAPS THAT EXPLAIN EVERYTHING ABOUT THE WORLD (2015). 95 LORI, supra note 82, at 94. 96 Organization of African Unity, Border Disputes Among African States, AHG/res. 16(I), Assembly of Heads of State and Government Meeting in Its First Ordinary Session in Cairo, UAR, 17–21 July (1964). 97 See Frontier Dispute (Burk. Faso v. Niger), 2013 ICJ Rep. 134, paras. 16–19 (Apr. 16) (sep. op., Yusuf, J.). J p p 4 On the continuing legacy of artiﬁcial borders, in particular in Africa and the Middle East, see TIM MARSHALL, SONERS OF GEOGRAPHY: TEN MAPS THAT EXPLAIN EVERYTHING ABOUT THE WORLD (2015). 3 See Järve & Poleshchuk, supra note 85, at 1; Krūma, supra note 86, at 10–11. g y y 97 See Frontier Dispute (Burk. Faso v. Niger), 2013 ICJ Rep. 134, paras. 16–19 (Apr. 16) (sep. op., Yusuf, J.). 93 See Järve & Poleshchuk, supra note 85, at 1; Krūma, supra note 86, at 10–11. J p p 94 On the continuing legacy of artiﬁcial borders, in particular in Africa and the Middle East, see TIM MARSHALL, PRISONERS OF GEOGRAPHY: TEN MAPS THAT EXPLAIN EVERYTHING ABOUT THE WORLD (2015). 95 LORI, supra note 82, at 94. 96 Organization of African Unity, Border Disputes Among African States, AHG/res. 16(I), Assembly of Heads of State and Government Meeting in Its First Ordinary Session in Cairo UAR 17 21 July (1964) 1. Freezing Time However, it has also been defended as a pragmatic solution to avoid armed conﬂicts over territorial dis- putes by putting existing territorial borders in a “holding pattern” to facilitate peaceful reso- lution of territorial disputes between African states.97 While these geographical borders and the populations they demarcate have for the most part proved stable in post-colonial states, decolonization of constituent populations has instead been attempted through changes in temporal borders, negatively impacting populations that are considered non-native and plac- ing them at risk of statelessness. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 253 MANUFACTURING STATELESSNESS One way to accomplish this resetting of the population to a previous self that preceded the colonial encounter is domestic constitutional and legal arrangements and state practice that make citizenship contingent on membership in “tribes” that pre-date colonial population transfers. A striking instance of the resulting statelessness was made visible in the African Commission on Human and People’s Rights decision holding that Côte d’Ivoire had violated the right to nationality of members of the Dioula ethnic community.98 The complaint hinged on the 1961 Ivorian Nationality Code’s adoption of the jus soli principle for grant of nation- ality deﬁning an Ivorian national as an individual who could trace her ancestry to an Ivorian at the time of independence. However, since the Code failed to deﬁne who counted as an “Ivorian,” or as a foreigner, this gave the state the discretion to introduce discriminatory nationality practices under the doctrine of “Ivoirité.” This doctrine was based on the concept of an authentic Ivorian heritage whereby indigenous Ivorians were to be distinguished from “foreign” immigrants from the North who were not entitled to Ivorian nationality.99 Labeling the Dioula people as foreign migrants, state authorities refused to issue them with documents verifying their legal status such as birth certiﬁcates and identity cards and destroyed previously issued documents. 98 Open Society Justice Initiative v. Côte d’Ivoire, Comm. 318/06, 17th Extraordinary Sess., Feb. 19–28, 2015. 99 Id., paras. 53–54, 116–18. 100 Id., paras. 119–20. 101 UNHCR Press Release, Côte d’Ivoire Adopts Africa’s First Legal Process to Identify and Protect Stateless People (Sept. 4, 2020), at https://www.unhcr.org/news/press/2020/9/5f51f33b4/cote-divoire-adopts-africas- ﬁrst-legal-process-identify-protect-stateless.html. 102 For a detailed history of the political history of Côte d’Ivoire’s nationality laws, see MANBY, supra note 70, at 207–20. 99 Id., paras. 53–54, 116–18. 8 Open Society Justice Initiative v. Côte d’Ivoire, Comm. 318/06, 17th Extraordinary Sess., Feb. 19–28, 5. 9 Id 53 54 116 18 100 Id., paras. 119–20. 1. Freezing Time The Commission held that this discriminatory interpretation of the nationality law by the state authorities led to thousands of residents who had been born in Côte d’Ivoire, and to parents who were also born in Côte d’Ivoire, becoming stateless.100 p g While the successful challenge to Côte d’Ivoire’s attempt to exploit the ambiguity in its nationality laws to create a class of stateless Dioula “immigrants” seems like a true victory for regional human rights institutions––in 2020, Côte d’Ivoire even became the ﬁrst African country to adopt a national Statelessness Determination Procedure101—it is impor- tant to bear in mind that this success story played out against the backdrop of favorable domestic political winds. The period between 2006, when the case was brought before the Commission, and 2015, the delivery of the decision, saw the election of President Alassane Ouattara, who had himself previously been accused of not being a native Ivorian citizen and faced signiﬁcant difﬁculties in proving his eligibility to run for political ofﬁce. Unsurprisingly, the reform of nationality laws was not only a legal, but equally a personal, project for President Ouattara, who introduced amendments to nationality laws, simpliﬁed procedures for access to nationality and the issuance of identity documents, and initiated Côte d’Ivoire’s accession to the Statelessness Conventions, measures that were undertaken before the Commission issued its decision.102 It is thus unclear what lessons international legal actors working to combat stateless- ness can learn from the Côte d’Ivoire example, an issue that this Article will revisit in Part III. Indeed, international and regional actors have struggled to devise an effective response to another case of statelessness also engineered by a countdown deadline: denial of citizenship to the Rohingya by the government of Myanmar. The 1982 Burmese Citizenship Law grants https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 254 citizenship to individuals belonging to one of the 135 “national groups” with permanent homes in the state territories before 1823. The government argues that the Rohingya are not “native” to Burma but illegal Bengali immigrants. 103 KELLY STAPLES, RETHEORISING STATELESSNESS: A BACKGROUND THEORY OF MEMBERSHIP IN WORLD POLITICS 139 (2012). 104 Human Rights Council, Res. A/HRC/RES39/2, para. 22 (Oct. 3, 2018). 105 Situation in the People’s Republic of Bangladesh/Republic of the Union of Myanmar, Case No. ICC-01/19-27, Decision Pursuant to Article 15 of the Rome Statute on the Authorisation of an Investigation into the Situation in the People’s Republic of Bangladesh/Republic of the Union of Myanmar, Pre-Trial Chamber III (Nov. 14, 2019). 106 Application of the Convention on the Prevention and Punishment of the Crime of Genocide (Gamb. v. Myan.), Request for Provisional Measures, 2020 ICJ Rep. 3 (Jan. 23). 107 Myanmar: UN Expert Says Current International Efforts Failing, Urges “Change of Course,” UN NEWS (Sept. 22, 2021), at https://news.un.org/en/story/2021/09/1100752. 108 Verena Hölzl, Three Years After Rohingya Exodus, Mismatched Expectations of Justice, NEW HUMANITARIAN (Aug. 24, 2020), at https://www.thenewhumanitarian.org/news-feature/2020/08/24/Bangladesh-Myanmar- Rohingya-international-justice. 1. Freezing Time Since they do not constitute one of the 135 recognized national races, they are not entitled to full Burmese citizenship.103 While there have been some formal legal efforts responding to Myanmar’s discriminatory treatment of the Rohingya prompted by the 2017 atrocities and forced displacement of thousands of Rohingya, they have mostly focused narrowly on the question of accountability for international crimes com- mitted against the Rohingya. Three such proceedings have been running in parallel before differ- ent institutions. The ﬁrst is the Independent Investigative Mechanism for Myanmar set up in 2018 with the mandate to “collect, consolidate, preserve and analyse evidence of the most serious international crimes and violations of international law committed in Myanmar since 2011. . .,” such that this evidence can be used in criminal proceedings.104 Simultaneously, the prosecutor of the International Criminal Court (ICC) has opened investigations into the Bangladesh/Myanmar situation focusing on crimes against humanity, though these will only cover alleged crimes against the Rohingya which took place, at least in part, on the territory of Bangladesh as a state party to the Rome Statute of the ICC.105 Third, proceedings have been instituted before the International Court of Justice (ICJ) by the Gambia against Myanmar alleging violations of the 1948 Genocide Convention. In January 2020, the ICJ ruled in favor of the Gambia’s request for pro- visional measures, ordering Myanmar to comply with its obligations under the Convention, report on their implementation, and take measures to preserve evidence.106 These proceedings have thus far failed to ameliorate the situation of the Rohingya, in par- ticular as concerns the resolution of their statelessness. As late as September 2021, the UN special rapporteur on the situation of human rights in Myanmar, accused the junta regime that came into power following the military coup of February 2021 of continuing to “deny the existence of the Rohingya ethnic minority.”107 Indeed, legal advocates fear that a failure to manage expectations as to what these proceedings can deliver in terms of a path to citizenship for the Rohingya can end up backﬁring: “[i]f you allow the Rohingya to believe that the inter- national courts are in a position to grant them citizenship directly, then these processes are set up to be perceived as failures before they have even started.”108 Part III reﬂects on what this experience might mean for future international litigation efforts to address statelessness. 104 Human Rights Council, Res. A/HRC/RES39/2, para. 22 (Oct. 3, 2018). 105 03 KELLY STAPLES, RETHEORISING STATELESSNESS: A BACKGROUND THEORY OF MEMBERSHIP IN WORLD POLITICS (2012). 04 H R h C l R A/HRC/RES39/2 22 (O 3 2018) 05 Situation in the People’s Republic of Bangladesh/Republic of the Union of Myanmar, Case No. C-01/19-27, Decision Pursuant to Article 15 of the Rome Statute on the Authorisation of an estigation into the Situation in the People’s Republic of Bangladesh/Republic of the Union of anmar, Pre-Trial Chamber III (Nov. 14, 2019). 2. Excising Time 2. Excising Time 2. Excising Time For those who do not pass through the gates of the nation at the time of its birth, the enti- tlement to become part of the political community is contingent on demonstrating ties with the community. A key element in proving these ties is through residence in the state. While states use various criteria such as family and social relationships, linguistic skills, and knowl- edge of culture, institutions, and social practices to measure prospective citizens’ embedded- ness in and loyalty to the community, these are typically over and above the requirement of physical presence over an extended period of time in the community. Time can be assigned this political value due to its ability to serve as an ostensibly neutral proxy for hard-to-measure criteria such as ﬁdelity, cultural assimilation, and civic virtue.109 Different lengths of resi- dence can be imbued with different political values and used as a form of exchange to accord or deny the right of citizenship depending on the duration of residence.110 Indeed, while cri- teria such as citizenship tests or demanding language requirements are often criticized for their exclusionary character, it is generally assumed that it is reasonable and fair for a state to use time as a factor to distribute the precious good of citizenship. What matters for citizenship however, is not always the physical, chronological time that an individual may have spent on the territory, but rather how the state counts that time. The legal value assigned to time can be used strategically to exclude, suspend, or fast-track poten- tial applicants in the queue for citizenship.111 Not only can states discount time spent illegally on the territory for establishing residence, but they can also enact and interpret legal concepts to erase the physical time for which an individual has lived within their boundaries for the purposes of nationality acquisition.112 The Dominican case of the Haitian migrants and Dominicans of Haitian descent is a prominent example of statelessness resulting from the distinction between the passage of chronological time on state territory and its legal counting for the purposes of citizenship enti- tlement and represents one of the few instances of statelessness to have been successfully lit- igated before an international court, the IACtHR. The case of Girls Yean and Bosico v. 109 COHEN, supra note 78, at 8. 110 Noora Lori, Migration, Time, and the Shift Toward Autocracy, in THE SHIFTING BORDER: LEGA CARTOGRAPHIES OF MIGRATION AND MOBILITY 121–22 (2020). 111 Id. at 119. 112 Id. at 119, 122–23. 113 The Girls Yean v. Dominican Republic, supra note 69. 114 American Convention on Human Rights, Nov. 22, 1969, OASTS No. B-32, 1144 UNTS 123. 1. Freezing Time As the above examples show, zero-option laws premised on time as currency typically rely on highly juridical arguments invoking notions of state continuity, illegal occupation, and reclaiming of sovereign authority. And those that are associated with decolonization and https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 255 2022 MANUFACTURING STATELESSNESS the rhetoric of return to an authentic, true, polity may even invite sympathy in some quarters, complicating efforts to identify and address the statelessness that ensues in their wake. 113 The Girls Yean v. Dominican Republic, supra note 69. 114 American Convention on Human Rights, Nov. 22, 1969, OASTS No. B-32, 1144 UNTS 123. 109 COHEN, supra note 78, at 8. 110 2. Excising Time Dominican Republic concerned the Dominican Republic’s refusal to issue birth certiﬁcates to the applicants who had been born on its territory, thereby denying them the nationality of the Dominican Republic which recognizes the principle of jus soli for grant of citizenship.113 The court held that this denial was a violation, among others, of the right to nationality under Article 20 of the American Convention on Human Rights.114 According to the court, the right to nationality was a fundamental, non-derogable right under the Convention and a prerequisite for the exercise of other Convention https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW 256 Vol. 116:2 rights.115 While the state retained authority over the determination of nationality questions, its discretion in this respect was circumscribed by the duty to guarantee to individuals the equal protection of law and the obligation to avoid statelessness.116 Moreover, in light of the peremptory norm on equal protection and non-discrimination, states were under an obli- gation to refrain from enacting discriminatory laws and practices regulating nationality and to take active measures to promote equal protection under the law.117 States were also prohib- ited from adopting laws and practices for the grant of nationality that lead to an increase in statelessness. Statelessness could arise “from the lack of a nationality, when an individual does not qualify to receive this under the State’s laws, owing to arbitrary deprivation or the granting of a nationality that, in actual fact, is not effective.”118 Applying these international norms to the law and practice of the Dominican Republic on the issuance of birth certiﬁcates, the court noted that the Dominican Constitution followed the principle of jus soli for the grant of nationality with the exception of children of diplomats and foreigners who are “in transit.” State authorities in the Dominican Republic considered Haitian migratory workers to be in transit and thus treated their children who were born in the Dominican Republic as not entitled to Dominican nationality. Relying on domestic court interpretations of the phrase “in transit” and the international obligation of non-discrimina- tion irrespective of an individual’s migratory status, the court held that an individual’s migra- tory status was not sufﬁcient justiﬁcation for denying them the right to nationality. 115 The Girls Yean v. Dominican Republic, supra note 69, paras 137–38. 116 Id., para. 140. 117 Id., para. 141. 118 Id., para. 142. 119 Id., paras. 150–56. 120 Id., paras. 165–71. 121 Dinah Shelton & Alexandra Huneeus, In Re Direct Action of Unconstitutionality Initiated Against the Declaration of Acceptance of the Jurisdiction of the Inter-American Court of Human Rights, 109 AJIL 866, 868 (2015). 120 Id., paras. 165–71. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2. Excising Time Further, this status could in any case not be transferred to the children of migrants and individuals had a right to nationality by birth of the state where they were born if they did not have the right to any other nationality.119 y y In this case, the state had applied procedures for the issuance of a birth certiﬁcate in a man- ner that was arbitrary and—especially in light of the vulnerable situation of Haitian migrants and Dominican nationals of Haitian origin—discriminatory, placing the applicants in a sit- uation of statelessness and extreme vulnerability. The state was therefore obligated to take afﬁrmative steps to ensure that the applicants could access birth registration procedures that were reasonable, and in conditions of equality, so as to fully exercise their right to nationality.120 The judgment and various remedial and reparative measures ordered by the court failed, however, to have the desired impact on nationality legislation in the Dominican Republic. Indeed, the situation became even worse with a 2013 ruling of the Dominican Constitutional Court ordering executive review and retroactive withdrawal of the Dominican citizenship of children born on the territory of the Dominican Republic whose parents who did not have a regular migration status in the country, rendering stateless tens of thousands of Dominicans of foreign descent.121 One year later, this led to the IACtHR https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press MANUFACTURING STATELESSNESS 257 2022 judgment in the case of Expelled Dominicans and Haitians v. Dominican Republic, addressing the arbitrary detention and expulsion of Haitians and Dominicans of Haitian descent from the Dominican Republic and the barriers to registering and obtaining nationality for individ- uals of Haitian descent born in the Dominican Republic.122 As part of its legal analysis, the court undertook an extensive factual analysis of the discrimination faced by Haitian migrants and Dominicans of Haitian descent, which also impacted their ability to obtain ofﬁcial iden- tity documents. 2. Excising Time The attainment of a birth certiﬁcate––which was a requirement for obtaining nationality––was difﬁcult both due to the social conditions in which these births took place as well as discriminatory ofﬁcial practices that accompanied efforts to register births with the authorities and the suspicion that attached to the subsequent use of these documents.123 p q The court then turned to the right to nationality under Article 20 of the Convention and reiterated its holding in Girls Yean and Bosico on the scope and importance of the right, adding that if the state is not certain that a child born in its territory would be able to obtain the nation- ality of another state, whether for formal legal reasons, or due to de facto obstacles, it is obligated to grant nationality in order to avoid statelessness at birth.124 Notwithstanding the interpretation of the nationality laws by the Dominican Constitutional Court, “basic standards of reasonable- ness” had to be met when evaluating the state’s obligations under the American Convention. Domestic constitutional amendments and court cases had held that migrants in an irregular sit- uation should be considered as “aliens in transit” irrespective of the length of their physical pres- ence on the territory as distinguished from the period of ten days that applied to the separate category of “transient aliens.”125 In line with its previous decision in Girls Yean and Bosico, and contrary to the decision of the Constitutional Court, the court thus held that “to consider a person . . . in transit . . . the State must respect a reasonable time frame, and be coherent with the fact that an alien who develops ties in a State cannot be compared to a transient or to a person in transit.”126 Furthermore, since the state had not proved that these individuals would in fact be able to acquire the nationality of another state––in this case, Haiti––its decision to deprive them of the nationality of the Dominican Republic risked rendering them stateless and violated their right to nationality and related Convention rights, including the right to recognition of juridical personality, the right to a name, and the right to identity.127 The ruling, which was hailed as an important moment for putting issues of arbitrary dep- rivation of nationality on the international legal and political map, has, however, failed to have the desired impact not only internationally, but also on home territory. 122 Expelled Dominicans and Haitians v. Dominican Republic, supra note 69. 123 Id., paras. 163–66. 124 Id., paras. 253–61. 125 Id., paras. 282–88. 126 Id., para. 294. 127 Id. paras. 286–99. 128 See Bridget Wooding, Seizing New Opportunities to Address Statelessness in the Dominican Republic, EUR. NETWORK STATELESSNESS BLOG (Nov. 19, 2020), at https://www.statelessness.eu/updates/blog/seizing-new- opportunities-address-statelessness-dominican-republic. 2. Excising Time The Dominican Republic’s executive branch moved immediately to reject the ruling and subsequent ofﬁcial public statements have reiterated the ﬁction that the Dominican Republic has no stateless persons and that the existing legal framework is adequate for protecting those who might be at risk of statelessness.128 Even the limited legal measures to address statelessness https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 258 introduced by the Dominican Republic following the severe criticism of the 2013 constitu- tional decision, and that were judged to be ﬂawed and insufﬁcient in Expelled Dominican, have been sought to be repealed by nationalist groups.129 Procedures for obtaining the rele- vant identity documents continue to be characterized by both structural discrimination against Haitian migrants and Dominicans of Haitian descent and additional barriers imposed by individual ofﬁces and ofﬁcials at their discretion.130 According to some estimates, the Dominican Republic remains the “single largest case of statelessness in Latin America.”131 p g g A more successful regional intervention, albeit one that was dependent on domestic legal efforts for resolution of statelessness, was the pilot judgement of the European Court of Human Rights (ECtHR) concerning the situation of stateless persons in Slovenia.132 This class of stateless persons was the outcome of a strategic legalistic effort by the Republic of Slovenia to mark time differently for eligibility for citizenship following its independence from the former Yugoslavia. Under the new citizenship law of Slovenia, citizens of other for- mer Yugoslav republics who were permanent Slovenian residents could acquire Slovenian cit- izenship by lodging an application within six months provided they had been Slovenian permanent residents on the day Slovenia held a plebiscite on independence. This effectively excluded from citizenship citizens of other former Yugoslav republics who had habitually resided and worked in Slovenia but who were not registered as immigrants in Slovenia and therefore did not count as permanent residents. Moreover, even registered immigrants in Slovenia who did not apply for citizenship within the six-month period or those whose appli- cations for citizenship were rejected were deemed foreigners rather than residents under the provisions of the Aliens Act. Their records were struck from the register of permanent resi- dents and transferred to the aliens register without any notice. 129 Dominicanos por Derechos, The Institute on Statelessness and Inclusion & The Center for Justice and International Law, Joint Submission to the Human Rights Council at the 32nd Session of the Universal Periodic Review: The Dominican Republic, paras. 22–23, 31 (July 12, 2018), available at https://ﬁles.institutesi.org/ UPR32_DominicanRepublic.pdf. 129 Dominicanos por Derechos, The Institute on Statelessness and Inclusion & The Center for Justice and International Law, Joint Submission to the Human Rights Council at the 32nd Session of the Universal Periodic Review: The Dominican Republic, paras. 22–23, 31 (July 12, 2018), available at https://ﬁles.institutesi.org/ UPR32_DominicanRepublic.pdf. 130 Id., paras. 24–32. 131 Wooding, supra note 128. 132 Kurić and Others v. Slovenia, 2012-IV Eur. Ct. H.R. 1 [hereinafter Kurić 2012]. 133 Vlasta Jalušič & Jasminka Dedić , (The) Erasure – Mass Human Rights Violation and Denial of Responsibility: The Case of Independent Slovenia, 9 HUM. RTS. REV. 93, 94–96 (2008). 134 Kurić 2012, supra note 132; Kurić and Others v. Slovenia, Eur. Ct. H.R. 2014. Wooding, supra note 128. 132 Kurić and Others v. Slovenia, 2012-IV Eur. Ct. H.R. 1 [hereinafter Kurić 2012]. 133 Vlasta Jalušič & Jasminka Dedić , (The) Erasure – Mass Human Rights Violation and Denial of Responsibility: The Case of Independent Slovenia, 9 HUM. RTS. REV. 93, 94–96 (2008). 134 Kurić 2012, supra note 132; Kurić and Others v. Slovenia, Eur. Ct. H.R. 2014. 131 Wooding, supra note 128. 132 130 Id., paras. 24–32. 135 Committee of Ministers, Resolution CM/ResDH(2016)112, Execution of the Judgments of the European Court of Human Rights, Kurić and Others Against Slovenia, 1257th Meeting of the Ministers’ Deputies (May 25, 2016), at http://hudoc.echr.coe.int/eng?i¼001-163580. 136 Ginger Hervey, Justice Evades Slovenia’s “Erased” Citizens, POLITICO (Mar. 28, 2017), at https://www.polit- ico.eu/article/justice-evades-slovenia-erased-citizens-yugoslavia; Anja Vladisavlj, Status Revoked: Slovenia’s “Erased” Recall Long Struggle for Justice, BALKAN TRANSITIONAL JUST. (Feb. 26, 2021), at https://balkaninsight. com/2021/02/26/status-revoked-slovenias-erased-recall-long-struggle-for-justice. 137 See Mariane C. Ferme, Introduction: Localizing the State, 86 ANTHROPOLOGICAL Q. 957, 958 (2013). 138 LORI, supra note 82, at 6. 139 Roos Pijpers, Waiting for Work: Labour Migration and the Political Economy of Borders, in THE ASHGATE RESEARCH COMPANION TO BORDER STUDIES 417, 432 (Doris Wastl-Walter ed., 2011). 140 Barry Schwartz, Waiting, Exchange, and Power: The Distribution of Time in Social Systems, 79 AM. J. SOC. 841, 844, 847 (1974). 2. Excising Time This population of mainly non- ethnic Slovenes, some of whom had been born in Slovenia and others who had been living there for several decades, became known as the “erased,” becoming de facto aliens or stateless persons illegally residing in Slovenia.133 Following signiﬁcant civil society mobilization, this erasure was challenged and held to be unconstitutional by the Slovenian domestic courts, following which Slovenia amended its laws to permit more of those whose records had been deleted to apply for permanent resi- dence. The ECtHR deemed these remedies to be inadequate and as perpetuating the state of limbo for a signiﬁcant percentage of the erased. It awarded the applicants non-pecuniary damages for their rights violations resulting from the situation of legal uncertainty and vul- nerability, ordered Slovenia to establish a domestic compensation scheme, and subsequently handed down a just satisfaction judgment for pecuniary damages.134 In 2016, the Council of Europe’s Committee of Ministers, which is responsible for supervising the implementation of the ECtHR’s judgements, declared itself satisﬁed that all the measures required had been https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 MANUFACTURING STATELESSNESS 259 adopted and closed its examination of the case.135 However, critics argue that a sizeable num- ber of the erased were unable to restore their legal status and were disqualiﬁed from claiming compensation. These include individuals who were deported from, or denied re-entry into, Slovenia pursuant to the loss of status. There is also little political will or momentum to attend to these cases any longer.136 As Part III will argue, the varying impact of progressive rulings by the IACtHR and ECtHR on statelessness on the ground holds important lessons for actors pursuing statelessness litigation before supranational courts. Similar to the act of freezing time, excising time does not involve physically expunging per- sons from the territory of the state. Rather, physical erasure is achieved through temporal era- sure whereby the state manipulates chronological time by making the temporal credit for the physical time spent on the territory contingent on the particular legal status of the individual during this period. 35 Committee of Ministers, Resolution CM/ResDH(2016)112, Execution of the Judgments of the European urt of Human Rights, Kurić and Others Against Slovenia, 1257th Meeting of the Ministers’ Deputies (May 25, 6), at http://hudoc.echr.coe.int/eng?i¼001-163580. 6 139 Roos Pijpers, Waiting for Work: Labour Migration and the Political Economy of Borders, in THE ASHGATE RESEARCH COMPANION TO BORDER STUDIES 417, 432 (Doris Wastl-Walter ed., 2011). 140 Barry Schwartz, Waiting, Exchange, and Power: The Distribution of Time in Social Systems, 79 AM. J. SOC. 841, 844, 847 (1974). 3. Dead Time Between those who pass through the gates of citizenship and those who are turned away lies another category of persons who are effectively stateless: those who are kept waiting indeﬁ- nitely at the gate. States control and tamper with the temporal register not only through freez- ing and excising time but equally through its suspension. Like Kafka’s “man from the country,” individuals waiting at the temporal borders of citizenship may wait patiently at the border their entire life, pestering, cajoling, and bribing the guards to let them in, only to discover near the end that the gate had been meant for them alone and would cease to exist with their death.137 While waiting can be the result of incompetence and bureaucratic delays on the part of the state, waiting can also be a deliberate state tactic to continuously defer citizenship.138 p Conceptualizing waiting, not as a “by-product of state institutions and bureaucracies” but as a “management technique that is not outside but fully part of the state,”139 highlights the ways in which the state can use delays, deferrals, and postponements to exercise power over putative citizens. While waiting is a universal human experience, each individual experiences waiting in his own way depending on his position in the social hierarchy. Waiting time is unequally distributed with those at the top of the social system waited upon by those at the bottom who do the most waiting.140 Waiting can be both a form of inducing compliance and a form of sanctioning by the state. As Auyero’s ethnographic account of welfare clients waiting in the welfare ofﬁce shows, the arbitrariness and uncertainty that characterizes this https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 Vol. 3. Dead Time 116:2 260 waiting zone reinforces poor people’s conviction that instead of negotiating with the author- ities, their only hope for welfare relief is to be patient and comply with the authorities’ dic- tates.141 Beyond the feelings of dependence and subordination that waiting engenders, an individual who is kept waiting indeﬁnitely without any indication of how long this wait may last or whether it will have any concrete outcome may even experience waiting as a form of punishment.142 p The de facto statelessness of individuals who ﬁnd themselves in this condition of state engi- neered precariousness can be seen in Noora Lori’s superb analysis of migrant populations who occupy a limbo status in the UAE either because their naturalization cases are continuously adjourned by the state or because they are assigned visa statuses that are intended to be tem- porary but are continuously renewed without, however, bringing them any closer to eligibility for citizenship.143 Lori refers to the kafala system, the guest worker program of the UAE which brings temporary contractual laborers to the UAE with the intention of meeting short term labor demands in a particular sector. While these temporary contractual laborers are expected to leave once these short-term needs are met and are hence not eligible to apply for permanent residence or citizenship, in practice, their residency is constantly extended through labor contracts that are renewed by domestic sponsors. This has a created a class of non-citizen residents who have been settled in the UAE—at times for several genera- tions—while formally being considered “temporary” by the UAE.144 In the absence of any formal rights of permanent residence or citizenship, these “guests” to the UAE are deportable at the discretion of the government notwithstanding their ties to the country.145 g g In addition to excluding guest workers from citizenship, the UAE also employs delaying tactics to indeﬁnitely postpone the citizenship of otherwise eligible individuals. Since all UAE residents, including UAE born residents, who cannot trace their lineage to the Arab tribes of 1925 are obligated to go through a naturalization process, one way to defer citizenship is by refusing to either approve or deny the naturalization application. 141 Javier Auyero, Patients of the State: An Ethnographic Account of Poor People’s Waiting, 46 LATIN AM. RES. REV. 5, 21–22 (2011). 142 Schwartz, supra note 140, at 862. 143 LORI, supra note 82, at 5–6. 144 Id. at 137–39. 145 Id. at 133. 146 Id. at 160–61. 147 Id. at 6–12. 148 See Ferme, supra note 137, at 958. 149 See Ayelet Shachar, The Shifting Border: Legal Cartographies of Migration and Mobility, in THE SHIFTING BORDER, supra note 110, at 4–6. 150 See Weissbrodt & Collins, supra note 49, at 256. 151 See Alison Kesby, The Shifting and Multiple Border and International Law, 27 OXFORD J. LEG. STUD. 101, 102–03 (2007). 152 COHEN, supra note 78, at 29–30. 153 See Kesby, supra note 151, at 110. 154 Shachar, supra note 149, at 43. B. The Spatiality of Statelessness B. The Spatiality of Statelessness The modern nation-state is premised on the notion of stability of territorial borders that are ﬁxed in both time and place. While there have been numerous legal attempts to address the statelessness that can result when these borders come under stress during periods of state dis- solution, succession, and transfer of territory, what is seldom acknowledged is that, even in normal times, the state’s boundaries can be discontinuous, contingent, and unstable.148 Rather than ﬁxed lines on a map that ﬁt neatly together to demarcate the exclusive domains of territorially circumscribed sovereign authority, state borders constitute a landscape of time and space that is portable and porous, that the state can continuously make and unmake through legal constructs.149 Scholarship on statelessness has failed to grasp the extent to which this legal fortiﬁcation and reinvention of the spatial border can be used to redeﬁne the principles along with political membership can be allocated, distributed, and rescinded by states. One example of this faith in spatial stability are legal advocacy efforts seeking to persuade states to adopt the jus soli principle for citizenship as one of the primary pathways for avoiding statelessness.150 While citizenship based on jus soli would go some way to prevent statelessness in certain countries, it nonetheless presupposes a clearly demarcated physical ter- ritory with stable borders rather than one characterized by a multitude of shifting borders that are negotiated and renegotiated during processes of admission to political membership.151 The modern nation-state is premised on the notion of stability of territorial borders that are ﬁxed in both time and place. While there have been numerous legal attempts to address the statelessness that can result when these borders come under stress during periods of state dis- solution, succession, and transfer of territory, what is seldom acknowledged is that, even in normal times, the state’s boundaries can be discontinuous, contingent, and unstable.148 3. Dead Time While the naturalization process can legally be initiated after seven years of residence, in practice, naturalization appli- cations of even individuals who have lived in one of the emirates for decades continue to be in abeyance, incentivizing them to persist in their engagement with the state in the hope of an eventual resolution.146 From the state’s perspective, the tactic of indeﬁnite postponement is inﬁnitely superior to one of outright citizenship denial. Not only is this permanently “temporary” status invisible to the international legal framework on statelessness, but persuading would-be citizens to engage in a perpetual waiting game where their patience and labor may be rewarded with the beneﬁts of citizenship serves the state’s economic purposes while allowing it to avoid dif- ﬁcult or unpopular political judgments on state membership, identity, and inclusion.147 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 261 MANUFACTURING STATELESSNESS 2022 148 See Ferme, supra note 137, at 958. 51 See Alison Kesby, The Shifting and Multiple Border and International Law, 27 OXFORD J. LEG. STUD. 101, –03 (2007). 155 See Willem van Schendel, Stateless in South Asia: The Making of the India-Bangladesh Enclaves, 61 J. ASIAN STUD. 115, 139 (2002). 156 Id. at 116–17. 157 Id. at 124–26. 158 Hosna J. Shewly, Abandoned Spaces and Bare Life in the Enclaves of the India–Bangladesh Border, 32 POL. GEO. 23, 24, 27 (2013). 159 Md. Azmeary Ferdoush & Reece Jones, The Decision to Move: Post-Exchange Experiences in the Former Bangladesh–India Border Enclaves, in ROUTLEDGE HANDBOOK OF ASIAN BORDERLANDS 255, 257–59 (Alexander Horstmann, Martin Saxer & Alessandro Rippa eds., 2018). 160 Id. at 263. 1. Perforating Borders In dissecting the time-space nexus in the construction of political borders, Cohen draws on the little known historical usage of the word “deadline,” which originally did not signify a time limit for the completion of a task, but rather the perimeter around a military prison that the prisoner was forbidden to cross on pain of getting shot. The deadline was thus a territorial limit, which permitted the state to make life-and-death decisions over the bodies under its control.152 Modern state territorial boundaries too can serve a life-and-death func- tion by acting as a sorting device by which the state categorizes individuals into citizens who fall within these boundaries and the foreigners without who have no entitlement to the pro- tection of the state and its political institutions. However, territorial borders are not merely a function of geography but also of the law and legal institutions that the state sets up to deﬁne and police these borders.153 In the evocative language of Ayelet Shachar, to prevent individ- uals from crossing—literally and metaphorically—through its borders, the state may even go to the lengths of excising its own territory and creating exceptional zones, as if it were “per- forating itself geographically.”154 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 262 Vol. 116:2 THE AMERICAN JOURNAL OF INTERNATIONAL LAW The borderlands of states are one such zone of exception where spatiality can be legally reconﬁgured to produce citizens and stateless foreigners. Contestations over citizenship in borderlands may be a legacy of artiﬁcially constructed borders during colonization, state dis- solution and transfer of territory, or population ﬂows due to political instability and market imperatives. The curious case of persons living in “enclaves” and consigned to de facto statelessness is one instance of this contestation. Largely ignored as “footnotes to the history of state forma- tion,”155 enclaves are segments of a state’s territory that are fully enclosed by another state’s territory. Though enclaves were somewhat frequent in the early period of Westphalian nation-state formation, modern enclaves are few and far between. 55 See Willem van Schendel, Stateless in South Asia: The Making of the India-Bangladesh Enclaves, 61 J. ASIAN D. 115, 139 (2002). 56 Id 116 17 1. Perforating Borders Though they now possess certain formal markers of citizenship such as voter identiﬁcation cards, the government has largely failed to provide them with land documentation and rehabilitation measures crucial to their https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 263 MANUFACTURING STATELESSNESS livelihoods, resulting in continuing difﬁculties with accessing the rights and privileges atten- dant on citizenship.161 Though enclaves may constitute a rather exceptional phenomenon of perforated borders, bor- derlands also yield other forms of state-manufactured statelessness and liminality. The “hill tribes” of Thailand, the ofﬁcial category used by the Thai state to designate various ethnic groups living in the uplands of the Thai borderland, are another example of subjects without citizen- ship.162 The highlanders have been constructed by the Thai state as “wild non-Thai others” liv- ing in the peripheral uplands as a way of reinforcing the Thai-ness of lowland Thais who constitute the core of the nation-state.163 Labeling the highlanders variously as insurgents, opium producers, and forest destroyers who threatened the security of the state, the government ofﬁcially classiﬁed and registered them as “hill tribes” in the mid-twentieth century, followed by severe restrictions on their mobility and land use throughout the twentieth century.164 Following the ofﬁcial classiﬁcation of hill tribes, Thailand made various efforts to accurately count and identify this population for administrative purposes. Numerous population surveys and registration campaigns were carried out to document and register highlanders and special color coded identity cards were issued to grant them semi-permanent residency. 161 See Shiv Sahay Singh, 5 Years After Land Border Agreement, Former Enclave Dwellers in Dire Straits, HINDU (Aug. 3, 2020). 162 Mika Toyota, Subjects of the Nation Without Citizenship: The Case of “Hill Tribes” in Thailand, in MULTICULTURALISM IN ASIA 110, 111 (Will Kymlicka & Baogang He eds., 2005). 163 Id. at 115. 164 Amanda Flaim, Daniel Ahlquist & Lindy Williams, How Statelessness, Citizenship, and Out-migration Contribute to Stratiﬁcation Among Rural Elderly in the Highlands of Thailand, 99 SOCIAL FORCES 323, 336 (2020). 165 Amanda Flaim, Problems of Evidence, Evidence of Problems: Expanding Citizenship and Reproducing Statelessness Among Highlanders in Northern Thailand, in CITIZENSHIP IN QUESTION: EVIDENTIARY BIRTHRIGHT AND STATELESSNESS 147, 159–61 (Benjamin N. Lawrance & Jacqueline Stevens eds., 2017). 166 Toyota, supra note 162, at 118–19. 167 Flaim, supra note 165, at 161. 168 Id. at 162. 169 Flaim, Ahlquist & Williams, supra note 164, at 337. 1. Perforating Borders Until recently, the vast majority of enclaves in the world existed on the borderlands of India and Bangladesh, a result of the independence and partition of British India into India and what was then East Pakistan (now Bangladesh) in 1947.156 Forming ﬁssures in the territories of each of these states, they came to be treated as non-state spaces, inhabited by islands of putative citizens who were ungoverned by either the state of their formal citizenship (the home state) or the state within whose territory they were resident (the host state). Not only did each state often deny access to ofﬁcials of the other state to their own enclave for administrative purposes, but inhabitants of the enclaves were also largely trapped inside the enclaves, unable to move freely outside the enclave and cut off from the life of the host state.157 Lacking all the trappings of citizenship such as a birth certiﬁcate, identity card or passport, or access to state facilities and social ser- vices, enclave inhabitants were abandoned by both their home state and the host state in the non-state space of the enclave.158 Although an initial attempt was made to absorb these enclaves into the territory of the sur- rounding territorial state through the 1958 Nehru-Noon Accords between India and Pakistan, the issue of the enclaves became mired in political opposition given the volatile rela- tionship between India and Bangladesh. It was only in 2015 that the two countries imple- mented a decades-old agreement to exchange the enclaves, with enclave residents being given the choice to acquire the citizenship of their host state or “return” to the home state of their formal citizenship.159 While the exchange has formally resolved the statelessness of enclave inhabitants, the former status of the enclaves as abandoned spaces means that the work of constructing the apparatus of the state around these new citizens who lack both affective and material ties to the states of their citizenship will prolong their limbo status for some time to come.160 Recent reports show that the effects of the nearly seven decades of stateless- ness experienced by enclave residents still persist. 169 Flaim, Ahlquist & Williams, supra note 164, at 337. 168 Id. at 162. 164 Amanda Flaim, Daniel Ahlquist & Lindy Williams, How Statelessness, Citizenship, and Out-migra Contribute to Stratiﬁcation Among Rural Elderly in the Highlands of Thailand, 99 SOCIAL FORCES 323, 336 (20 1. Perforating Borders However, due to lack of knowledge of the terrain, lack of resources and administrative capacity, and prob- lems with processing the data, these surveys were far from complete and many highland villages were never found or registered, excluding inhabitants from identity documents signifying rights of residence.165 The administrative non-existence of highland minority villages in the ofﬁcial territory of the Thai state has thus produced several generations of highlanders who the state does not even register as hill tribes, thus excluding them from Thai citizenship.166 And even for the highlanders who were registered, this did not grant them automatic cit- izenship on the basis of either jus soli or jus sanguinis, but only entitled them to subsequent citizenship by naturalization.167 In the absence of proof of registration, the primary way for stateless highlanders who have been physically resident in Thailand for generations to qualify for Thai citizenship is through ofﬁcially documented proof of birth on the territory before the date of February 26, 1992, which the majority of them lack.168 As scholars note, the Thai state has largely refused to acknowledge the reasons for the lack of this documentation and has persisted in demanding evidence that most elderly highlanders do not possess, resulting in a situation where they have no path to citizenship.169 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW 264 Vol. 116:2 Even for the highlanders who do possess the appropriate state-produced documentation, ethnographic accounts reveal that the documents themselves are often riddled with inconsis- tencies and subject to the unpredictable evaluation of state ofﬁcials, that prevents them from evidencing citizenship.170 Given the constantly changing rules and policies and a prolifera- tion of different categories of registration numbers and identity cards that distinguish between various kinds of non-citizens, errors in registration practices by state ofﬁcials are not uncom- mon. Once produced, these errors can not only ensnare the individual in a bureaucratic maze of attempting to acquire the appropriate paperwork that evidences his claim to citizenship, but also highlight the fragility of these documents whose worth may always be challenged, rejected, or revoked.171 Not everyone has to bear the brunt of this hermeneutics of suspicion. 170 Janepicha Cheva-Isarakul, “Diagnosing” Statelessness and Everyday State Illegibility in Northern Thailand, 1 STATELESSNESS & CITIZENSHIP REV. 214, 216–27 (2019). 171 Id. at 233–35. 172 Flaim, Ahlquist & Williams, supra note 164, at 337, 361. 173 Toyota, supra note 162, at 128. 174 See ÉTIENNE BALIBAR, WE, THE PEOPLE OF EUROPE?: REFLECTIONS ON TRANSNATIONAL CITIZENSHIP 109 (2004) (characterizing the border as a “limit institution”). 175 Étienne Balibar, Europe as Borderland, at 4 (The Alexander von Humboldt Lecture in Human Geography, University of Nijmegen) (Nov. 10, 2004), available at http://gpm.ruhosting.nl/avh/Europe%20as% 20Borderland.pdf. 1. Perforating Borders The ethno-nationalist construction of the Thai state means the burden of enduring stateless- ness falls disproportionately on ethnic minorities like the highlanders who have long been subject to state discrimination.172 In addition to not being able to access the rights attached to citizenship, they are also at risk of being classiﬁed as unauthorized foreign workers and refugee populations from Burma, with whom they share ethnic and kinship ties, and labeled as illegal migrants.173 The legal conﬁguration of territory in borderlands represents state attempts to pin down and delineate the ambiguous nature of belonging of the populations that inhabit and traverse these territories. In the process, inhabitants of these non-state spaces may be rendered outsid- ers in the name of state building, labor migration enforcement, and domestic security. 170 Janepicha Cheva-Isarakul, “Diagnosing” Statelessness and Everyday State Illegibility in Northern Thailan STATELESSNESS & CITIZENSHIP REV. 214, 216–27 (2019). 171 Id. at 233–35. 173 Toyota, supra note 162, at 128. 2. Hardening Borders In order for the territorial border to identify and categorize people into insiders and out- siders with certainty, the border itself must give the illusion of being the stable eye in the storm of ever-changing population movements within and outside its limits.174 In bringing persons within the territorial fold and thereby the political authority of the state, the state engages in the production of collective identities that are developed and strengthened in opposition to external others. Since this exercise in building collective identity is often pre- mised on a ﬁctional unity among “insider” citizens, the presence of outsiders within poses a threat to the nation-state’s territorial and political authority. In the words of Balibar, the attempt at consolidation is tinged with the ever-present danger that “outsiders or “nomadic subjects,” in the broad sense, resist territorialization, remain located outside the normative “political space,” in the land of (political) nowhere.”175 While Balibar’s reference to nomadic subjects is intended to broadly refer to all internal others within the state’s physical bound- aries, his play on words highlights the different categories of people who pose a particular https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 265 MANUFACTURING STATELESSNESS challenge to the state’s territorial concept of citizenship: groups that are excluded from its territorial imaginary and nomadic peoples.176 The bidoon (literally those “without nationality”) of Kuwait are one group that has fallen victim to a “hyper-territorialised and hyper-localised” delineation of the boundaries of Kuwaiti membership.177 The 1959 Nationality Law based national membership on the con- cept of a settled population: entitlement to automatic citizenship was limited to individuals who could prove uninterrupted residence in the territory of the emirate since 1920 and their descendants (through the male line). However, the conception of territory in 1920 was not one of the “national” territory––since the borders of Kuwait were yet to be deﬁned in 1920–– but the walls of the old town and surrounding oases.178 The initial beneﬁciaries of Kuwaiti citizenship were thus an eclectic mix of town dwellers––known as hadar in contrast to the badû (bidoon) or the desert dwellers––that included the royal family and merchants, but also slaves and artisans. 176 See Kesby, supra note 151, at 111 (using Balibar’s theory to dissect states’ closure of geographical borders to the Roma who are widely portrayed as a non-territorial nomadic people). 178 Id. at 232–33. 33 180 Claire Beaugrand, Borders and Spatial Imaginaries in the Kuwaiti Identity, 23 GEOPOLITICS 544, 555–56 (2018). 181 See Amnesty International, Kuwait: Mandate of Abusive Government Body in Charge of Stateless Bidun People Extended (Nov. 24, 2020), at https://www.amnesty.org/en/latest/news/2020/11/kuwait-mandate-of-abusive-gov- ernment-body-in-charge-of-stateless-bidun-people-extended. 182 See Tom Gunnar Hoogervorst, Ethnicity and Aquatic Lifestyles: Exploring Southeast Asia’s Past and Present Seascapes, 4 WATER HIST. 245 (2012). 177 Claire Beaugrand, Statelessness & Administrative Violence: Bidūns’ Survival Strategies in Kuwait, MUSLIM WORLD 228, 233 (2011). 178 176 See Kesby, supra note 151, at 111 (using Balibar’s theory to dissect states’ closure of geographical borders to the Roma who are widely portrayed as a non-territorial nomadic people). 177 Claire Beaugrand, Statelessness & Administrative Violence: Bidūns’ Survival Strategies in Kuwait, MUSLIM WORLD 228, 233 (2011). 178 Id. at 232–33. 179 Id. at 233. 180 Claire Beaugrand, Borders and Spatial Imaginaries in the Kuwaiti Identity, 23 GEOPOLITICS 544, 555–56 (2018). 181 See Amnesty International, Kuwait: Mandate of Abusive Government Body in Charge of Stateless Bidun People Extended (Nov. 24, 2020), at https://www.amnesty.org/en/latest/news/2020/11/kuwait-mandate-of-abusive-gov- ernment-body-in-charge-of-stateless-bidun-people-extended. 182 See Tom Gunnar Hoogervorst, Ethnicity and Aquatic Lifestyles: Exploring Southeast Asia’s Past and Present Seascapes, 4 WATER HIST. 245 (2012). 179 Id. at 233. 180 Claire Beaugrand, Borders and Spatial Imaginaries in the Kuwaiti Identity, 23 GEOPOLITICS 544, 555 (2018). 181 g , p g y, , (2018). 181 See Amnesty International, Kuwait: Mandate of Abusive Government Body in Charge of Stateless Bidun People Extended (Nov. 24, 2020), at https://www.amnesty.org/en/latest/news/2020/11/kuwait-mandate-of-abusive-gov- ernment-body-in-charge-of-stateless-bidun-people-extended. 182 See Tom Gunnar Hoogervorst, Ethnicity and Aquatic Lifestyles: Exploring Southeast Asia’s Past and Present 4 4 ( ) 2. Hardening Borders In addition, groups that had close networks with this sedentary pop- ulation of town-dwellers through frequent presence in the territory, such as individuals living in the peripheral settlements, were included in this qualiﬁed citizenship.179 p p q p The inexperienced Committees of Nationality who were in charge of applying these nation- ality criteria to evaluate claims of citizenship had to rely mainly on oral testimonies of individuals and their community members rather than documentary evidence for proof of settlement. In the case of the bidoon who had applied for citizenship, the Committees were confronted with the dilemma that though the bidoon belonged to tribes that had settled within the “territorial limits” of the Kuwaiti desert, they could not claim to have lived within the town walls. As such, the work of the Committees that was premised on an urban concept of citizenship with a clear divide between the hadar and the badû was unable to account for the bidoons’ claim to citizenship, a geographic and mental map that continues to inﬂuence the perception ofthe bidoon as internal others within Kuwait.180 Though the Kuwaiti government has made repeated promises over the years to ostensibly “resolve” the bidoon situation, its ofﬁcial agency tasked with this mandate–– the Central System for the Remedy of the Situation of Illegal Residents established in 2010––has persistently labeled the bidoon as “illegal residents,” reportedly refusing to issue them with iden- tity documents unless they confess to their “true” nationalities.181 The mental map of the modern nation-state in which groups such as the bidoon are trapped is often not only territorially, but also terrestrially, rigid: the borders are drawn on land rather than water, excluding the diverse ways of identity formation and belonging that are based on seascapes.182 Members of groups who inhabit the maritime interstices of https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 266 these terra ﬁrma shaped boundaries ﬁnd themselves without recognition in any of the terri- tories claimed by nation-states. These groups, variously known as “sea gypsies” or “sea nomads,” are often arbitrarily lumped together with “irregular migrants” notwithstanding their prolonged presence in maritime regions that straddle the borders of nation-states.183 p g p g The Bajau Laut of Malaysia are one such sea-faring nomadic population inhabiting an area that was traditionally part of the Sulu state and is now apportioned between Malaysia and the Philippines. 2. Hardening Borders Occupying the lowest rung in the ethnic hierarchy and exploited by other dominant ethnic groups, the boat-dwelling Bajau are considered a pariah people.184 Common to the var- ious origin myths of the Bajau as a separate people is the commission of an act of sacrilege due to which they were cast out to sea and condemned to live the life of nomads as a people without religion and without a territorial home.185 This traditional Bajau nomadic existence and way of life underwent a signiﬁcant transformation after World War II when a combination of market forces and government pressure led to the settlement of the majority of the Bajau and the adop- tion of a terrestrial lifestyle.186 However, a smaller percentage of the “sea Bajau” continue to have a semi-nomadic lifestyle, living in water-villages in peripheral and intertidal zones and even on house-boats plying the waters on Sabahs’s eastern coast, at times all the way to the places of their ancestral origin in the Philippine waters. Most of them lack documentary proof of their residence in Malaysia and are considered as illegal residents by the Malaysian state. Denied access to the rights attendant upon citizenship in Malaysia and lacking recognition by any other country, they have been faced with a protracted situation of statelessness over successive generations.187 p g This rejection––cultural as well as political––of the Bajau Laut is characteristic of the sym- bolic rejection of peripatetic minority lifestyles by dominant sedentary majorities in modern nation-states.188 The Bajau Laut, like other nomadic groups inhabiting the state’s peripheries, threaten both the state’s political imaginary by rejecting the cultural model of citizenship asso- ciated with modernity and its security and immigration apparatus focused on policing move- ments within and beyond territorial boundaries.189 183 Greg Acciaioli, Helen Brunt & Julian Clifton, Foreigners Everywhere, Nationals Nowhere: Exclusion, Irregularity, and Invisibility of Stateless Bajau Laut in Eastern Sabah, Malaysia, 15 J. IMMIGRANT & REFUGEE STUD. 232, 233 (2017). 184 Carol Warren, Consciousness in Social Transformation: The Bajau Laut of East Malaysia, 5 DIALECTICAL ANTHROPOLOGY 227, 228 (1980). 185 Clifford Sather, Commodity Trade, Gift Exchange, and the History of Maritime Nomadism in Southeastern Sabah, 6 NOMADIC PEOPLES 20, 36 (2002). 186 Julian Clifton, Greg Acciaioli, Helen Brunt, Wolfram Dressler, Michael Fabinyi & Sarinda Singh, Statelessness and Conservation: Exploring the Implications of an International Governance Agenda, 19 TILBURG L. REV. 81, 85 (2014). 187 Acciaioli, Brunt & Clifton, supra note 183, at 236–37. 188 Sather, supra note 185, at 35. 189 Acciaioli, Brunt & Clifton, supra note 183, at 242–43. 190 See Spiro, supra note 26, at 721–23. 189 Acciaioli, Brunt & Clifton, supra note 183, at 242–43. 183 Greg Acciaioli, Helen Brunt & Julian Clifton, Foreigners Everywhere, Nationals Nowhere: Exclusion, Irregularity, and Invisibility of Stateless Bajau Laut in Eastern Sabah, Malaysia, 15 J. IMMIGRANT & REFUGEE STUD. 232, 233 (2017). 184 190 See Spiro, supra note 26, at 721–23. 3. Reinventing Borders Scholars writing on nationality and citizenship have remarked on the emergence of habit- ual (territorial) residence as a norm that is considered to constrain states’ ability to deny indi- viduals the right to citizenship.190 However, this emphasis on the coincidence of habitual residence and eligibility for citizenship understates the extent to which states are increasingly https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 MANUFACTURING STATELESSNESS 267 engaged in decoupling territorial presence from political membership when doing so serves their political interests. Most of the literature on this separation focuses on the “ladders” rather than the “chutes”––to borrow Ayelet Shachar’s vivid metaphor––of access to political membership, that is, the ability of elites to buy their way into the citizenship of states with whom they share no historical, socio-cultural, or even residential ties.191 These investor cit- izenship, golden passport, or cash-for-passport programs as they are variously labeled, allow states to fast-track the citizenship of those who they perceive as creating value for the state by dispensing with the traditional criteria for political membership and in the absence of any evidence of affective ties to the nation, even as states close the doors to “unworthy” internal others, including long term residents.192 One such ingenious scheme that severs the connection between territory and citizenship is the creation of a class of “offshore citizens” by governments such as the UAE and Kuwait.193 Noora Lori’s incisive ethnographic account of the arrangement between the UAE and the Union of Comoros, one of the world’s smallest and poorest countries, paints a striking picture of this practice of off-shoring. As Lori discusses, the process whereby the UAE purchased Union of Comoros passports to turn its own resident minorities into stateless “citizens” of Comoros was a carefully orchestrated process by the UAE Ministry of Interior. Beginning in 2008, the Ministry launched a statelessness registration drive and issued stateless identity cards to stateless individuals and those with pending naturalization applications. Persons with a stateless ID card were then issued Comoros passports with the understanding that the for- eign passports were a temporary documentation measure to “regularize” their legal residence as foreigners in the UAE pending the eventual grant of UAE citizenship.194 Through this outsourcing arrangement, long-term resident minorities in the UAE have been transformed into temporary residents, permanently at risk of deportation. 191 See Shachar, supra note 149, at 242, 246. 192 See the discussion in Peter Spiro, Cash-for-Passports and the End of Citizenship, in DEBATING TRANSFORMATIONS OF NATIONAL CITIZENSHIP 17 (Rainer Bauböck ed., 2018). These schemes have been criticized by, among others, the European Commission, which launched infringement proceedings against Malta and Cyprus, two of the states with investor citizenship schemes. 191 See Shachar, supra note 149, at 242, 246. 192 See the discussion in Peter Spiro, Cash-for-Passports and the End of Citizenship, in DEBATING TRANSFORMATIONS OF NATIONAL CITIZENSHIP 17 (Rainer Bauböck ed., 2018). These schemes have been criticized by, among others, the European Commission, which launched infringement proceedings against Malta and Cyprus, two of the states with investor citizenship schemes. 193 See LORI, supra note 82. 194 Id. at 203–04. 195 Id. at 212, 231. 196 Id. at 6–9, 196, 213. 197 Id. at 12. 196 Id. at 6–9, 196, 213. 195 Id. at 212, 231. 197 Id. at 12. 198 Zahra Albarazi & Yoana Kuzmova, Trafﬁcking in (Non)-Citizenship in Kuwait and the UAE, in ROUTLEDGE HANDBOOK OF CITIZENSHIP IN THE MIDDLE EAST AND NORTH AFRICA 349, 357 (Roel Meijer, James N. Sater & Zahra R. Babar eds., 2020). 199 Amnesty International, The State of the World’s Human Rights 378 (2021), available at https://www. amnesty.org/en/wp-content/uploads/2021/06/English.pdf. 200 LORI, supra note 82, at 37. 201 See Daniel Kanstroom, Afterword, in CITIZENSHIP IN QUESTION, supra note 165, at 241–42. 3. Reinventing Borders However, since these “passports of convenience” do not entitle passport recipients to call on the Union of Comoros for diplomatic protection or to reside in Comoros, it is unclear where they could be deported if the UAE were minded to expel them.195 This risk may in fact never materialize given that the limbo status of offshore citizenship is not foreseen as a path- way to expulsion, but rather as a form of conditional inclusion. Counting putative citizens as temporarily resident foreigners meets the UAE’s security objective of comprehensively doc- umenting its minority population and its economic interest in being able to call upon its labor, while simultaneously postponing any resolution of the political contestation around who belongs to the body politic.196 At the same time, the artiﬁcial attribution of the citizen- ship of Comoros to this population allows the UAE to avoid having to explicit deny them Emirati citizenship and the formal statelessness that would ensue.197 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 268 Off-shoring citizenship enables the UAE to perform the alchemy of converting would-be citizens to foreign residents without any modiﬁcation of its territorial boundaries or any movement of population within and beyond these borders. A similar program introduced by Kuwait to outsource the bidoon to the Comoros was reportedly abandoned following international outcry, though a more cynical explanation is that carving a path for residence in the state would have undermined Kuwait’s stance that the bidoon should return to the countries of their true nationality.198 Details of the current status of the UAE’s scheme, which did not receive the same level of international visibility, are difﬁcult to come by, but the latest reports suggest that Emirati holders of Comoros passports have found it either dif- ﬁcult or impossible to renew these passports, and in the latter case, have yet again found them- selves without any identity documents.199 Scholars have noted that rather than being provoked by any real crisis, the UAE’s stateless- ness problem was prompted by the UAE’s “own attempts to exhaustively count and categorize all residents in the country without expanding the boundaries of the citizenry.”200 As Part III will argue, the possibility that state identity regularization drives can create new forms of exclusion should inform international policymaking on statelessness. C. Statelessness as Administrative Practice As much as being a citizen of somewhere, anywhere, is a matter of meeting the criteria for citizenship, fulﬁlling these eligibility conditions counts for little if one lacks the means to prove it. The citizenship law of modern nation-states demands that individuals can give an account of their standing in the nation not in terms of identity or political membership, but in the form of evidence that the state considers legitimate. Moreover, the law assumes that this evidence is accessible to all putative citizens on equal terms. An individual who cannot furnish this ofﬁcially sanctioned proof of his citizenship does not exist in the eyes of the nation; or rather, he exists, but not as a citizen, but as an alien.201 Since citizenship is constituted not merely through affective ties, performance, and ritual, but through documents, it is equally through documents––and their denial––that statelessness can be constructed. 98 Zahra Albarazi & Yoana Kuzmova, Trafﬁcking in (Non)-Citizenship in Kuwait and the UAE, in ROUTLEDGE NDBOOK OF CITIZENSHIP IN THE MIDDLE EAST AND NORTH AFRICA 349, 357 (Roel Meijer, James N. Sater & ra R. Babar eds., 2020). 99 1. Archival Erasure Can personal identity be obliterated through the destruction of an archive? Prior to the advent of documentation practices of state legibility, this could have been dismissed as a the- oretical question with little impact on the identity and everyday life of a member in the polit- ical community. In the context of the modern nation-state, identity documentation has become a form of “administrative ordering” with both emancipatory and regressive conse- quences: identity documents are both vital for the welfare state to provide and expand access to social services as well as an instrument of surveillance and control for the state to “discipline https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 269 MANUFACTURING STATELESSNESS and punish” subjects.202 Ofﬁcial recognition of one’s legal status through documents such as birth certiﬁcates, identity cards, and passports can empower individuals and collectives to assert claims against the state or against other members of the political community.203 At the same time, through providing the criteria for state legibility, documents can also be used to exclude, create, manipulate, and obscure identities, molding them to serve the differ- ent purposes of the state and in the process even shaping how individuals perceive themselves.204 The consequences of making a population visible through identity documentation is that one’s membership in the state can be worth little more than the paper that it is written on. The case of “the erased” in Slovenia mentioned previously is a particularly chilling instance of the state’s ability to transform community members into foreigners through paperwork, and with little in the way of warning.205 Many of the erased had failed to apply for citizenship in the six- month window for submitting applications either because they were given inaccurate infor- mation on the documents they would need to supply, or because they had practical concerns relating to inheritance and retirement in their countries of origin. Even more importantly, they were under the illusion that they would continue to retain their social rights as perma- nent residents with long term ties to Slovenia.206 The erasure of their records and loss of rights thus came as a shock: residents were asked to appear in person before ofﬁcial authorities and the documents pertaining to their residence were either conﬁscated or destroyed.207 Equally poignant is the case of “non-citizens” in Latvia and Estonia, whose identity was not erased, but rather reconﬁgured through documentation. 202 See Wendy Hunter & Robert Brill, “Documents, Please”: Advances in Social Protection and Birth Certiﬁcation in the Developing World, 68 WORLD POL. 191, 202 (2016); Jane Caplan & John Torpey, Introduction, in DOCUMENTING INDIVIDUAL IDENTITY, supra note 12, at 1, 5. 203 Caplan & Torpey, Introduction, supra note 202, at 6. 204 See Timothy Longman, Identity Cards, Ethnic Self-Perception, and Genocide in Rwanda, in DOCUMENTING INDIVIDUAL IDENTITY, supra note 12, at 345, 346–47 (on the relationship between colonial Belgian documentation practices and construction of ethnic identity in Rwanda and its eventual role in the Rwandan genocide). 205 See text at note 133 supra. 206 Jelka Zorn, From Erased and Excluded to Active Participants in Slovenia, in STATELESSNESS AND THE BENEFITS OF CITIZENSHIP: A COMPARATIVE STUDY 50, 52 (Brad K. Blitz & Maureen Lynch eds., 2009). 207 Brad K. Blitz, Statelessness and the Social (De)Construction of Citizenship: Political Restructuring and Ethnic Discrimination in Slovenia, 5 J. HUM. RTS. 453, 462–63 (2006). 208 Krūma, supra note 86, at 9. 209 Constitutional Court Case 2004-15-0106, Ofﬁcial Gazette No. 40, Mar. 9, 2005. 210 Available at https://index.statelessness.eu/country/netherlands. p 209 Constitutional Court Case 2004-15-0106, Ofﬁcial Gazette No. 40, Mar. 9, 2005. 210 204 See Timothy Longman, Identity Cards, Ethnic Self-Perception, and Genocide in Rwanda, in DOCUMENTING INDIVIDUAL IDENTITY, supra note 12, at 345, 346–47 (on the relationship between colonial Belgian documentation practices and construction of ethnic identity in Rwanda and its eventual role in the Rwandan genocide). 205 See text at note 133 supra. 206 Jelka Zorn, From Erased and Excluded to Active Participants in Slovenia, in STATELESSNESS AND THE BENEFITS OF CITIZENSHIP: A COMPARATIVE STUDY 50, 52 (Brad K. Blitz & Maureen Lynch eds., 2009). 207 Brad K. Blitz, Statelessness and the Social (De)Construction of Citizenship: Political Restructuring and Ethnic Discrimination in Slovenia, 5 J. HUM. RTS. 453, 462–63 (2006). 208 208 Krūma, supra note 86, at 9. 210 Available at https://index.statelessness.eu/country/netherlands. 202 See Wendy Hunter & Robert Brill, “Documents, Please”: Advances in Social Protection and Birth Certiﬁcation in the Developing World, 68 WORLD POL. 191, 202 (2016); Jane Caplan & John Torpey, Introduction, in DOCUMENTING INDIVIDUAL IDENTITY, supra note 12, at 1, 5. p 203 Caplan & Torpey, Introduction, supra note 202, at 6. 1. Archival Erasure While this status gives Latvian non- citizens a range of rights and privileges beyond stateless persons––for example, a special pass- port guaranteeing them the right of return, the right of residence, and the right to diplomatic protection—they have restricted political rights and are prohibited from occupying certain ofﬁcial positions.208 The lack of international pressure to clarify the international law status of Latvian non-citizens has received a boost with the 2005 Latvian Constitutional Court rul- ing that non-citizens cannot be classiﬁed as either stateless persons or as citizens, but occupy a sui generis status that should not be considered akin to Latvian citizenship.209 g Much worse, from the point of view of access to state protection and rights, is the case of individuals registered as “nationality unknown” in the Netherlands. A designation applied to thousands of persons in the Dutch civil registration records,210 “nationality unknown” is 202 See Wendy Hunter & Robert Brill, “Documents, Please”: Advances in Social Protection and Birth Certiﬁcation in the Developing World, 68 WORLD POL. 191, 202 (2016); Jane Caplan & John Torpey, Introduction, in DOCUMENTING INDIVIDUAL IDENTITY, supra note 12, at 1, 5. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 270 premised on the individual’s inability to prove a negative, namely, they do not hold any nationality. Unlike the category of statelessness, which would provide a pathway to citizen- ship in the Netherlands, “nationality unknown” leaves the individual in limbo, subject to detention and without any prospect of acquiring Dutch nationality.211 This practice was the subject of a petition before the UN Human Rights Committee (HRC) ﬁled by a child born in the Netherlands to a Chinese mother who had been trafﬁcked to the Netherlands as a minor and classiﬁed as an “illegal alien.”212 Since the mother was unable to acquire Chinese citizenship due to lack of birth registration in China and was also unable to conclusively prove premised on the individual’s inability to prove a negative, namely, they do not hold any nationality. 211 UN Hum. Rts. Comm., Views Adopted by the Committee Under Article 5 (4) of the Optional Protocol, Concerning Communication No. 2918/2016, para. 2.4, Comm. No. 2918/2016, CCPR/C/130/D/2918/2016 (Jan. 20, 2021). 1. Archival Erasure Unlike the category of statelessness, which would provide a pathway to citizen- ship in the Netherlands, “nationality unknown” leaves the individual in limbo, subject to detention and without any prospect of acquiring Dutch nationality.211 This practice was the subject of a petition before the UN Human Rights Committee (HRC) ﬁled by a child born in the Netherlands to a Chinese mother who had been trafﬁcked to the Netherlands as a minor and classiﬁed as an “illegal alien.”212 Since the mother was unable to acquire Chinese citizenship due to lack of birth registration in China and was also unable to conclusively prove that her son did not have any other nationality, the Dutch authorities refused to change his status from “nationality unknown” to “stateless,” thus excluding him from the international protections applicable to stateless children.213 Mother and son were compelled to live in a center for unsuccessful asylum applicants and under constant risk of being deported.214 The HRC held that in depriving the petitioner of his right as a minor to acquire a nationality, the Netherlands had violated his right to a nationality under Article 24(3) of the International Covenant on Civil and Political Rights (ICCPR). In addition, the Netherlands had an obli- gation under the Statelessness Convention to “‘determine whether a child would otherwise be stateless as soon as possible so as not to prolong a child’s status of undetermined nationality.’”215 y The HRC decision signals a signiﬁcant international victory for resolving cases of child- hood statelessness in the Netherlands, in that it marks the beginning of a series of steps that would have to be undertaken in order to eventually acquire Dutch nationality. It was made possible by over a decade of strategic litigation by international and local NGOs and experts (including the Open Society Justice Initiative, which has actively pursued cases of statelessness before regional human rights mechanisms) coupled with an administrative court decision and a report by the Dutch Advisory Committee on Migration Affairs repri- manding the Dutch government for its failure to establish a stateless determination proce- dure.216 As Part III will argue, strategic litigation efforts that combine domestic and international expertise to mobilize a range of legal actors may provide one avenue to counter statelessness creep. 216 See Laura Bingham & Jelle Klass, A Victory for Human Rights in Zhao v. the Netherlands (the “Denny Case”): Nationality from Birth, Without Exceptions, EUI GLOBAL CITIZENSHIP OBSERVATORY (Jan. 19, 2021), at https:// globalcit.eu/a-victory-for-human-rights-in-zhao-v-the-netherlands-the-denny-case-nationality-from-birth-with- out-exceptions. 215 Id., paras. 8.3, 8.5. 212 Id., paras. 2.1–2.2. 213 213 Id., para. 8.5 214 Id., para. 2.9. 2. Paper Non-citizens While administrative erasure is a clinically efﬁcient way for a state to simply “disappear” various citizens from its books, a more insidious way to turn citizens into stateless persons is through “paper-izing” informal identity and the criteria for belonging. Documents such as ID https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 MANUFACTURING STATELESSNESS 271 cards and passports capture “the citizen himself as an effect of his ‘mise en carte,’”217 in the process thwarting or rendering obsolete previously important sources of recognition and iden- tity.218 The state’s supreme authority to evaluate and enforce identities through the ofﬁcial truths embodied in its documents authoritatively forecloses the possibility of ofﬁcially unrec- ognized identities, including those that may challenge the falsehoods that are sanctiﬁed through ofﬁcialdom.219 These documentary errors and inconsistencies are not always an out- come of a weakly bureaucratic state, but are at times an actively produced form of marginality in service of the state’s political purposes.220 Citizenship that emerges through the law, its institutions, and forensic documentation—so-called administrative citizenship—can be a highly regulated and exclusionary institution that renders stateless already marginalized groups in society.221 g p One of the most ambitious attempts at the “weaponization of documentary citizenship” is the effort to enumerate and comprehensively document the population of the Indian state of Assam through the implementation of a National Register of Citizens (NRC).222 The NRC has a long and tortured history in Indian politics, buffeted by waves of migration across the border between India and Bangladesh, leading to local Assamese anxieties about the inﬂux of “illegal” Bangladeshi immigrants into the state across the porous border between the two countries.223 The NRC process that commenced in 2015 is intended to update the 1951 NRC, which was set up as a population enumeration exercise following the ﬁrst national cen- sus of independent India. Based on the Citizenship Act, 1955 and The Citizenship (Registration of Citizens and Issue of National Identity Cards) Rules, 2003, it is ostensibly a highly legalistic exercise to identify Indian citizens and weed out “illegal immigrants,” deﬁned as those who can prove that they or their ancestors entered Assam before March 24, 1971, the eve of Bangladesh’s declaration of independence from Pakistan. This proof con- sists of being able to furnish “legacy data”: individuals can either submit documents evidenc- ing that their names were included in the 1951 NRC or on the electoral rolls before March 24, 1971. 222 Sahana Ghosh & Radhika Moral, The Slipperiness of Documents: Notes from India’s Eastern Borderlands (2020), at https://www.law.ox.ac.uk/research-subject-groups/centre-criminology/centreborder-criminologies/ blog/2020/02/slipperiness. 221 Kamal Sadiq, Limits of Legal Citizenship: Narratives from South and Southeast Asia, in STATELESSNESS AND THE BENEFITS OF CITIZENSHIP, supra note 206, at 165–66. In their recent article, Michelle Foster and Jade Roberts also discuss the phenomenon of “documentary citizenship” and its politicization by the state to enact exclusion from within in the context of the NRC exercise in India. See Foster & Roberts, supra note 6, at 228. 24 See Praveen Donthi, How Assam’s Supreme Court-mandated NRC Project Is Targeting and Detaining Bengali slims, Breaking Families, CARAVAN (July 1, 2018). 223 See Talha Abdul Rahman, Identifying the “Outsider”: An Assessment of Foreigner Tribunals in the Indian State of Assam, 2 STATELESSNESS & CITIZENSHIP REV. 112, 113–18 (2020). 224 217 Jane Caplan, Illegibility: Reading and Insecurity in History, Law and Government, 68 HIST. WORKSHOP J. 99, 103 (2009). 218 Id 219 Caplan, supra note 217, at 104. 220 217 Jane Caplan, Illegibility: Reading and Insecurity in History, Law and Government, 68 HIST. WORKSHOP J. 99, 103 (2009). 218 Id. 219 Caplan, supra note 217, at 104. 220 Id. 221 Kamal Sadiq, Limits of Legal Citizenship: Narratives from South and Southeast Asia, in STATELESSNESS AND THE BENEFITS OF CITIZENSHIP, supra note 206, at 165–66. In their recent article, Michelle Foster and Jade Roberts also discuss the phenomenon of “documentary citizenship” and its politicization by the state to enact exclusion from within in the context of the NRC exercise in India. See Foster & Roberts, supra note 6, at 228. 222 Sahana Ghosh & Radhika Moral, The Slipperiness of Documents: Notes from India’s Eastern Borderlands (2020), at https://www.law.ox.ac.uk/research-subject-groups/centre-criminology/centreborder-criminologies/ blog/2020/02/slipperiness. 223 See Talha Abdul Rahman, Identifying the “Outsider”: An Assessment of Foreigner Tribunals in the Indian State of Assam, 2 STATELESSNESS & CITIZENSHIP REV. 112, 113–18 (2020). 224 See Praveen Donthi, How Assam’s Supreme Court-mandated NRC Project Is Targeting and Detaining Bengali Muslims, Breaking Families, CARAVAN (July 1, 2018). 2. Paper Non-citizens In addition, they can submit documentary proof, such as birth certiﬁcates or bank documents, of their family links to parents and ancestors who are included in the legacy data.224 The requirement to provide documentary proof of residence stretching back several decades ignores the reality of documentation practices in a country like India, where a https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 272 signiﬁcant percentage of the population lives its life bereft of any state-produced identity and where it is relatively easy to obtain fraudulent “ofﬁcial” documents to serve particular pur- poses.225 Indeed, many minority families that returned to Assam after the guarantee of safe passage under the 1950 Nehru–Liaquat Agreement did so only after the 1951 census and NRC exercise—that is said to have been carried out in less than a month—was com- pleted.226 Thus, not only does the emphasis on inclusion in the 1951 NRC exclude various individuals who had been unable to register at the time, but given the geography of Assam where property and documents are regularly lost to natural disasters such as ﬂoods and land erosion, any paperwork paints at best a partial snapshot of the population. In addition, several classes of people in Assam, such as local Indigenous communities never saw the need to acquire documents that were considered an alien, colonial, process and others, such as women who were married as minors and transgender persons, would ﬁnd it nearly impossible to prove documentary links to biological family members who qualify as citizens.227 It is not only those who lack the documentary proof required by the NRC exercise that have found themselves excluded from the ﬁnal register, but also those who possess the right documents, but have the misfortune of having them recorded incorrectly. 225 See KAMAL SADIQ, PAPER CITIZENS: HOW ILLEGAL IMMIGRANTS ACQUIRE CITIZENSHIP IN DEVELOPING COUNTRIES 26–28 (2008). 226 Rahman, supra note 223, at 115. 227 Ditilekha Sharma, Determination of Citizenship Through Lineage in the Assam NRC is Inherently Exclusionary, 54 ECON. & POL. WEEKLY (2019). 228 Ghosh & Moral, supra note 222. 229 Rahman, supra note 223, at 123. 230 Malini Sur, In the Name of Indian Citizenship? Criminalizing Statelessness at the India-Bangladesh Border, U. OXFORD FACULTY LAW BLOG (2020), at https://www.law.ox.ac.uk/research-subject-groups/centre-criminology/ centreborder-criminologies/blog/2020/02/name-indian. 231 Rahman, supra note 223, at 128–36. 232 Arijit Sen & Leah Verghese, Weaponising Citizenship in India, U. OXFORD FACULTY LAW BLOG (2020), at https://www.law.ox.ac.uk/research-subject-groups/centre-criminology/centreborder-criminologies/blog/2020/ 02/weaponising. 231 Rahman, supra note 223, at 128–36. 225 See KAMAL SADIQ, PAPER CITIZENS: HOW ILLEGAL IMMIGRANTS ACQUIRE CITIZENSHIP IN DEVELOPING COUNTRIES 26–28 (2008). p 232 Arijit Sen & Leah Verghese, Weaponising Citizenship in India, U. OXFORD FACULTY LAW BLOG (2020), at https://www.law.ox.ac.uk/research-subject-groups/centre-criminology/centreborder-criminologies/blog/2020/ 02/weaponising. 226 Rahman, supra note 223, at 115. 227 2. Paper Non-citizens Since the transcribing, recording, and translation of the documents was done manually, modeling from previous error rates in the processing of such data in India reveals that around 5 percent of those with correct documents could have been excluded from citizenship due to something as minor as a spelling error.228 p g The NRC was published in August 2019 and the 1.9 million residents who were excluded from the register and classiﬁed as “D” or “doubtful” Voters, were given 120 days to appear before “Foreigners Tribunals,” quasi-judicial bodies tasked with determining whether they are Indian nationals or illegal migrants who could be detained and subject to expulsion.229 Several of those labeled as illegal migrants challenged the NRC process arguing that the authorities had in fact never attempted to verify their status or that they had been the victims of fraud by those who wanted to lay claim to their property.230 The proceedings themselves were widely criticized as lacking independent and well-qualiﬁed judicial ofﬁcers and proce- dural safeguards.231 Not only was the burden of proof placed on the supposed illegal immi- grant to establish their right to citizenship, but any inconsistencies or errors in documents or testimonies were treated with suspicion, leading to persons being declared “foreigners” and placed in detention centers.232 p 232 Arijit Sen & Leah Verghese, Weaponising Citizenship in India, U. OXFORD FACULTY LAW BLOG (2020), at https://www.law.ox.ac.uk/research-subject-groups/centre-criminology/centreborder-criminologies/blog/2020/ 02/weaponising. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 273 MANUFACTURING STATELESSNESS India is not the only country that has seen the production of statelessness as a consequence of a national effort at population enumeration and categorization. 2. Paper Non-citizens As scholars have noted, statelessness can be the intended as well as unintended result of state identity regularization drives, that are increasingly being undertaken by states also following sustained international pressure from agencies such as the United Nations and the World Bank that have promoted identity documents, including digital IDs, as a tool for social inclusion.233 In some cases, however, the rollout of these identiﬁcation programs have been plagued with logistical and technical failures, resulting in the exclusion of already marginalized groups in society such as women or the elderly as in the case of Uganda.234 More troubling, however, for the purposes of this Article has been the deliberate effort by states to exclude minorities from these regis- tration processes, as in the case of the Haitian migrants and Dominicans of Haitian descent mentioned previously. Recent scholarship has sought to draw a link between the advocacy of legal identity documents by international actors and the measures taken by the Dominican Republic to exclude these populations from accessing this documentation, rendering their entitlement to the rights of citizenship even more precarious.235 As states undertake efforts to consolidate and strengthen their processes for identity management, individuals and groups who do not fall within the state’s political imaginary may either be systematically left out of documentation processes, or assigned the label of “stateless” persons, thus ﬁxing what was previously a ﬂuid and malleable identity.236 This is an issue to which Part III returns with a cautionary message for international policy actors. 238 See, e.g., Committee on Economic, Social & Cultural Rights, Concluding Observations: Macedonia, para. 32, UN Doc. E/C.12/MKD/CO/1 (Jan. 15, 2008) (asking Macedonia to remove administrative obstacles to the acquisition of citizenship by the Roma); CERD, Concluding Observations: Croatia, para. 17, UN Doc. 233 See, e.g., World Bank, Inclusive and Trusted Digital ID Can Unlock Opportunities for the World’s Most Vulnerable (Aug. 14, 2019), at https://www.worldbank.org/en/news/immersive-story/2019/08/14/inclusive- and-trusted-digital-id-can-unlock-opportunities-for-the-worlds-most-vulnerable. 234 Center for Human Rights and Global Justice, Initiative for Social and Economic Rights, & Unwanted Witness, Chased Away and Left to Die: How a National Security Approach to Uganda’s National Digital ID Has Led to Wholesale Exclusion of Women and Older Persons (2021), available at https://chrgj.org/wp-content/ uploads/2021/06/CHRGJ-Report-Chased-Away-and-Left-to-Die.pdf. 235 See EVA HAYES DE KALAF, LEGAL IDENTITY, RACE, AND BELONGING IN THE DOMINICAN REPUBLIC: FROM CITIZEN TO FOREIGNER (2021). 236 LORI, supra note 82, at 201–03. 237 See Flaim, supra note 165, at 148. 238 See, e.g., Committee on Economic, Social & Cultural Rights, Concluding Observations: Macedonia, para. 32, UN Doc. E/C.12/MKD/CO/1 (Jan. 15, 2008) (asking Macedonia to remove administrative obstacles to the acquisition of citizenship by the Roma); CERD, Concluding Observations: Croatia, para. 17, UN Doc. 233 See, e.g., World Bank, Inclusive and Trusted Digital ID Can Unlock Opportunities for the World’s Most Vulnerable (Aug. 14, 2019), at https://www.worldbank.org/en/news/immersive-story/2019/08/14/inclusive- and-trusted-digital-id-can-unlock-opportunities-for-the-worlds-most-vulnerable. 233 See, e.g., World Bank, Inclusive and Trusted Digital ID Can Unlock Opportunities for the World’s Most Vulnerable (Aug. 14, 2019), at https://www.worldbank.org/en/news/immersive-story/2019/08/14/inclusive- and-trusted-digital-id-can-unlock-opportunities-for-the-worlds-most-vulnerable. 234 Center for Human Rights and Global Justice, Initiative for Social and Economic Rights, & Unwanted Witness, Chased Away and Left to Die: How a National Security Approach to Uganda’s National Digital ID Has Led to Wholesale Exclusion of Women and Older Persons (2021), available at https://chrgj.org/wp-content/ uploads/2021/06/CHRGJ-Report-Chased-Away-and-Left-to-Die.pdf. 235 See EVA HAYES DE KALAF, LEGAL IDENTITY, RACE, AND BELONGING IN THE DOMINICAN REPUBLIC: FROM CITIZEN TO FOREIGNER (2021). 237 See Flaim, supra note 165, at 148. 238 236 LORI, supra note 82, at 201–03. 35 See EVA HAYES DE KALAF, LEGAL IDENTITY, RACE, AND BELONGING IN THE DOMINICAN REPUBLIC: FROM IZEN TO FOREIGNER (2021). 234 Center for Human Rights and Global Justice, Initiative for Social and Economic Rights, & Unwanted Witness, Chased Away and Left to Die: How a National Security Approach to Uganda’s National Digital ID Has Led to Wholesale Exclusion of Women and Older Persons (2021), available at https://chrgj.org/wp-content/ uploads/2021/06/CHRGJ-Report-Chased-Away-and-Left-to-Die.pdf. 235 CERD/C/HRV/CO/8 (Mar. 24, 2009) (asking Croatia to remove administrative barriers and assist persons such as those of Roma, Serb, or Bosniak origin, who have limited access to mandatory documentation); CERD, Concluding Observations on the Thirteenth to Fifteenth Periodic Reports of Suriname, para. 20, UN Doc. CERD/C/SUR/CO/13-15 (Aug. 28, 2015) (calling on Suriname to remove administrative barriers and discrim- inatory practices to prevent statelessness, particularly in relation to birth registration). 244 WENDY HUNTER, UNDOCUMENTED NATIONALS: BETWEEN STATELESSNESS AND CITIZENSHIP 48–49 (2019 245 Id. at 50. 240 Samantha Balaton-Chrimes, Indigeneity and Kenya’s Nubians: Seeking Equality in Difference or Sameness?, 51 J. MOD. AFR. STUD. 331, 332, 338 (2013). 241 Abraham Korir Sing’Oei, Promoting Citizenship in Kenya: The Nubian Case, in STATELESSNESS AND THE BENEFITS OF CITIZENSHIP: A COMPARATIVE STUDY, supra note 206, at 37, 38–39. 242 Id 39 CERD/C/HRV/CO/8 (Mar. 24, 2009) (asking Croatia to remove administrative barriers and assist persons such as those of Roma, Serb, or Bosniak origin, who have limited access to mandatory documentation); CERD, Concluding Observations on the Thirteenth to Fifteenth Periodic Reports of Suriname, para. 20, UN Doc. CERD/C/SUR/CO/13-15 (Aug. 28, 2015) (calling on Suriname to remove administrative barriers and discrim- inatory practices to prevent statelessness, particularly in relation to birth registration). 239 The Nubian Community in Kenya v. The Republic of Kenya, Comm. 317/2006, 17th Extraordinary Sess., Feb. 19–28, 2015. 240 Samantha Balaton-Chrimes, Indigeneity and Kenya’s Nubians: Seeking Equality in Difference or Sameness?, 51 J. MOD. AFR. STUD. 331, 332, 338 (2013). 241 Abraham Korir Sing’Oei, Promoting Citizenship in Kenya: The Nubian Case, in STATELESSNESS AND THE BENEFITS OF CITIZENSHIP: A COMPARATIVE STUDY, supra note 206, at 37, 38–39. 242 Id. at 39. 243 Balaton-Chrimes, supra note 240, at 339–40. 244 WENDY HUNTER, UNDOCUMENTED NATIONALS: BETWEEN STATELESSNESS AND CITIZENSHIP 48–49 (2019). 245 Id. at 50. 39 The Nubian Community in Kenya v. The Republic of Kenya, Comm. 317/2006, 17th Extraordinary Sess., . 19–28, 2015. 40 Abraham Korir Sing Oei, Promoting Citizenship in Kenya: The Nubian Case, in STATELESSNESS AND THE EFITS OF CITIZENSHIP: A COMPARATIVE STUDY, supra note 206, at 37, 38–39. 42 Id. at 39. 240 Samantha Balaton-Chrimes, Indigeneity and Kenya’s Nubians: Seeking Equality in Difference or Sameness?, 51 J. MOD. AFR. STUD. 331, 332, 338 (2013). 241 Abraham Korir Sing’Oei, Promoting Citizenship in Kenya: The Nubian Case, in STATELESSNESS AND THE BENEFITS OF CITIZENSHIP: A COMPARATIVE STUDY, supra note 206, at 37, 38–39. 242 Id. at 39. 243 3. Bureaucratic Statelessness Even for individuals who are entitled to claim citizenship in a state, access to citizenship is not merely a matter of formal laws or policy; rather, conferral of citizenship involves the con- ferral of belief by ofﬁcials tasked with administering these laws. The processing and evaluation of citizenship claims is thus contingent not only on the strength of the evidence that the claimant can provide, but also on the individual judgment and discretion exercised by the ofﬁcial in charge. This valuation will not always produce citizenship but may reinforce or pro- long statelessness.237 Recent legal developments at the international and regional human rights bodies display greater awareness and heightened scrutiny of the role of bureaucracy in the creation of state- lessness.238 The case of the Nubians of Kenya before the African Commission on Human and https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 274 Peoples’ Rights illustrates the uphill battle faced by communities and groups seeking to prove evidentiary statelessness based on everyday bureaucratic state practices.239 Described as a “subject race turned ethnic stranger,” the Nubians were conscripted from Egypt and Sudan into the British colonial armed forces and migrated to Kenya as part of the British col- onization effort in East Africa. Having been categorized as “detribalized natives” who were subject to native local laws during the colonial era, the Nubians found themselves in a polit- ically challenging and legally uncertain post-independence environment due to their previous association with the colonial administration.240 The Nubians were in principle entitled to claim Kenyan nationality at the time of Kenya’s independence in 1963 on the basis of the jus sanguinis principle. However, in practice, many of them were excluded from obtaining citizenship since Kenya did not include the Nubians amongst one of the forty-two ofﬁcial ethnic groups and few Nubians had documentary proof that would allow them to establish citizenship by descent from individuals living in Kenya over two generations.241 Peoples’ Rights illustrates the uphill battle faced by communities and groups seeking to prove evidentiary statelessness based on everyday bureaucratic state practices.239 Described as a “subject race turned ethnic stranger,” the Nubians were conscripted from Egypt and Sudan into the British colonial armed forces and migrated to Kenya as part of the British col- onization effort in East Africa. 3. Bureaucratic Statelessness The Commission held that the exercise of administrative decision making in the issuance of identity documents was not only https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 MANUFACTURING STATELESSNESS 275 arbitrary and burdensome for the Nubians, but that the overt discrimination against the Nubians on the basis of their ethnic and religious identity was a clear violation of their dignity and the right to equality and non-discrimination under the African Charter.246 In addition, these discriminatory practices, procedural hurdles, and delays in the acquisition of identity documents prevented the Nubians from exercising the rights associated with citizenship and rendered them effectively stateless, violating the right to recognition of legal status guar- anteed under Article 5 of the Charter.247 While the decision has led to a few concrete changes at the domestic level, including the presence of Nubians on the vetting committees and acceleration in birth registrations and issuance of identity documents, administrative discretion in the process has been retained. The applicant still bears the burden of proof to establish citizenship before the vetting com- mittees, which operate with few procedural rules and without any possibility of an appeal against their decisions.248 Neither is the situation of bureaucratic statelessness experienced by the Nubians entirely exceptional, whether in Kenya or beyond. In the African context, scholars have argued that notwithstanding ostensibly neutral citizenship laws, various groups associated with the colonial legacy or living in border regions have had their citizenship status challenged and been required to prove their entitlement to nationality.249 A less well-known example from Kenya itself are the Makonde, descendants of migrant farm workers who were brought to Kenya from Mozambique beginning in the 1930s to work on sisal plantations. 3. Bureaucratic Statelessness Having been categorized as “detribalized natives” who were subject to native local laws during the colonial era, the Nubians found themselves in a polit- ically challenging and legally uncertain post-independence environment due to their previous association with the colonial administration.240 The Nubians were in principle entitled to l i K ti lit t th ti f K ’ i d d i 1963 th b i f th Given the difﬁculties in establishing their citizenship based on descent, the Nubians have been relegated to seeking citizenship status through naturalization and registration. These processes lack detailed procedural safeguards and rules to orient administrative decision mak- ing, resulting in signiﬁcant discretion being exercised by administrative ofﬁcials.242 Perceived by many Kenyans, including Kenyan ofﬁcials, as “foreigners” lacking a recognized homeland and land tenure in the manner of the ofﬁcial tribes, the Nubians have been subjected to addi- tional vetting procedures to obtain important documents such as national ID cards, which are amongst the most important ofﬁcial markers of Kenyan citizenship.243 The vetting proce- dures have been criticized as highly discriminatory and prone to corruption, with the Muslim identity of the Nubians being treated as a ground for suspicion and demands for addi- tional documentary proof of ancestral ties to Kenya, such as birth certiﬁcates of parents and grandparents.244 Commentators have noted that since these acts of discrimination that pro- duce evidentiary statelessness do not stem from any identiﬁable law or ofﬁcial policy but are rather the result of fragmented decisions made by state agents on an individual basis, they are harder to prove and challenge before courts and other bodies.245 The African Commission’s decision in the Nubian Community in Kenya is one of the rare examples where these claims were in fact successful, though only after signiﬁcant advocacy and a string of failed efforts at the domestic level. 246 Nubian Community in Kenya, supra note 239, paras. 133–35. 247 Id., paras 148–51. 248 See Sing’Oei, supra note 241, at 42–43; MANBY, supra note 70, at 193. 249 Bettina Ng’weno & L. Obura Aloo, Irony of Citizenship: Descent, National Belonging, and Constitutions in the Postcolonial African State, 53 L. & SOC. REV. 141, 166–67 (2019). 250 MANBY, supra note 70, at 184. 251 Ng’weno & Aloo, supra note 249, at 167. 252 See Abdi Latif Dahir, Kenya’s New Digital IDs May Exclude Millions of Minorities, N.Y. TIMES (Jan. 28, 2020), at https://www.nytimes.com/2020/01/28/world/africa/kenya-biometric-id.html. 3. Bureaucratic Statelessness While they were not automatically entitled to Kenyan citizenship at the time of independence due to the restrictive requirements of jus sanguinis, they would have been eligible for citizen- ship through registration, a procedure that most of them failed to go through due to lack of information, cost, or the assumption that they will be returned to Mozambique.250 After decades of being denied or facing discrimination in the acquisition of birth certiﬁcates and identity documents, it is only after extensive lobbying that they obtained a directive for their naturalization and registration as Kenyan citizens in 2016, a move that was interpreted as politically expedient for the government given the impending national elections.251 As recently as last year, the Kenyan biometric ID scheme that aims to provide every Kenyan with a unique identiﬁcation number was accused of “digitizing discrimination” by systemati- cally making it more difﬁcult for minorities such as Nubians and Kenyan Somalians to apply for a digital ID.252 251 Ng’weno & Aloo, supra note 249, at 167. 5 252 See Abdi Latif Dahir, Kenya’s New Digital IDs May Exclude Millions of Minorities, N.Y. TIMES (Jan. 2020), at https://www.nytimes.com/2020/01/28/world/africa/kenya-biometric-id.html. 50 MANBY, supra note 70, at 184. 247 Id., paras 148–51. III. RESPONDING TO STATELESSNESS While, as Part II has described, international and regional actors have begun to take steps to combat state intentionality in the creation of statelessness, these efforts have been geared https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 276 toward the minutiae of combating country-speciﬁc cases of statelessness. What they have failed to appreciate is that an exclusionary tactic or method that might seem peculiar to the circumstances of a certain minority group, in the context of a particular state, is in fact an instance of the larger phenomenon of statelessness as statecraft. This creeping form of statelessness does not come breathing ﬁre and brimstone, but rather takes the form of manip- ulating the criteria for inclusion in the political constituency based on seemingly impartial criteria underpinning citizenship entitlement.253 These ostensibly neutral criteria become difﬁcult to evaluate or challenge given the fact of citizenship as an inherently exclusionary institution that simultaneously claims to be theoretically open to all but is nonetheless pre- mised on an act of boundary-drawing between community members and outsiders.254 The Article does not claim to provide a comprehensive answer to the larger question of “who has a claim to be included in a democratic polity,” an issue that has confounded not only international lawyers, but also political theorists and philosophers.255 Rather, it argues that state prerogative to exclude individuals from political membership and in the process ren- der them stateless, can be subject to fundamental international law norms such as non-dis- crimination. A prima facie discrimination claim should arise in situations of statelessness involving the denial of citizenship rights to a class of individuals notwithstanding the exis- tence of a close connection with the state such that they are not only affected by its laws and policies, but also depend on it for their well-being and autonomy.256 In principle, this would include claims based on “birthright citizenship,” that is citizenship through descent or through birth on the territory, but also on the fact of habitual or prolonged residence on the territory of a state,257 an element that characterizes many of the situations discussed in Part II. 256 This is an amalgam of principles for democratic inclusion proposed by Rainer Bauböck. These principles are both complex and contested in citizenship theory (see DEMOCRATIC INCLUSION: RAINER BAUBÖCK IN DIALOGUE, supra note 255). This Article does not propose to enter into these debates or develop a new political theory of citizenship. Rather, these principles are put forward as intuitively appealing grounds for inclusion that moreover resemble international law’s emphasis on a genuine and effective link for the attribution of nationality. 58 Council of Europe Convention on the Avoidance of Statelessness in Relation to State Succession, supra note Art. 5; ILC Draft Articles, supra note 57, Art. 5. Lori, supra note 110, at 132. 254 See LINDA BOSNIAK, THE CITIZEN AND THE ALIEN: DILEMMAS OF CONTEMPORARY MEMBERSHIP 102 (2006) (noting how “citizenship talk . . . trades in both universalism and particularism”). 255 For a masterful recent attempt to develop principles that respond to this question, see Rainer Bauböck, Democratic Inclusion: A Pluralist Theory of Citizenship, in DEMOCRATIC INCLUSION: RAINER BAUBÖCK IN DIALOGUE 3 (2017). 256 This is an amalgam of principles for democratic inclusion proposed by Rainer Bauböck. These principles are both complex and contested in citizenship theory (see DEMOCRATIC INCLUSION: RAINER BAUBÖCK IN DIALOGUE, supra note 255). This Article does not propose to enter into these debates or develop a new political theory of citizenship. Rather, these principles are put forward as intuitively appealing grounds for inclusion that moreover resemble international law’s emphasis on a genuine and effective link for the attribution of nationality. 257 See Bauböck, supra note 255, at 66. 258 Council of Europe Convention on the Avoidance of Statelessness in Relation to State Succession, supra note 56, Art. 5; ILC Draft Articles, supra note 57, Art. 5. 257 See Bauböck, supra note 255, at 66. 253 Lori, supra note 110, at 132. 4 255 For a masterful recent attempt to develop principles that respond to this question, see Rainer Bauböck, Democratic Inclusion: A Pluralist Theory of Citizenship, in DEMOCRATIC INCLUSION: RAINER BAUBÖCK IN DIALOGUE 3 (2017). 253 Lori, supra note 110, at 132. 254 See LINDA BOSNIAK, THE CITIZEN AND THE ALIEN: DILEMMAS OF CONTEMPORARY MEMBERSHIP 102 (2006) (noting how “citizenship talk . . . trades in both universalism and particularism”). Lori, supra note 110, at 132. 254 See LINDA BOSNIAK, THE CITIZEN AND THE ALIEN: DILEMMAS OF CONTEMPORARY MEMBERSHIP 102 (2006) (noting how “citizenship talk . . . trades in both universalism and particularism”). 255 For a masterful recent attempt to develop principles that respond to this question, see Rainer Bauböck, D ati I l i A Pl ali t Th f Citi hip i DEMOCRATIC INCLUSION RAINER BAUBÖCK IN Lori, supra note 110, at 132. 254 See LINDA BOSNIAK, THE CITIZEN AND THE ALIEN: DILEMMAS OF CONTEMPORARY MEMBERSHIP 102 (2006) (noting how “citizenship talk . . . trades in both universalism and particularism”). 255 For a masterful recent attempt to develop principles that respond to this question, see Rainer Bauböck, Democratic Inclusion: A Pluralist Theory of Citizenship, in DEMOCRATIC INCLUSION: RAINER BAUBÖCK IN DIALOGUE 3 (2017). 256 Thi i l f i i l f d i i l i d b R i B bö k Th i i l III. RESPONDING TO STATELESSNESS The salience of habitual residence as evidence of a substantial and real connection between the individual and the state has already been recognized in instruments on state succession where it functions as the default basis for attribution of citizenship.258 p Even if the individual is able to demonstrate this close connection with the putative state of nationality, they would still face an uphill battle establishing that the exclusion from citizen- ship violates the prohibition of non-discrimination such that international intervention is appropriate. As the examples in Part II highlight, while current state practices of sorting pop- ulations into boxes of citizens and stateless others exhibit continuities with traditional dis- criminatory modes of exclusion, rather than using “suspect categories of discrimination” such as race, religion, or ethnic origin that are explicitly mentioned in various international https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 277 MANUFACTURING STATELESSNESS and regional human rights instruments, states rely on the purportedly unbiased and stable criteria of geography, time, and documentation, to enact and implement statelessness.259 Theoretically, these legal tools are equally applicable to any individual seeking entry into the gates of citizenship. Quantitative measures such as time that are perceived as scientiﬁcally objective have a special resonance in liberal polities with a commitment to equal treatment of similarly situated individuals. As Cohen recounts, “[c]locks and calendars exhibit no indepen- dent bias.”260 This makes chronological time ideally suited as a proxy to avoid the arbitrar- iness and subjectivity associated with qualitative criteria for political membership such as those that measure affective ties, loyalty, or cultural assimilation. However, as this Article has shown, temporal rules and standards are not always impartial or egalitarian. In practice, states apply them selectively to achieve the same objectives as measures that would immedi- ately invite international oversight or condemnation.261 The same is true of states’ manipulation of legal geography and documentation practices, which can render citizenship indeterminate through denial and revocation of nationality but also through suspending individuals in the liminal space between citizenship and stateless- ness.262 Through the miracle of the written word, documents such as identity cards can ascribe a status that far from substantiating the truth, “sometimes plunge[s] their referents into a reality that is incommensurable with their sense of self,”263 by transforming citizens into stateless persons. 259 See Ayelet Shachar, The Multiple Sites of Justice: A Reply, in THE SHIFTING BORDER, supra note 110, at 242 (making a similar argument in the context of migration control through the legal device of “shifting borders”). 260 COHEN, supra note 78, at 11. 261 See id. 262 Jacqueline Stevens, Introduction, in CITIZENSHIP IN QUESTION, supra note 165, at 1, 3–4. 263 Barbara Yngvesson & Susan Bibler Coutin, Backed by Papers: Undoing Persons, Histories, and Return, 33 AM. ETHNOLOGIST 177, 184 (2006). 264 Jacqueline Bhabha, The Politics of Evidence: Roma Citizenship Deﬁcits in Europe, in CITIZENSHIP IN QUESTION, supra note 165, at 43, 45–46. 265 See id. 64 Jacqueline Bhabha, The Politics of Evidence: Roma Citizenship Deﬁcits in Europe, in CITIZENSHIP IN ESTION, supra note 165, at 43, 45–46. 65 S id 259 See Ayelet Shachar, The Multiple Sites of Justice: A Reply, in THE SHIFTING BORDER, supra note 110, at 242 (making a similar argument in the context of migration control through the legal device of “shifting borders”). 260 COHEN, supra note 78, at 11. 261 S d J q , , Q , p , , 63 Barbara Yngvesson & Susan Bibler Coutin, Backed by Papers: Undoing Persons, Histories, and Return, 33 AM. HNOLOGIST 177, 184 (2006). 64 62 Jacqueline Stevens, Introduction, in CITIZENSHIP IN QUESTION, supra note 165, at 1, 3–4. 266 See, e.g., Biao v. Denmark, App. No. 38590/10, para. 114 (Eur. Ct. H.R. May 24, 2016) (interpreting the ECHR); Kambole v. United Republic of Tanzania, App. No. 018/2018, Judgment, paras. 69–72 (Afr. Ct. H.P.R. July 15, 2020) (interpreting the African Charter). 267 See Hugh Collins & Tarunabh Khaitan, Indirect Discrimination Law: Controversies and Critical Questions, in FOUNDATIONS OF INDIRECT DISCRIMINATION LAW 1, 2 (Hugh Collins & Tarunabh Khaitan eds., 2018). 268 See Tarunabh Khaitan, Indirect Discrimination, in THE ROUTLEDGE HANDBOOK OF THE ETHICS OF DISCRIMINATION 30 (Kasper Lippert-Rasmussen ed., 2017). III. RESPONDING TO STATELESSNESS State machinery and techniques for operationalizing citizenship that are facially procedural can in fact embody substantive value judgments on who has to contend with the hermeneutics of suspicion when accessing and adducing documentary proof of membership. For some more than others, the road to citizenship is paved with bureaucratic hurdles and the “legalized illegality” of routine exclusion by ofﬁcial agencies.264 g g g This Article has a simple—albeit ambitious—prescriptive message: instead of continuing to rely on ad hoc and piecemeal legal and advocacy efforts, international law needs to develop a legal roadmap—one that not only equals but surpasses the state’s creativity—to avoid, pre- vent, and respond to statelessness. This roadmap must be responsive to sophisticated legal concepts, interpretations, and practices that give the state the leeway to disenfranchise and exclude without running up directly against international law norms that constrain stateless- ness. It thus needs to recognize and challenge the ways in which states exclude, not only or even predominantly through explicitly prohibited grounds of discrimination, but through tampering with the standard universal criteria for political inclusion. And it needs to be suf- ﬁciently agile to keep pace with the shifting landscape of statelessness production that is con- stantly shaped and reshaped by the state’s legal imagination.265 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 278 Much like the multiple sites at which statelessness is produced, this roadmap too will need concerted legal, institutional, and advocacy efforts by a range of international, regional, and local actors. This Part explores three such potential instruments rooted in international law doctrine, policies, and litigation strategies that may be pursued by actors who play different roles in international norm-building. These efforts, experimental as they are, emphasize a multi-actor collaborative process guided by ethical incrementalism which respects the com- plexity of the conditions for political belonging in societies. Legal and political measures to tackle the production of statelessness must not only be grounded in normative principles such as non-discrimination, but also in pragmatic considerations as to their real-world conse- quences and potential for backlash. An ethically incremental approach would allow for the development of new legal and policy tools over time through a process of continuous learning and feedback on the understanding that small steps carried out responsibly and effectively can lead to genuine social change. A. The Promise of Indirect Discrimination The prohibition of discrimination is one of the core guarantees enshrined in international as well as regional human rights treaties. Notwithstanding variations in language across different instruments, the prohibition is understood to encompass both “direct” and “indirect” discrim- ination, either expressly or through interpretation.266 While the dividing line between the two forms of discrimination is neither uniform nor always clear, in general, “direct discrimination” refers to deliberate or intentional unjustiﬁed differential treatment based on a suspect category, whereas the notion of indirect discrimination is concerned with seemingly neutral laws, policies, and practices that have a disproportionately adverse impact on a protected category.267 Prominent theorists of discrimination law argue that discrimination is a highly mutable and adaptable phenomenon, becoming even more elusive when under attack—in other words, pre- cisely the kinds of scenarios outlined in this Article—and indirect discrimination norms are the law’s way of “playing catch up” with its plasticity.268 Indeed, nearly all of the rare legally success- ful challenges to the production of statelessness—as in the case of the Kenyan Nubians, the Slovenian erased, and the Dominicans of Haitian descent—have relied in part on the prohibition on discrimination, albeit without clearly distinguishing between the direct and indirect forms, as one of the underlying rights violations. Given the promise that the law of indirect discrimination in particular holds for challenging exclusionary but facially neutrally state policies and techniques of the kind discussed in Part II, why has it not emerged as a ubiquitous instrument for courts and other human rights bodies to challenge statelessness? Part of the problem lies in the nebulousness of indirect discrimination itself. As Victor Madrigal-Borloz, the UN Independent Expert on Protection against Violence and Discrimination Based on Sexual Orientation and Gender Identity has recently stated, “concep- tualizations of indirect discrimination are largely absent from the doctrine and case law of United https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 279 MANUFACTURING STATELESSNESS Nations (“UN”) human rights treaty bodies and special procedures.”269 Another recent study analyzing the individual communications of the HRC giving effect to the prohibition of discrim- ination under Article 26 of the ICCPR argues that the HRC has paid lip-service to the notion, with its approach in concrete cases being far from even. 274 See Indirect Discrimination and Sexual Orientation and Gender Identity: October 2020 Workshop Proceedi at 18–19 (Harvard Human Rights Program Research Working Paper Series). 269 Victor Madrigal-Borloz, The Theory of Indirect Discrimination: Application to the Lived Realities of Lesbian, Gay, Bisexual, Trans, and Other Gender Diverse (LGBT) Persons, 34 HARV. HUM. RTS. L.J. 295 (2021). 270 Niels Petersen, The Implicit Taxonomy of the Equality Jurisprudence of the UN Human Rights Committee, 34 LEIDEN J. INT’L L. 421, 429–430 (2021). 271 See Deborah Hellman, Indirect Discrimination and the Duty to Avoid Compounding Injustice, in FOUNDATIONS OF INDIRECT DISCRIMINATION LAW, supra note 267, at 105, 105–06 (discussing disagreements between theorists as to the distinction between direct and indirect discrimination). 272 Collins and Khaitan, supra note 267, at 25–26. 273 See Gerald L. Neuman, Questions of Indirect Discrimination on the Basis of Religion, 34 HARV. HUM. RTS. L.J. 177, 180 (2021). 274 See Indirect Discrimination and Sexual Orientation and Gender Identity: October 2020 Workshop Proceedings, at 18–19 (Harvard Human Rights Program Research Working Paper Series). 273 See Gerald L. Neuman, Questions of Indirect Discrimination on the Basis of Religion, 34 HARV. HUM. RTS. 177, 180 (2021). 4 269 Victor Madrigal-Borloz, The Theory of Indirect Discrimination: Application to the Lived Realities of Lesbian, Gay, Bisexual, Trans, and Other Gender Diverse (LGBT) Persons, 34 HARV. HUM. RTS. L.J. 295 (2021). 270 Niels Petersen, The Implicit Taxonomy of the Equality Jurisprudence of the UN Human Rights Committee, 34 LEIDEN J. INT’L L. 421, 429–430 (2021). 271 See Deborah Hellman, Indirect Discrimination and the Duty to Avoid Compounding Injustice, in A. The Promise of Indirect Discrimination The Committee’s abstract pronounce- ment that distinctions between groups that are based on “objective and reasonable grounds” can be justiﬁed has led to contradictory outcomes in analogous fact situations.270 j y g There are several possible explanations for this reluctance to engage deeply with the sub- stance and implications of the doctrine of indirect discrimination by human rights bodies. Conceptually and normatively, it is contested why indirect discrimination constitutes a moral wrong in the ﬁrst place. Theorists disagree, for instance, on whether direct and indirect discrimination share a common moral foundation, such as equal respect for persons, or if indi- rect discrimination does not wrong an individual in the same way but may nonetheless be prohibited on grounds of distributive justice so as to promote equality of opportunity and social welfare that takes into account the unfair distribution of beneﬁts and opportunities for some groups of people. Other scholars suggest that indirect discrimination constitutes a distinct moral wrong because it compounds existing societal injustice.271 Still others suggest that rather than constituting an independent moral wrong, indirect discrimination should be viewed as a pragmatic instrument to achieve objectives pursued by the law of direct discrim- ination: it can smoke out pretextual discrimination, serve as evidence of discriminatory intent, and strengthen the effectiveness of norms prohibiting direct discrimination by also sanction- ing policies and practices that are unconsciously or accidentally discriminatory.272 These nor- mative differences as to what makes indirect discrimination wrongful may impact the law’s conception of when it should be considered wrongful and whose perspective—the one of the alleged perpetrator or that of the alleged victim(s)––will be given weight.273 j y g There are several possible explanations for this reluctance to engage deeply with the sub- stance and implications of the doctrine of indirect discrimination by human rights bodies. Conceptually and normatively, it is contested why indirect discrimination constitutes a moral wrong in the ﬁrst place. Theorists disagree, for instance, on whether direct and indirect discrimination share a common moral foundation, such as equal respect for persons, or if indi- rect discrimination does not wrong an individual in the same way but may nonetheless be prohibited on grounds of distributive justice so as to promote equality of opportunity and social welfare that takes into account the unfair distribution of beneﬁts and opportunities for some groups of people. A. The Promise of Indirect Discrimination Other scholars suggest that indirect discrimination constitutes a distinct moral wrong because it compounds existing societal injustice.271 Still others suggest h h h i i i d d l i di di i i i h ld b g p p g g g For advocates as well as the human rights institutions themselves, labeling something “indirect” rather than “direct” discrimination then poses a Catch-22. If indirect discrimina- tion does not signal the same kind of “bad faith” on the part of the policymaker, as does its direct counterpart, then it may not imply the kind of blameworthiness that advocates want to call out and target. On the other hand, it is precisely because of the “softer” condemnation of state policy without the corresponding moral judgment that a ruling of indirect discrimina- tion may be more palatable to states and reduce the potential for political backlash. This may be the case even though indirect discrimination highlights the ways in which exclusionary policies reﬂect and amplify, not just individual, but structural discrimination, and thus may necessitate transformational, institutional reform.274 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW 280 Vol. 116:2 This dilemma is illustrated by the co-evolution of the doctrines of direct and indirect dis- crimination in the jurisprudence of the ECtHR. The court ﬁrst formally recognized indirect discrimination in the form of disproportionately prejudicial impact on a protected group pur- suant to a neutral policy in the case of D.H. and Others v. the Czech Republic, a case dealing with the disproportionate impact of a general educational rule on Roma children in the Czech Republic. The court ruled that prima facie evidence of this discrimination could be produced through statistical evidence showing disproportionate prejudicial impact on the protected group compared to a similarly situated group. It would nonetheless be open to the state to justify the indirect discrimination by demonstrating that the policy pursued a legitimate aim through measures that were both proportionate and necessary.275 While DH and Others could have been a turning point in the ECtHR’s approach to dis- crimination, it has fallen short of that promise. 275 D.H. v. The Czech Republic, 2007-IV Eur. Ct. H.R., para. 196. 276 See Mathias Möschel, The Strasbourg Court and Indirect Race Discrimination: Going Beyond the Education Domain, 80 MOD. L. REV. 121, 124–125 (2017). 277 See Samantha Besson, Evolutions in Non-discrimination Law within the ECHR and the ESC Systems: It Takes Two to Tango in the Council of Europe, 60 AM. J. COMP. L. 147, 167–71 (2012). 278 See Barbara Havelková, Judicial Scepticism of Discrimination at the ECtHR, in FOUNDATIONS OF INDIRECT DISCRIMINATION LAW, supra note 267, at 83, 84, 98–99. 279 Id. at 98. 280 Id. at 101. A. The Promise of Indirect Discrimination For one, the court does not always distinguish between direct and indirect discrimination in its case law, making it difﬁcult to trace the impact of the latter, in particular since there have been few pronouncements on indirect discrimination outside the context of education segregation.276 There is also a lack of mean- ingful precision in the standard for justiﬁed discrimination—what, for instance, is a legitimate aim?—which is further compounded by the margin of appreciation accorded to states in the application of the European Convention on Human Rights (ECHR). Scholars claim that even though the court requires “very weighty reasons” to justify differential treatment on pro- tected grounds such as gender, race, and religion, this heightened protection is often diluted by the court’s deference to states under the margin of appreciation.277 Even so, the court’s embrace of indirect discrimination has invited a steady stream of objec- tions from defendant states as well as individual judges of the ECtHR in their separate and dissenting opinions, a pressure that the court has withstood thus far, but for reasons that may not be entirely benign.278 In a fascinating study dissecting the court’s racial discrimination jurisprudence, Barbara Havelková contrasts the court’s willingness to infer structural preju- dice from statistical evidence in indirect discrimination cases and its failure to infer racial motive in arguably more egregious cases of direct discrimination. Havelková queries whether this divergent approach is because “[f]inding structural bias can be more palatable than ﬁnd- ing racist motives.”279 Seen in this light, rather than a progressive tool for furthering anti-dis- crimination norms, indirect discrimination appears to be a way for the court to avoid making uncomfortable pronouncements on racist bias. Indeed, as Havelková goes on to argue, “in several of the post-DH discrimination cases, the Court did not merely not require a proof of intent, but seemed to reassure the defendant state that their ﬁnding of discrimination was not meant to imply the government had racist intentions.”280 https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 281 MANUFACTURING STATELESSNESS As these developments highlight, much conceptual excavation and doctrinal ﬁne-tuning remains to be done for international and regional norms on indirect discrimination to realize their potential, in particular when it comes to challenging the state’s prerogative on matters that relate to membership in the political community. 281 See Collins & Khaitan, supra note 267, at 21–25 (discussing the distinction between the two categories of discrimination in a number of different jurisdictions). 282 Open Society Justice Initiative, Strategic Litigation Impacts: Insights from Global Experience, at 25 (2018). 283 Adam Weiss, The Essence of Strategic Litigation, in STRATEGIC LITIGATION: BEGRIFF UND PRAXIS 27, 28 (Alexander Graser & Christian Helmrich eds., 2019). 284 See, e.g., Cathryn Costello, Strategic Litigation to Vindicate the Rights of Refugees and Migrants: Pyrrhic Perils and Painstaking Progress, in LEGAL CASES THAT CHANGED IRELAND (Ivana Bacik & Mary Rogan eds., 2016). A. The Promise of Indirect Discrimination What kinds of evidence would be needed to argue that ostensibly neutral policies such as demanding registration requirements that have served to disenfranchise Thai hill tribes or documentation drives that have led to the UAE’s practice of off-shore citizenship for minority groups, constitute instances of prohibited discrimination? Would it be more strategic to deliberately frame challenges to manufactured statelessness as not implicating malign intentions, but rather state failure to pay sufﬁcient attention to the prejudicial impact of its neutral policies on a protected group? Alternatively, would it be better to consciously blur the boundaries between direct and indi- rect discrimination by rethinking whether direct discrimination must always require deliber- ate intent to discriminate? In domestic legal systems, for example, jurisdictions such as Canada have chosen to abandon the distinction between the two categories of discrimination whereas those such as the UK treat the distinction as one of degree rather than kind, with proof of deliberate intent to discriminate not required for either form of discrimination.281 281 See Collins & Khaitan, supra note 267, at 21–25 (discussing the distinction between the two categories of discrimination in a number of different jurisdictions). B. Strategic Litigation as a Way Station As the examples of state-choreographed statelessness in Part II revealed, some of the partial successes in constraining statelessness practices in cases such as the Dioula ethnic community in Côte d’Ivoire and those “without nationality” in the Netherlands have come from actors bringing claims before regional human rights courts and bodies. Indeed, many would be clas- siﬁed as instances of “strategic litigation,” where, in the words of one of its main proponents, the Open Society Justice Initiative (OSJI), the claimant pursues “legal action in a court that is consciously aimed at achieving rights-related changes in law, policy, practice, and/or public awareness above and beyond relief for the named plaintiff(s).”282 Transnational and international strategic litigation is a relatively recent phenomenon with its fair share of both supporters and detractors. For one, there are inherent challenges with identifying a “test case” for the purposes of strategic litigation efforts. Or as Adam Weiss, the managing director of European Roma Rights Centre puts it, “strategic litigation has a lot in common with the notion of “black swans”” and “[w]hat makes a case strategic can only be seen in retrospect.”283 There is always a risk that the lawyer(s) attempting to engage in the risk versus rewards exercise of the potentially beneﬁcial outcome gets the calculation wrong, leading to an unfavorable outcome that ends up reversing, instead of accelerating, the campaign for legal reform. And even cases that result in victories might only incentivize states to come up with workarounds that avoid the precedent set by a negative ruling.284 Moreover, overreliance on one court as a site for demanding rights comes with its own problems and can increase the possibility of backlash from states and domestic non-state actors such as https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 282 conservative parties or lobby groups.285 And ﬁnally, there is the ever-present question of resources: even if lawyers could be conﬁdent of choosing wisely, not all of them have the insti- tutional or ﬁnancial capacity to pursue strategic litigation. 285 See, e.g., Daniel Thym, The End of Human Rights Dynamism? Judgments of the ECtHR on “Hot Returns” and Humanitarian Visas as a Focal Point of Contemporary European Asylum Law and Policy, 32 INT’L J. REFUGEE L. 569 (2020) (analyzing the European courts’ recent turn away from rights-enhancing judgments in the context of migration litigation in light of the changing political context and perceptions of their legitimacy). 286 B. Strategic Litigation as a Way Station It is not surprising to see studies highlighting that strategic litigation before supranational courts is dominated by a few players who tend to be larger well-funded organizations or smaller organizations that can partner with them.286 Notwithstanding these limitations within which strategic litigation operates, scholarship as well as practice, including in the context of statelessness, point to ways in which strategic lit- igation can act as “a pebble that paves the road for certain [rights-enhancing] changes.”287 The earlier example of OSJI’s successful claim before the African Commission on Human and People’s Rights challenging Côte d’Ivoire’s denial of nationality to members of the Dioula ethnic community is a good illustration of some of the elements that would need to be in place to maximize the chances of strategic litigation translating into positive legal change. The ﬁrst is acute sensitivity to the political and social environment that is especially important for politically explosive issues such as citizenship. In the case of Côte d’Ivoire, as previously mentioned, President Ouattara’s ascendency to political ofﬁce meant that the shifting political winds were likely to favor the Commission’s decision. Strategic litigators seeking to combat statelessness can, however, also play a part in preparing the ground so as to increase the likelihood of compliance with supranational decisions through mechanisms such as robust local civil society mobilization and bringing “preparatory” cases at the national level that feed into and reinforce the supranational ruling.288 Complementary rulings by pro- gressive and independent domestic courts combined with legitimation through national human rights actors might help counter the perception of supranational meddling in sover- eign matters related to the grant of nationality. Equally important is what happens in the aftermath of a successful ruling, even when it falls on fertile political ground. 286 See HEIDI NICHOLS HADDAD, THE HIDDEN HANDS OF JUSTICE: NGOS, HUMAN RIGHTS, AND INTERNATIONAL COURTS 61–62, 104–05 (2018) (surveying the types of NGOs that typically engage in strategic litigation at the ECtHR and the IACtHR). 287 Open Society Justice Initiative, Strategic Litigation Impacts: Roma School Desegregation, at 72 (2016) (quot- ing a Czech Ministry of Justice ofﬁcial on the impact of the ECtHR’s ruling in DH on the Czech legal landscape). 288 See MORITZ BAUMGÄRTEL, DEMANDING RIGHTS: EUROPE’S SUPRANATIONAL COURTS AND THE DILEMMA OF MIGRANT VULNERABILITY 134 (2019). 289 See HADDAD, supra note 286, at 105; STRATEGIC LITIGATION IMPACTS, supra note 282, at 61. 290 Freek van der Vet, Transitional Justice in Chechnya: NGO Political Advocacy for Implementing Chechen Judgments of the European Court of Human Rights, 38 REV. CENT. & E. EUR. L. 363, 376 (2013). 90 Freek van der Vet, Transitional Justice in Chechnya: NGO Political Advocacy for Implementing Chechen gments of the European Court of Human Rights, 38 REV. CENT. & E. EUR. L. 363, 376 (2013). 287 Open Society Justice Initiative, Strategic Litigation Impacts: Roma School Desegregation, at 72 (2016) (quot- ing a Czech Ministry of Justice ofﬁcial on the impact of the ECtHR’s ruling in DH on the Czech legal landscape). 288 See MORITZ BAUMGÄRTEL, DEMANDING RIGHTS: EUROPE’S SUPRANATIONAL COURTS AND THE DILEMMA OF MIGRANT VULNERABILITY 134 (2019). 285 See, e.g., Daniel Thym, The End of Human Rights Dynamism? Judgments of the ECtHR on “Hot Returns” and Humanitarian Visas as a Focal Point of Contemporary European Asylum Law and Policy, 32 INT’L J. REFUGEE L. 569 (2020) (analyzing the European courts’ recent turn away from rights-enhancing judgments in the context of migration litigation in light of the changing political context and perceptions of their legitimacy). 286 See HEIDI NICHOLS HADDAD, THE HIDDEN HANDS OF JUSTICE: NGOS, HUMAN RIGHTS, AND INTERNATIONAL COURTS 61–62, 104–05 (2018) (surveying the types of NGOs that typically engage in strategic litigation at the ECtHR and the IACtHR). 287 Open Society Justice Initiative, Strategic Litigation Impacts: Roma School Desegregation, at 72 (2016) (quot- ing a Czech Ministry of Justice ofﬁcial on the impact of the ECtHR’s ruling in DH on the Czech legal landscape). 288 See MORITZ BAUMGÄRTEL, DEMANDING RIGHTS: EUROPE’S SUPRANATIONAL COURTS AND THE DILEMMA OF MIGRANT VULNERABILITY 134 (2019). 289 See HADDAD, supra note 286, at 105; STRATEGIC LITIGATION IMPACTS, supra note 282, at 61. 290 Freek van der Vet, Transitional Justice in Chechnya: NGO Political Advocacy for Implementing Chechen Judgments of the European Court of Human Rights, 38 REV. CENT. & E. EUR. L. 363, 376 (2013). 89 See HADDAD, supra note 286, at 105; STRATEGIC LITIGATION IMPACTS, supra note 282, at 61. 90 87 Open Society Justice Initiative, Strategic Litigation Impacts: Roma School Desegregation, at 72 (2016) (quot- a Czech Ministry of Justice ofﬁcial on the impact of the ECtHR’s ruling in DH on the Czech legal landscape). 88 B. Strategic Litigation as a Way Station The importance of strategic litigators actively engaging with imple- mentation in the post-judgment phase has been borne out in the context of supranational courts, such as the ECtHR and the IACtHR.289 For example, research on the experience of litigating Chechen cases at the ECtHR has shown the crucial role played by NGOs in the post-litigation phase in actively lobbying the Committee of Ministers tasked with supervising the implementation of the court’s judgments by submitting memoranda and creating net- works with Council of Europe ofﬁcials.290 In Côte d’Ivoire, following the Commission’s deci- sion on denial of nationality, OSJI worked closely with civil society actors to build local and https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 283 2022 MANUFACTURING STATELESSNESS regional networks that would facilitate the decision’s implementation. This included partner- ing with the UNHCR ofﬁce in Abidjan to help set up the Civil Society against Statelessness (CICA), a coalition of local NGOs with the mandate to act as a focal point for liaising with government actors as well as providing feedback and information to the Commission on the implementation of measures to combat statelessness. B. Strategic Litigation as a Way Station The OSJI itself also assisted these efforts by making submissions to the Commission outlining the steps that the Ivorian government had taken to implement the decision.291 p Given the dearth of international and supranational jurisprudence on the conditions for the acquisition and denial of nationality, strategic litigation on statelessness can thus trigger medium- to long-term legal reforms, including in cases that result in losses, if litigators are able to incrementally move the courts’ jurisprudence in a progressive direction or jumpstart domestic mobilization.292 In the same vein, instead of putting all their eggs in one litigation basket, strategic litigators on statelessness may adopt a more pragmatic “topographical” pos- ture, whereby they simultaneously pursue remedies for tackling statelessness through differ- ent legal and political mechanisms at the domestic, regional, and international levels.293 The idea is that combining overlapping and mutually reinforcing liability regimes will not only prove to have a greater deterrent effect on states but also make it that much harder for them to introduce policies and measures that are designed to evade the precedential effect of a negative ruling in any particular legal regime.294 As cases such as the Dioula, those with- out nationality in the Netherlands, and the Kenyan Nubians demonstrate, strategic litigators on statelessness would be well advised to move beyond a court-centric focus and also consider initiating cases before “softer” mechanisms such as human rights treaty bodies. They should also explore the prospects of bringing nationality claims through international procedures that have seldom been tested. For example, it has been suggested that India’s discriminatory citizenship laws discussed earlier could be challenged under the CERD Convention by a state initiating a complaint before the CERD Committee through the interstate mechanism.295 291 Alpha Sesay & Amon Dongo, Côte d’Ivoire’s Statelessness Problem: Utilizing Multiple Tools to Support Implementation of Judgments, in IMPLEMENTING HUMAN RIGHTS DECISIONS: REFLECTIONS, SUCCESSES, AND NEW DIRECTIONS 29 (2021). 295 See Priya Pillai, State Responsibility for Citizenship in India: Lessons from Myanmar, and the CERD Inter-State Communication Mechanism, OPINIO JURIS (Dec. 21, 2019), at http://opiniojuris.org/2019/12/21/state-responsi- bility-for-citizenship-in-india-lessons-from-myanmar-and-the-cerd-inter-state-communications-mechanism. 296 A il bl h // h /ib l 292 See BAUMGÄRTEL, supra note 288, at 129 (arguing, in the context of migration, that even cases that are des- tined to fail may turn into “successes without victories”). 296 Available at https://www.unhcr.org/ibelong. 293 Nikolas Feith Tan & Thomas Gameltoft-Hansen, A Topographical Approach to Accountability for Human Rights Violations in Migration Control, 21 GERMAN L.J. 335, 337 (2020) (outlining the contours of a topographical approach to strategic litigation). pp 294 Id. 291 Alpha Sesay & Amon Dongo, Côte d’Ivoire’s Statelessness Problem: Utilizing Multiple Tools to Support Implementation of Judgments, in IMPLEMENTING HUMAN RIGHTS DECISIONS: REFLECTIONS, SUCCESSES, AND NEW DIRECTIONS 29 (2021). 292 See BAUMGÄRTEL, supra note 288, at 129 (arguing, in the context of migration, that even cases that are des- tined to fail may turn into “successes without victories”). 293 Nikolas Feith Tan & Thomas Gameltoft-Hansen, A Topographical Approach to Accountability for Human Rights Violations in Migration Control, 21 GERMAN L.J. 335, 337 (2020) (outlining the contours of a topographical approach to strategic litigation). 294 Id. 295 See Priya Pillai, State Responsibility for Citizenship in India: Lessons from Myanmar, and the CERD Inter-State Communication Mechanism, OPINIO JURIS (Dec. 21, 2019), at http://opiniojuris.org/2019/12/21/state-responsi- bility-for-citizenship-in-india-lessons-from-myanmar-and-the-cerd-inter-state-communications-mechanism. 296 Available at https://www.unhcr.org/ibelong. C. Rethinking Legal Identity Documentation While formal legal actions are certainly one potential avenue to counter manufactured statelessness, no less important are policy and advocacy measures championed by interna- tional actors, in particular, those taken by the UNHCR as the nodal agency for tackling state- lessness. In 2014, the UNHCR launched the #IBelong Campaign with the ambitious aim to end statelessness in a decade.296 The campaign represents a major shift in the UNHCR’s https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 284 posture on statelessness on which it has historically been reluctant to adopt an interventionist stance for fear of compromising political support for its central activities concerning refugees and asylum seekers.297 To fulﬁll this mandate, the UNHCR has developed the Global Action Plan to End Statelessness: 2014–2024, which sets out ten actions that must be taken in order to end statelessness, including formal legal measures with respect to which it has already achieved some success, such as advocating for the removal of gender discrimination in laws governing nationality, improving data on statelessness, and encouraging accession to the Statelessness Conventions. Other prominent recommendations relate to ensuring birth reg- istration and the issuance of nationality documentation to persons who would be entitled to it.298 The UNHCR’s emphasis on the acquisition of identity documentation to address stateless- ness has been echoed in the 2018 Global Compact on Refugees’ program of action, which advocates civil and birth registration and documentation to reduce the risk of statelessness.299 It is also in line with the championing of legal identity and registration documentation by major international human rights and development agencies on the premise that they symbol- ize universal membership and confer a standardized equal identity on the bearer.300 This is exempliﬁed in the language of the Sustainable Development Goals (SDG) setting out the tar- get to “provide legal identity for all, including birth registration” by 2030.301 However, as this Article has argued, identiﬁcation practices and documentation are a double-edged sword that can be used to both include and exclude. While the ascription of legal identity and its doc- umentary proof can certainly enable access to essential goods and services such as health, edu- cation, housing, and ﬁnance, and promote the efﬁcient functioning of social welfare programs,302 as scholars have noted, the very same document may carry different meanings depending on the political context and purposes for which it is employed. 297 STAPLES, supra note 103, at 147. 298 Other measures involve resolving existing situations of statelessness, granting protection to stateless migrants, preventing childhood statelessness and loss of nationality on discriminatory grounds and in cases of state succession. Global Action Plan to End Statelessness, supra note 7. 299 Global Compact on Refugees, § 2.8, UN Doc. A/73/12 (Part II) (Aug. 2, 2018). 300 See Samantha Balaton-Chrimes, Statelessness, Identity Cards and Citizenship as Status in the Case of the Nubians of Kenya, 18 CITIZENSHIP STUD. 15, 18, 20 (2014). 301 UN General Assembly, Transforming Our World: The 2030 Agenda for Sustainable Development, Goal 16.9, UN Doc. A/RES/70/1 (Oct. 21, 2015). 302 See Bronwen Manby, The Sustainable Development Goals and “Legal Identity for All”: “First, Do No Harm,” 139 WORLD DEV. 2, 3 (2021) (summarizing the economics and policy literature on the beneﬁts of identity documentation). 303 DAVID LYON, IDENTIFYING CITIZENS: ID CARDS AS SURVEILLANCE 3 (2009). 304 See Rachel E. Rosenbloom, From the Outside Looking In: U.S. Passports in the Borderlands, in CITIZENSHIP IN QUESTION, supra note 165, at 132, 140. 303 DAVID LYON, IDENTIFYING CITIZENS: ID CARDS AS SURVEILLANCE 3 (2009). 304 304 See Rachel E. Rosenbloom, From the Outside Looking In: U.S. Passports in the Borderlands, in CITIZENSHI QUESTION, supra note 165, at 132, 140. 297 STAPLES, supra note 103, at 147. 298 p 98 Other measures involve resolving existing situations of statelessness, granting protection to stateless rants, preventing childhood statelessness and loss of nationality on discriminatory grounds and in cases of e succession. Global Action Plan to End Statelessness, supra note 7. C. Rethinking Legal Identity Documentation Identity documents “may be carried with pride, indifference, reluctance or even fear, depending on the political conditions and the history of using such documents in the country in question.”303 Identity cards, in particular, have traditionally carried a range of negative connotations in countries where they have been used as a form of surveillance and control and to deny and restrict the rights of marginalized groups.304 As the examples in Part II have indicated, the drive to document and enumerate popula- tions has at times had the paradoxical effect of rendering stateless individuals who fall out of the carefully constructed categories of citizens and “foreigners.” Previous studies had https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 285 MANUFACTURING STATELESSNESS 2022 examined the ways in which countries such as the Dominican Republic have implemented legal and non-legal measures to “forcibly displace” Dominican born persons of Haitian descent by forcibly attributing to them the putative nationality of their “native land” or risk being placed in a limbo state of statelessness.305 More recent scholarship has speciﬁcally highlighted the link between the identity documentation drive in the Dominican Republic and its impact on the exclusion of Dominicans of Haitian descent from citizenship. The push by international actors such as the UN, the World Bank, and the Inter-American Development Bank for the Dominican Republic to undertake population identiﬁcation and civil registration measures in order to facilitate the delivery of social welfare programs had the paradoxical effect of unsettling the long established entitlements of Dominicans of Haitian descent.306 International actors such as the World Bank provided ﬁnancial and tech- nical support to implement registration policies at the macro-level but left the Dominican Republic free to determine the criteria for citizenship acquisition and eligibility for legal iden- tity documentation. Thus, even Dominicans of Haitian descent who possessed the appropri- ate paperwork suddenly found themselves unable to renew their documentation or were faced with their conﬁscation.307 In the Kenyan case, the new digital identity system, “Huduma Namba,” intended as a comprehensive registration system to guarantee legal identity and facilitate access and entitle- ment to government services and welfare programs, would theoretically have functioned as an inclusionary instrument that recognizes the status of marginalized communities such as the Nubians and Somali Kenyans as equal citizens. 305 See Kristy A. Belton, Rooted Displacement: The Paradox of Belonging Among Stateless People, 19 CITIZENSHIP STUD. 907 (2016). 306 HAYES DE KALAF, supra note 235. 307 Eve Hayes de Kalaf, Making Foreign: Legal Identity, Social Policy and the Contours of Belonging in the Contemporary Dominican Republic, in WELFARE AND SOCIAL PROTECTION IN CONTEMPORARY LATIN AMERICA 101, 107 (Gibran Cruz-Martinez ed., 2019). 308 Holly Ritson, “We Are Not Data Points”: Highlights from our Conversation on the Kenyan Digital ID System, NYU CENTER FOR HUMAN RIGHTS AND GLOBAL JUSTICE (Nov. 9, 2020), at https://chrgj.org/2020/11/09/we-are- not-data-points-highlights-from-our-conversation-on-the-kenyan-digital-id-system. 309 Nubian Rights Forum & 2 others v. Attorney General & 6 others; Child Welfare Society & 9 Others (Interested Parties) [2020] eKLR, at http://kenyalaw.org/caselaw/cases/view/189189. 310 Open Society Justice Initiative, Litigation, Nubian Rights Forum et al. v. the Honourable Attorney General of Kenya et al. (“NIIMS Case”), at https://www.justiceinitiative.org/litigation/nubian-rights-forum-et-al-v-the- honourable-attorney-general-of-kenya-et-al-niims-case. 311 Manby, supra note 302, at 7. 312 See Bronwen Manby, Naturalization in African States: Its Past and Potential Future, 25 CITIZENSHIP STUD. 514, 525 (2021). 313 See Christoph Sperfeldt, Legal Identity in the Sustainable Development Agenda: Actors, Perspectives and Trends in an Emerging Field of Research, 26 INT’L J. HUM. RTS. 1, 14–16 (2021). C. Rethinking Legal Identity Documentation However, as advocates have argued, the Huduma Namba system is merely “layered over a history of exclusion” faced by these com- munities and replicates many of the endemic discriminatory practices in their access to iden- tity documents. Not only may Kenyans from border communities, such as Somali Kenyans, be required to undergo vetting for community membership by local chiefs before being able to access the ID system, but the difﬁculties and delays in being able to prove this membership has driven some applicants to register as refugees simply to be able to gain access to services.308 Multiple domestic legal actions have been brought before Kenyan domestic courts, chal- lenging, amongst other things, the broad discretion vested in registration authorities to demand additional forms of documentary proof of nationality from members of the margin- alized groups as a violation of the right to equality and non-discrimination. However, in its January 2020 ruling, the Kenyan High Court held that the law establishing the digital iden- tity system did not, on its face, establish any distinction between marginalized groups such as the Nubians and other Kenyans, and the evidence adduced before the court was insufﬁcient to prove discrimination. Thus, while it recognized the possibility of the risk of exclusion of some groups and stressed the need for a regulatory mechanism to mitigate this risk, the court https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 286 rejected the discrimination claim.309 Civil society actors such as the OSJI have nonetheless persisted in their advocacy efforts calling out the digital identity system as a form of indirect discrimination that violates the Kenyan constitution by penalizing marginalized groups who face difﬁculties accessing legal identity documentation required for registration.310 g g g Scholarship analyzing the impact of identity documentation drives shows that far from being outliers, cases such as the Dominican Republic and Kenya are emblematic of the poten- tial of new registration systems to reinforce discriminatory citizenship practices. C. Rethinking Legal Identity Documentation The intro- duction of a new nationality identity card in Mauritania, for instance, has been claimed to be part of the government’s strategy to revoke the citizenship of black Mauritanians and as a form of “biometric genocide.” Similarly, concerns have been raised on the establishment of the Sudanese civil registration system and ID card as a tool to identify individuals of southern Sudanese descent and deny them the right to citizenship.311 These examples point to the depressing possibility that, instead of eliminating statelessness, the push by international actors to encourage or even require states to ascribe a legal status—and the accompanying identity document—to an individual, may inadvertently end up facilitating its entrenchment. y y y p g This does not imply that measures to access legal identity and documentation are automat- ically suspect; rather they suggest the need for agencies such as the UNHCR to recognize the Janus-faced nature of identity documentation as reinforcing the binary divisions between cit- izens and non-citizen others, at times to the detriment of those who the state wants to exclude from its imaginary.312 Instead of furthering the goal of “leaving no one behind,” the promo- tion of legal identity documentation and registration without sufﬁcient regard to the politics of documentation and evidence can end up reproducing marginalization and statelessness. International human rights and development actors should thus be cautious of an uncritical embrace of legal identity documentation and insist on advance and robust scrutiny of the legal framework and social context in which they will be implemented in order to prevent them from becoming tools of coercion and exclusion.313 313 See Christoph Sperfeldt, Legal Identity in the Sustainable Development Agenda: Actors, Perspectives and Tr in an Emerging Field of Research, 26 INT’L J. HUM. RTS. 1, 14–16 (2021). CONCLUSION The 1920s ﬁctional protagonist of The Death Ship has much in common with the ﬂesh- and-blood stateless person today. Stripped of his nationality and legal identity in the territory he seeks to call home and unable to prove either his nationality or the lack of it, the stateless person inhabits an existential precariousness that knows no escape. As this Article has argued, international and regional courts and human rights bodies have been doggedly pursuing piecemeal efforts to challenge manufactured statelessness and, in the rare case, have done so with some success. However, even these progressive rulings have been characterized by https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press 2022 287 MANUFACTURING STATELESSNESS adhocism and failed to ignite a “justice cascade” where states are routinely being called on to answer for the liminal no-man’s land to which they have consigned thousands of people where none of the protections of citizenship apply. Moreover, as the cases of the Dominicans of Haitian descent and the Kenyan Nubians reveal, their actual impact on the reality of state- lessness on the ground is questionable. States have either ﬂouted the rulings, adopted an “à la carte” approach to implementing the least politically costly aspects of the rulings, surrepti- tiously backtracked on initial compliance measures, or found creative workarounds to avoid the international legal precedent. In parallel, agencies such as the UNHCR have championed policy interventions to resolve statelessness by emphasizing the need for a state-centered response grounded in changes to nationality laws, documentation and registration practices, and improved data collection, to prevent and eliminate statelessness. However, some of these strategies that emphasize formal, technical reforms will do little to address the main pathways through which states manufac- ture statelessness in the shadow of the law. And others, such as identity documentation and registration drives may even prove to be counterproductive and exacerbate covert stateless- ness. Comprehensive identity management systems intended to serve as a “single source of truth,” as claimed by Kenya’s Huduma Namba—and often supported by technical and ﬁnan- cial assistance measures by international human rights and development agencies—can be layered upon existing forms of exclusion or give rise to new ones. This Article has argued that states’ agility in countering halting efforts by international legal actors calls for an international legal roadmap that can go toe-to-toe with the various and evolving techniques the state implements to manufacture statelessness. 314 See Gibney, supra note 27, at 53–55 (on the various reasons motivating states to intentionally create statelessness). CONCLUSION This roadmap requires recognizing that rather than invite the wrath of international law by enacting overtly illegal or discriminatory rules and institutions, the state can instead achieve the same result by manipulating ostensibly egalitarian criteria for political membership such as time, territory, and administrative processes. Far from being neutral and unbiased, the effects of legal recon- ﬁgurations of time and space, and the requirements for empirical citizenship, are not ran- domly distributed but reﬂect pre-existing and extra-legal exclusions of certain types of people from the political imaginary of the nation-state. The ultimate result is thus often the same—statelessness that is caused by state-sponsored discrimination and inequality— but implemented through facially neutral conceptual and policy instruments that ﬂy under the radar of international law. This strategic statelessness is not evidence of the limits of the state’s political authority, but rather constitutive of it: strategy that allows the state to exploit the vulnerability and labor of stateless persons, strategy that questions the loyalty of certain groups and paints them as unworthy of citizenship, and strategy that uses exclusion as a polit- ical weapon to facilitate people-building.314 For international legal actors to build and implement this roadmap, they would need to learn how to think and see like a state. To do otherwise would be to risk introducing a doc- trinal or policy measure only to be outsmarted by the state carving out creative exceptions to its application. The Article has thus emphasized a multi-pronged approach that begins the process of constructing such a roadmap drawing on legal doctrine, litigation strategy, and pol- icy. The ﬁrst of these advocates developing the normative foundations and doctrinal contours https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press THE AMERICAN JOURNAL OF INTERNATIONAL LAW Vol. 116:2 288 of international and regional legal norms on indirect discrimination. As various studies have shown, these have either been vastly underexplored or unevenly applied in the jurisprudence of regional human rights courts and the human rights treaty bodies. Since indirect discrim- ination is generally seen as avoiding the attribution of bad faith or an evil motive to the state, this may be a more palatable way for international bodies to signal the illegal impact of dis- criminatory state policies on statelessness. Any doctrinal reform, however, can only be as good as the actors who use it to litigate state- lessness. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press CONCLUSION The Article has thus highlighted the potential of the “topographical” approach to strategic litigation on statelessness. This will involve international and local civil society actors exploring multiple legal fora—including those that may not be obvious choices for stateless- ness claims—to ﬁnd different jurisdictional hooks to simultaneously pursue remedies for state manufactured statelessness. Spreading the litigation risk between different fora may also pre- vent any particular court being singled out as a repeat player and potentially being subject to backlash from disgruntled states. And ﬁnally, the Article has recommended revisiting the policy push by agencies such as the UNHCR for legal identity documentation and registration as an instrument to tackle state- lessness. As the various examples ranging from the UAE, India, Kenya, the Dominican Republic, and other parts of the world undergoing rapid upgrades in identiﬁcation and reg- istration systems have shown, without advance assessment of the legal framework and polit- ical conditions in which these systems will be implemented, they can easily mutate into technologies of exclusion rather than inclusion. Vital as these reforms are, they make no claim to be a panacea for all the ingenious ways in which states may potentially manipulate facially neutral criteria of time, space, and documen- tation to produce statelessness. In this sense, they are not directed at achieving the interna- tional community’s aspirational goal of ending statelessness in a decade. Rather, they are intended to narrow, as much as possible, and within the existing limits of the international legal order, the circumstances in which states can discriminate against and exclude those who have a prima facie sufﬁciently close connection with the state to be able to claim citizenship in order to engineer and maintain statelessness. The ambition of these proposals is thus—to take a page out of the European Court of Human Rights’ playbook—one of ethical incremental- ism rather than radical overhaul. The hope, nonetheless, is that this decision to “hasten slowly” inspires more revolutionary counter-narratives on statelessness. These may involve rethinking the nature of citizenship as a politico-legal institution that is associated with a cer- tain set of rights, territory, and form of political authority. And to refashioning principles of citizenship allocation in a global political and legal order that systematically produces vast inequalities in the distribution of political power and resources. https://doi.org/10.1017/ajil.2022.2 Published online by Cambridge University Press
https://openalex.org/W2921120407	https://wnus.edu.pl/cto/file/article/view/15356.pdf	Polish	null	Report from the 5th National Conference on Process Law "Judicial Cooperation in the Field of Command". Faculty of Law and Administration of Warmia i Mazury in Olsztyn, April 24, 2018	Colloquia Theologica Ottoniana/Colloquia Theologica Ottoniania	2,018	cc-by-sa	1,384	* Ks. dr Krzysztof Oskar Pokorski – prezbiter archidiecezji szczecińsko-kamieńskiej; doktor na- uk prawnych; wiceoficjał w Metropolitalnym Sądzie Biskupim Archidiecezji Szczecińsko-Ka- mieńskiej; adiunkt w Wyższej Szkole Administracji Publicznej w Szczecinie; dziekan Wydziału Administracji Publicznej i Nauk Społecznych WSAP. E-mail: xkpokorski@gmail.com. COLLOQUIA THEOLOGICA OTTONIANA 2/2018, s. 259–263 COLLOQUIA THEOLOGICA OTTONIANA 2/2018, s. 259–263 COLLOQUIA THEOLOGICA OTTONIANA 2/2018, s. 259–263 DOI: 10.18276/cto.2018.2-15 SPRAWOZDANIE Z V OGÓLNOPOLSKIEJ KONFERENCJI PRAWA PROCESOWEGO „WSPÓŁPRACA SĄDÓW W ZAKRESIE DOWODZENIA” WYDZIAŁ PRAWA I ADMINISTRACJI UNIWERSYTETU WARMIŃSKO-MAZURSKIEGO W OLSZTYNIE 24 KWIETNIA 2018 ROKU Krzysztof Oskar Pokorski* Wydział Administracji Publicznej i Nauk Społecznych Wyższa Szkoła Administracji Publicznej Szczecin Na Wydziale Prawa i Administracji Uniwersytetu Warmińsko-Mazurskiego w Olsztynie, już po raz piąty, zorganizowano Ogólnopolską Konferencję Prawa Procesowego, która odbyła się 24 kwietnia 2018 roku, w Centrum Konferencyj- nym WPiA UWM. Patronat honorowy nad konferencją objął minister sprawiedli- wości, natomiast patronat naukowy objął Instytut na rzecz Kultury Prawnej „Ordo Iuris” oraz Adwokatura Polska. Organizatorami konferencji byli: Katedra Praw Człowieka, Prawa Europejskiego i Kanonicznego WPiA UWM, Katedra Postępo- wania Cywilnego WPiA UWM, Studenckie Koło Naukowe Prawa Wyznaniowe- go „Fides”, Koło Naukowe Postępowania Cywilnego „Contra” oraz Studenckie Koło Naukowe Prawników Kanonistów. Celem konferencji była analiza norm prawnych oraz praktyki w zakresie dowodzenia w postępowaniach sądowych i administracyjnych w Polsce zarów- no w systemie prawa państwowego, jak i w prawie kanonicznym. Tematyka 260 Krzysztof Oskar Pokorski konferencji została potraktowana wielopłaszczyznowo, ponieważ podczas etapu dowodzenia, w każdym systemie prawnym, ujawniają się liczne problemy i kwe- stie wymagające analizy prawnej. Nauka i praktyka prawa procesowego pozwala- ją stwierdzić, że gromadzenie, przedkładanie środków dowodowych w perspekty- wie ich prawnej dopuszczalności, mocy dowodowej i ich oceny, wymagają ciągłej refleksji naukowej i poszukiwań badawczych. Uroczystego otwarcia konferencji dokonał JM prof. zw. dr hab. Ryszard Gó- recki, rektor Uniwersytetu Warmińsko-Mazurskiego. W swoim przemówieniu wyraził wdzięczność organizatorom za cykliczne już podejmowanie zagadnień prawa procesowego. Wskazał również, że dynamika zamian w prawie polskim stwarza konieczność naukowej dyskusji i refleksji. Część merytoryczna tegorocznej konferencji rozpoczęła się sesją plenarną, aby następnie obradować w ramach aż dziewięciu paneli, podczas których głos zabrało ok. 90 prelegentów. Referaty prezentowane były nie tylko przez specjali- stów z różnych ośrodków naukowych, ale również przez sędziów, prokuratorów, adwokatów, a także sędziów i pracowników sądownictwa kościelnego. Sesji plenarnej przewodniczył ks. dr hab. Mieczysław Różański prof. UWM. Już pierwsze, wygłoszone podczas tej sesji referaty pokazały, że temat dowo- dzenia w prawie procesowym jest zagadnieniem bardzo szerokim. Ks. prof. zw. dr hab. Ryszard Sztychmiler (UWM) podjął się refleksji nad znaczeniem proce- sowym rekwizycji sądowych. Wskazał on, że sposób przeprowadzenia rekwizycji oraz ich poziom, nie zawsze są pomocne w dalszym procedowaniu. Apelował, aby przyjęte zlecenie wykonania sądowej rekwizycji wypełnić rzetelnie i z poczu- ciem odpowiedzialności. W tej samej sesji referat wygłosiła mec. Agata Rewerska, ze Szkoły Prawa Procesowego „Ad Exemplum”. Tytuł referatu brzmiał: „Błąd w ustaleniach fak- tycznych jako skutek błędu logicznego sądu (błąd samoistny)”. SPRAWOZDANIE Z V OGÓLNOPOLSKIEJ KONFERENCJI PRAWA PROCESOWEGO „WSPÓŁPRACA SĄDÓW W ZAKRESIE DOWODZENIA” WYDZIAŁ PRAWA I ADMINISTRACJI UNIWERSYTETU WARMIŃSKO-MAZURSKIEGO W OLSZTYNIE 24 KWIETNIA 2018 ROKU Poprowadzony z pasją i na wysokim poziomie merytorycznym referat, zakończony został oficjal- nym zgłoszeniem wniosku de lege ferenda do ministra sprawiedliwości. Kolejnym prelegentem był mec. Bartosz Lewandowski, który reprezento- wał Instytut na rzecz Kultury Prawnej „Ordo Iuris”, tytuł jego referatu brzmiał: „Przesłuchanie małoletniego pokrzywdzonego poniżej 15. roku życia w trybie art. 185a KPK – praktyczne problemy czynności obrońcy w sprawach karnych”. Korzystając z własnego doświadczenia zawodowego, mecenas wskazał na wie- le problemowych kwestii, które dotyczą przesłuchań osób poniżej 15 roku życia i dowodzenia w sprawach z ich udziałem. Prelegent ukazywał również, jak istotna dla dowodzenia jest rola biegłego psychologa i jak ważna jest prawidłowa komu- nikacja między biegłym, sędzią a reprezentantem pokrzywdzonego. 261 Sprawozdanie z V Ogólnopolskiej Konferencji Prawa… Sprawozdanie z V Ogólnopolskiej Konferencji Prawa… Wszystkie referaty sesji plenarnej były przyczynkiem do bardzo ciekawej i ożywionej dyskusji. Każdy z prelegentów odpowiadał kilkakrotnie na zadawa- ne pytania, które były dowodem, że podjęte kwestie nadal wymagają naukowej refleksji i wypracowania wskazań praktycznych. Część dyskusyjną zakończyło zaproszenie na przerwę i poczęstunek. Nie sposób opisać wszystkich wystąpień ze względu na ich liczbę, ale trzeba zauważyć, że tematyka prezentowanych referatów potwierdziła jak ważna i wy- magająca ciągłej dyskusji jest kwestia współpracy sądów w zakresie dowodzenia. Część referatów wprost dotyczyła współdziałania różnych sądów w prowadzeniu spraw. Przykładowo: „Zakres współpracy organów procesowych w postępowaniu w sprawach o wykroczenia”, „Transkrypcja uzasadnień orzeczeń kasatoryjnych sądów gospodarczych”, „Współpraca sądów w zakresie przeprowadzania dowo- dów w państwach Europy środkowo-wschodniej”, „Współpraca sądów admini- stracyjnych z organami administracji publicznej w zakresie dowodzenia”, „Akta sprawy rozwodowej i ich wykorzystanie w procesie o stwierdzenie nieważno- ści małżeństwa”, „Właściwości sądów w procesie cywilnym z uwzględnieniem zasady perpetuatio fori”, „Współpraca sądów w Unii Europejskiej w zakresie przeprowadzania dowodów w sporach transgranicznych”, „Europejski nakaz aresztowania”, „Międzynarodowa pomoc prawna w zakresie przeprowadzania dowodów w sprawach cywilnych na podstawie rozporządzenia Rady (WE) Nr 1206/2001”, czy „Współpraca sądów krajów członkowskich UE w postępowaniu spadkowym”. Warto także wskazać te referaty, które dotyczyły mocy dowodowej poszcze- gólnych środków dowodowych: „Nagranie jako dowód w sprawie rozwodowej”, „Wysłuchanie informacyjne a dowód z przesłuchania stron w postępowaniu cy- wilnym”, „Zakaz przesłuchania duchownego w świetle art. 178 kpk”, „Dopusz- czalność wykorzystania procedury Niebieskiej Karty w kanonicznym procesie małżeńskim”, „Wiarygodność zeznań świadków pozyskanych w drodze pomocy sądowej”, „Dowód z opinii biegłego psychologa i psychiatry w procesie kano- nicznym oraz prawie polskim”, „Dowody i ich charakter w postępowaniu karnym w przypadku plagiatu”, „SMS, E-mail i wydruk z Facebooka jako dowód w spra- wie sądowej”, „Sądowe uznanie za zmarłego jako dowód w procesie o nieważ- ność małżeństwa z kan. 1085 KPK”, „Moc dowodowa opinii biegłego prywatne- go w postępowaniu administracyjnym i cywilnym”, czy „Dowód z badań DNA w sprawach o ustalenie lub zaprzeczenie pochodzenia dziecka”. Warto zaznaczyć, że szczecińskie środowisko prawnicze było reprezentowane przez czterech prelegentów. Referaty wygłosili: dr hab. Kinga Flaga-Gieruszyń- ska, prof. US – „Zasada bezpośredniości a przeprowadzanie dowodu na odle- 262 Krzysztof Oskar Pokorski głość”, dr Aleksandra Klich (US) – „Stopień dowodów aspekcie oceny dowodów pozyskanych w drodze pomocy sądowej”, dr Karolina Ziemianin (US) – „Wiary- godność zeznań świadków pozyskanych w drodze pomocy sądowej” oraz ks. dr Krzysztof O. Sprawozdanie z V Ogólnopolskiej Konferencji Prawa… Pokorski (WSAP) – „System legalnej i swobodnej oceny środków dowodowych w kanonicznym procesie zwyczajnym”. Całość konferencji, oprócz dziewięciu paneli, podzielona została na części, po których możliwa była już prywatna dyskusja podczas wspólnej kawy czy posiłku. Referaty i dyskusje trwały od godziny 10.00 aż do 18.30, kiedy nastąpiło oficjal- ne zakończenie obrad. Wiele dyskusji nie zostało dokończonych z braku czasu, a wiele podjętych kwestii powinno stać się przyczynkiem do dalszych zmagań naukowych. Kolejna już Konferencja Prawa Kanonicznego w Olsztynie była dowodem jak potrzebna jest naukowa refleksja nad współpracą sądów nie tylko w kwestii do- wodzenia, ale każdej czynności, która jest częścią procedury procesowej. Docenić należy, że w dyskusję akademicką włączają się także praktycy – sędziowie, adwo- kaci. W ten sposób ożywa łacińska sentencja, która stwierdza, że „verba docent, exempla trahunt”. Słowa kluczowe: dowodzenie, środki dowodowe, moc dowodowa, współpraca sądów, proces Streszczenie Na Wydziale Prawa i Administracji Uniwersytetu Warmińsko-Mazurskiego w Olsztynie dnia 24 kwietnia br., obyła się V Ogólnopolska Konferencja Prawa Procesowego. Jej celem była analiza norm prawnych dotyczących dowodzenia w postępowaniach sądo- wych i administracyjnych w systemie prawa polskiego i kanonicznego oraz współpracy sądowej w tej materii. Liczni prelegenci, jak też wielopłaszczyznowość podejmowanych zagadnień, potwierdzili jak ważna i potrzebna jest naukowa dyskusja i refleksja nie tylko teoretyków prawa, ale także jego praktyków. Wiele podjętych zagadnień powinno stać się przyczynkiem do dalszych zmagań naukowych na kolejnych konferencjach. Słowa kluczowe: dowodzenie, środki dowodowe, moc dowodowa, współpraca sądów, proces Słowa kluczowe: dowodzenie, środki dowodowe, moc dowodowa, współpraca sądów, proces 263 Sprawozdanie z V Ogólnopolskiej Konferencji Prawa… Summary At the Faculty of Law and Administration of the University of Warmia and Mazury in Olsztyn on April 24, 2018, the 5th National Conference on Process Law was held. Its purpose was to analyze legal norms regarding the command in court and administrative proceedings in the system of Polish and canon law as well as judicial cooperation in this matter. Numerous speakers and multidimensionality of the issues confirmed how impor- tant and needed is a scientific discussion and reflection not only of theoreticians of the law but also of its practitioners. Many of the issues raised should become a contribution to further scientific struggles at subsequent conferences. Keywords: command, evidence, probative power, judicial cooperation, trial Translated by Mirosława Landowska
https://openalex.org/W4232997551	https://bmcresnotes.biomedcentral.com/track/pdf/10.1186/s13104-019-4708-z	English	null	Flight muscles degenerate by programmed cell death after migration in the wheat aphid, Sitobion avenae	Research Square (Research Square)	2,019	cc-by	5,710	Feng et al. BMC Res Notes (2019) 12:672 https://doi.org/10.1186/s13104-019-4708-z Feng et al. BMC Res Notes (2019) 12:672 https://doi.org/10.1186/s13104-019-4708-z BMC Research Notes BMC Research Notes © The Author(s) 2019. 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 Objective: Previous studies showed that flight muscles degenerate after migration in some aphid species; however, the underlying molecular mechanism remains virtually unknown. In this study, using the wheat aphid, Sitobion ave- nae, we aim to investigate aphid flight muscle degeneration and the underlying molecular mechanism. Results: Sitobion avenae started to differentiate winged or wingless morphs at the second instar, the winged aphids were fully determined at the third instar, and their wings were fully developed at the fourth instar. After migration, the aphid flight muscles degenerated via programmed cell death, which is evidenced by a Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay. Then, we identified a list of differentially expressed genes before and after tethered flights using differential-display reverse transcription-PCR. One of the differentially expressed genes, ubiquitin-ribosomal S27a, was confirmed using qPCR. Ubiquitin-ribosomal S27a is drastically up regulated follow- ing the aphids’ migration and before the flight muscle degeneration. Our data suggested that aphid flight muscles degenerate after migration. During flight muscle degeneration, endogenous proteins may be degraded to reallocate energy for reproduction. Keywords: Aphid, Sitobion avenae, Flight muscle, Ubiquitin-ribosomal S27a, Programmed cell death of JH initiated degeneration earlier than the innervated muscles in cricket [11]. (iii) specific proteins are induced despite the overall decreases of protein synthesis. Ubiq- uitin, a marker for programmed cell death (PCD), accu- mulates when aphid IFM undergoes degeneration [7, 8]. Although IFM degeneration has been suggested as an active PCD, the underlying molecular mechanism remains unclear.h Flight muscles degenerate by programmed cell death after migration in the wheat aphid, Sitobion avenae Honglin Feng1,2,4† , Xiao Guo1,3†, Hongyan Sun1,2, Shuai Zhang1, Jinghui Xi2, Jiao Yin1, Yazhong Cao1 and Kebin Li1 Correspondence: h.feng2@umiami.edu; kbli@ippcaas.cn †Honglin Feng and Xiao Guo contributed equally to this manuscript 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Science, NO. 2 Yuanmingyuan Xilu, Haidian District, Beijing 100193, China Full list of author information is available at the end of the article Introduction Many aphids develop wing polyphenism, and winged aphids explore new habitats by migration [1]. After winged aphids migrate to new host plants and/or the onset of larviposition, the indirect flight muscles (IFM) degenerate [2–6]. IFM degeneration has been previously depicted as regulated processes in insects including some aphids [7, 8]. The regulation of IFM degeneration involves multi- ple factors: juvenile hormone (JH); neural factors; and specific proteins. (i) JH treatment induces IFM degen- eration [8–10]. (ii) Denervated muscles in the presence The wheat aphid, Sitobion avenae, migrates from southern to northern China and causes ~ 10% wheat yield losses every year [12]. Here, we investigated S. avenae wing development and IFM degeneration, and identified some differentially expressed genes pre-/post-migration. We further analyzed the dynamic expression of ubiqui- tin-ribosomal S27a (RPS27a) during IFM degeneration. Correspondence: h.feng2@umiami.edu; kbli@ippcaas.cn †Honglin Feng and Xiao Guo contributed equally to this manuscript 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Science, NO. 2 Yuanmingyuan Xilu, Haidian District, Beijing 100193, China Full list of author information is available at the end of the article Feng et al. BMC Res Notes (2019) 12:672 Page 2 of 7 Feng et al. BMC Res Notes (2019) 12:672 Page 2 of 7 Page 2 of 7 Morphological examination We examined the external morphology of aphid thorax using scanning electron microscope (SEM). Aphids were fixed in 3% glutaraldehyde for 24 h and transferred to 1% osmic acid. Then aphids were saturated with ethanol, exchanged using isopentyl acetate, and dried in a Hitachi CO2 Critical Point Dryer. Aphids were then coated with gold in a sputter coater (Hitachi, IB-5) and imaged under a Hitachi S-570 SEM (Additional file 1: Figure S1). i g We examined the internal morphology of aphid thorax using histological staining. Aphids were fixed in 4% par- aformaldehyde for 4 h. The specimens were dehydrated in a serial of ethanol solutions (70%, 80%, 90%, 100%, 10 min/each), cleared in xylene, and embedded in paraf- fin. Serial sections were cut and stained with hematoxy- lin and eosin for imaging using a Carl Zeiss Primo Star Microscope (Additional file 1: Figure S1). Differential‑display reverse transcription‑PCR (DDRT‑PCR) Differential‑display reverse transcription‑PCR (DDRT‑PCR) To identify differentially expressed genes pre-/post- migration, we performed tethered flight using 3–4 days post-eclosion aphids. For pre-migration, aphids were tethered (not flighted) and flash-frozen. For post- migration, aphids were tethered and flighted for 24 h using a flight-mill program [14]; flighted aphids were flash-frozen. To investigate IFM degeneration, we collected the winged aphids every 24 h from eclosion (0 day), to migra- tion (5th day), to reproduction (8th day), until death was observed. For each timepoint, half of the aphids were collected for morphological, histological, and apoptosis examinations. The other half were dissected for qPCR following a freeze-drying procedure [13]. l Total RNA was extracted from single aphid using Easy-Spin Total RNA Rapid Extraction Kit (Biomad) and reverse-transcribed by PrimeScript™ 1st-Strand cDNA Synthesis Kit (Takara). For DDRT-PCR, we designed three one-base anchored oligo-dT 3′ primers and eight arbitrary 5′ primers (13-mers) according to the Gen- Hunter RNAimage DD Kit (3 × 8 pairs) (Additional file 2: Table S1). Each PCR reaction was composed of 10 μl 2× PCR mix, 1 μl 5′ primer, 1 μl 3′ primer, 2 μl cDNA, and 6 μl ddH2O. The PCR program was 94 °C for 1 min, fol- lowed by 30 cycles of 94 °C for 30 s, 48 °C for 1 min and 72 °C for 1 min, and a final extension step at 72 °C for 5 min. Then 2 μl PCR products of each reaction were used as template for a second round of PCR. The final PCR products were visualized using 6% SDS polyacryla- mide gel electrophoresis (Additional file 3: Figure S2). Differentially expressed genes were excised for sequenc- ing, obtained sequences were annotated on NCBI. Main text Methods Aphids Diaminobenzidine for 15 min, then counterstained with Hematoxylin for 3 min. Finally, specimens were washed with 100% ethanol, 100% xylene, and mounted for imaging. We generated an isogenic S. avenae population using a single wingless aphid. Aphids were raised on wheat seedlings at 22 °C with a 16 h/8 h light/dark photo- period. Winged aphids were obtained by manipulating aphid densities. Under low-density, one wingless aphid was reared on a joint-stage wheat to maintain the wing- less morph. Under high-density, 80 wingless adult aphids were reared on one ripe wheat to induce the winged morph [6]. Previous study suggested that feeding induces JH secre- tion, which triggers IFM degeneration [5]. Therefore, we examined the IFM degeneration on fasted aphid as a control. For fasting, winged aphids were transferred to water-soaked sponges. Every 6 h, aphids were transferred to rearing plants for 2 h to avoid death. RPS27a dynamic expression Th f The sequence of S. avenae RPS27a was obtained using a rapid amplification of cDNA ends method (3′-RACE). Partial S. avenae RPS27a sequence containing the start codon was amplified using Ub-F/Ub-R primers designed based on A. pisum RPS27a. The complete C-terminal of RPS27a was obtained using a RACE kit (Takara) with gene specific primers and the kit-provided outer/inner primers (Additional file 4: Table S2). To check the con- servation of RPS27a, the nucleotide and deduced amino acid sequences were aligned with homologous from other insects using DNAMAN (Additional file 5: Figure S3).i To examine apoptosis, we performed a TUNEL assay using an in situ apoptosis detection kit (Boster, China). Briefly, paraffin-embedded aphids were sliced into 5 μm sections, which were rehydrated in xylene for 20 min and a serial of ethanol solutions (100%, 90%, 80%, 70%, 10 min/each). Then all specimens were permeabilized using Proteinase K (1:200) for 10 min and quenched using 3% H2O2 for 10 min. Quenched specimen were labeled with TdT Labeling Reaction Mix (TdT:DIG- dUTP:Buffer = 1:1:18) for 2 h at 25 °C. After wash with 0.01 M TBS buffer, specimens were incubated with anti-DIG antibody (1:100) for 30 min. Specimens were then incubated with SABC (1:100) for 30 min and with i We quantified the RPS27a expression during IFM degeneration using qPCR in tissues including head, tho- rax, and abdomen. Briefly total RNA was extracted from Feng et al. BMC Res Notes (2019) 12:672 Feng et al. BMC Res Notes (2019) 12:672 Page 3 of 7 (Fig. 1d). To the 13th day, the IFM was degenerated com- pletely (Fig. 1e). The contractile fiber vanished, and the intact IFM was not visible. tissues pooled from ~ 10 aphids, then same amount of RNA (0.1 μg) for each sample was used for cDNA Syn- thesis. The qPCR reaction was 25 μl containing 12.5 μl 2*PCR SuperMix, 0.5 μl Passive Reference DYE, 0.5 μl of each qPCR primer (Additional file 4: Table S2), 1 μl cDNA and ddH2O. The qPCR program was 2 min at 95 °C followed by 45 cycles of 15 s at 95 °C, 15 s at 55.5 °C and 30 s at 72 °C, and then melt curve under 55–95 °C 0.5 °C + (80 cycles). To calculate gene expression, a cred- ible standard curve was constructed using a series of 10× dilutions of a standard sample. RPS27a dynamic expression Th f Each experiment included 3–4 technical replicates for each sample and repeated three times. Sitobion avenae RPS27a dynamic expressionh Sitobion avenae RPS27a dynamic expression The S. avenae RPS27a encodes a 150 aa protein (76aa ubiquitin + 74aa ribosomal protein). The sequences of RPS27a are highly conserved across different insects (84– 93.3%), and the divergence was mainly from the ribo- somal protein, whereas the ubiquitin monomers were almost identical (Additional file 5: Figure S3). Results Differentially expressed genes pre /post migration We identified 36 differentially expressed genes that were classified into three groups (Additional file 6: Table S3): (1) genes related to apoptosis: this group includes the widely studied apoptotic genes like p53 regulator [15] and the apoptosis marker, RPS27a [7, 8]; Some additional genes are related to apoptosis in specific conditions. Ras- related protein Rab-29B regulates lysosome integrity related to age associated defects [16]. (2) genes related to metabolism: this group includes genes in metabolism of amino acids (e.g. d-amino acid dehydrogenase), sugars (e.g. glucose transporter 1), and vitamins (e.g. 3,4-dihy- droxy-2-butanone-4-phosphate synthase). Genes impor- tant to energy re-allocation were also found differentially expressed, including exocyst complex component 3 [17] and serpin 3a [18]. (3) genes with unknown functions. IFM degeneration after migration is PCD In sections of aphid pterothorax stained in the TUNEL experiment, no apoptotic signals were observed in ala- tae aphid before the 7th day after eclosion (Fig. 2a). The first apoptotic signal appeared in aphids at the 7th day as brownish yellow grains (Fig. 2b), which is similar to the apoptosis signals in the positive controls (Fig. 2c, c′). In parallel, we did not find any apoptotic signals in the mus- cles of the 3rd, and 7th day fasted aphids (Fig. 2a′, b′). Flight muscle development in S. avenaeh The external and internal structures were similar in the winged and wingless aphids at the 1st instar; wing pri- mordia were observed in the internal structures of both morphs (Additional file 1: Figure S1A, a). From the 2nd instar, wing primordia developed and enlarged in the winged morph, but disappeared in the wingless morph (Additional file 1: Figure S1b, b′). At the 3rd instar of wing aphids, swollen structures appeared and later devel- oped into wing bud (Additional file 1: Figure S1C, C′). IFM fibers also differentiated; the corresponding area was occupied by fat bodies in the wingless morph hereafter (Additional file 1: Figure S1c, c′). To the 4th instar, wing buds enlarged into a plate shape and the wing epithelia were folded in a complicated structure, differentiating the forewings and hindwings (Additional file 1: Figure S1d′). To adults, the wings were fully developed; wing hair sen- silla were also seen in the winged morph (Additional file 1: Figure S1E, F). IFM degeneration after S. avenae migrationh RPS27a expres- sion significantly increased and was significantly higher in abdomen than head and thorax in the 6th day, which is 1 day post-migration and 1 day before the apoptotic signals were detected (Fig. 2c). After aphid migration, a sharp decrease of RPS27a occurred in the 7th day (Fig. 3), the day that apoptosis signals were detected (Fig. 2c). Discussion IFM degeneration has been mostly studied physiologi- cally in insects such as fruit fly, crickets, bugs, and some aphids [7, 19–25]. Here, we depicted the IFM develop- ment and degeneration in S. avenae and identified genes that may regulate IFM degeneration. Fig. 3 The dynamic expression of S. avenae ubiquitin-ribosomal S27a gene. The aphids we collected are from eclosion (0 day) to breeding finished (8th day). The expression in all three tissues always show stable and relatively lower, except a sharp increase in 5th day, with higher in abdomen than in head and thorax. Error bar = ± standard deviation Fig. 3 The dynamic expression of S. avenae ubiquitin-ribosomal S27a gene. The aphids we collected are from eclosion (0 day) to breeding finished (8th day). The expression in all three tissues always show stable and relatively lower, except a sharp increase in 5th day, with higher in abdomen than in head and thorax. Error bar = ± standard deviation raising from the 3rd day post-eclosion. RPS27a expres- sion significantly increased and was significantly higher in abdomen than head and thorax in the 6th day, which is 1 day post-migration and 1 day before the apoptotic signals were detected (Fig. 2c). After aphid migration, a sharp decrease of RPS27a occurred in the 7th day (Fig. 3), the day that apoptosis signals were detected (Fig. 2c). IFM degeneration after S. avenae migrationh The IFM of an alatae adult was plump at the 1st day after eclosion till the 5th day (Fig. 1a, b). From the 9th day, the IFM started to degenerate. The myofibrils appeared to be thin. The diameter of the myofibrils was reduced, and the volume of the interfibrillar sarcoplasmic region increased (Fig. 1c); the degradation continued through the 11th day i The expression of S. avenae RPS27a changed dynami- cally pre/post IFM degeneration. Initially after eclosion, RPS27a remained a constant low expression in head, thorax, and abdomen (Fig. 3). The expression started Fig. 1 Breakdown of flight muscles of alatae aphids. Mesothoraces of alatae aphids at various stages were fixed, sectioned at 5 μm thickness and stained on the a 1st day, b 5th day, c 9th day, d 11th day, and e 13th day after the final ecdysis flight muscles of alatae aphids. Mesothoraces of alatae aphids at various stages were fixed, sectioned at 5 μm thickness and ay, b 5th day, c 9th day, d 11th day, and e 13th day after the final ecdysis Feng et al. BMC Res Notes (2019) 12:672 Page 4 of 7 a b c a' b' c' Fig. 2 Cross-sections of the mesothoracic flight muscle at various stages. The apoptotic wing degeneration occurs at 7th day after the final ecdysis. All the mesothoraces were sectioned at 5 μm thickness. Images for the TUNEL assays are shown for the 3rd day (a, a′), and 7th day (b, b′) after the final ecdysis in both the normal and fasted aphid groups. In each of the group, a positive control sample were included to shown the cell apoptosis signals (c, c′). The arrowheads indicate apoptotic cells c a' b' c' b' a' c' Fig. 2 Cross-sections of the mesothoracic flight muscle at various stages. The apoptotic wing degeneration occurs at 7th day after the final ecdysis. All the mesothoraces were sectioned at 5 μm thickness. Images for the TUNEL assays are shown for the 3rd day (a, a′), and 7th day (b, b′) after the final ecdysis in both the normal and fasted aphid groups. In each of the group, a positive control sample were included to shown the cell apoptosis signals (c, c′). The arrowheads indicate apoptotic cells raising from the 3rd day post-eclosion. Supplementary information Supplementary information Supplementary information accompanies this paper at https:/ org/10.1186/s13104-019-4708-z. A trade-off has been proposed between IFM degenera- tion and reproduction with regard to energy allocation [26]. IFM degeneration is regulated in many insects, and the products are reallocated to reproduction [24–30]. Here, we identified two groups of genes that differen- tially expressed pre-/post- aphid migration (Additional file 6: Table S3). First group included apoptotic genes, like Ras-related Rab-29b, p53, and RPS27a, which sug- gested that aphid IFM degeneration is an active PCD. The second group included metabolic genes. Some of those genes are involved in the metabolism of energy building blocks (e.g. amino acids, sugars, and vitamins), while other genes are involved in energy transport (e.g. exocyst complex component-3). The differentially expressed genes indicated that aphid IFM degeneration is an active process that involves a trigger of PCD fol- lowed by a systematic reallocation of energies (e.g. from migration to reproduction) [5, 29, 31, 32]. Supplementary information ac org/10.1186/s13104-019-4708-z. Additional file 1: Figure S1. Morphological and histological examina- tions of wing development in aphid nymphs. (A, a) The external and internal structures in the first instar nymphs under HD and LD conditions are similar. Wing primordia were observed in the internal structures of nymphs in both conditions. (B, b, b′) In the second instar stage, the exter- nal structures continued to be similar in nymphs under HD and LD condi- tions. However, the wing primordia (arrow) developed and enlarged in the winged line and the wing primordia disappeared in the wingless line. (C, C′, c, c′) Swollen structures were observed on the third instar nymphs under HD conditions, while not in the LD conditions. Swollen structures were identified as the wing bud in the future winged aphids. In the inner structures, the wing primordia continually developed and enlarged in the winged lines. Flight muscles fibers also differentiated. The primordia disappeared in the wingless line, and the area in which the flight muscle develops was occupied by fat bodies. (D, D′, d, d′) In the fourth instar stage, wing buds enlarged into the shape of a plate. There were no wing buds in the wingless line. The flight muscles of the winged line increased in size and occupied half of the thoracic area. The wing epithelia of the wing buds were folded in a complicated structure. The folding patterns were different between the forewings and hindwings. Discussion IFM degeneration has been mostly studied physiologi- cally in insects such as fruit fly, crickets, bugs, and some aphids [7, 19–25]. Here, we depicted the IFM develop- ment and degeneration in S. avenae and identified genes that may regulate IFM degeneration. Fig. 3 The dynamic expression of S. avenae ubiquitin-ribosomal S27a gene. The aphids we collected are from eclosion (0 day) to breeding finished (8th day). The expression in all three tissues always show stable and relatively lower, except a sharp increase in 5th day, with higher in abdomen than in head and thorax. Error bar = ± standard deviation Fig. 3 The dynamic expression of S. avenae ubiquitin-ribosomal S27a gene. The aphids we collected are from eclosion (0 day) to breeding finished (8th day). The expression in all three tissues always show stable and relatively lower, except a sharp increase in 5th day, with higher in abdomen than in head and thorax. Error bar = ± standard deviation Feng et al. BMC Res Notes (2019) 12:672 Page 5 of 7 Feng et al. BMC Res Notes (2019) 12:672 Additional file 4: Table S2. Primers used for the amplification of RPS27a sequences. Additional file 5: Figure S3. Alignment (left) and phylogenetic analysis (right) of amino acids sequences of ubiquitin-ribosomal S27a from18 insects. The tree shown is an observed divergency tree inferred from align- ment. Statistical support for each individual node on the tree is shown above the nodes. GreenBox stands for Coleoptera, BlueBox stands for Lepi- doptera, YellowBox stands for Diptera and RedBox stands for Hemiptera. Al Agriotes lineatus (CAJ01876), Cg Carabus granulates (CAH04347), Md Micro- malthus delibis (CAJ01880), Tc Tribolium castaneum (XP_969023), Tb Tima- rcha balearica (CAJ01881), Bm Byoxsm mori (ABM55591), Px Plutella xylostella (P68202), Pd Papilio dardanus (CAH04128), Sf Spodoptera frugiperda (P68203), Se Spodoptera exigua (Deduced), Dm Drosophila melanogaster (NP_476778), Aa Aedes aegypti(AAS79344), Dc Diaphorina citri (ABG81958) Ga Graphoceph- ala atropunctata (DQ445503), Mh Maconellicoccus hirsutus(ABM55591), Of Oncopeltus fasciatus (ABN54483), AP Acyrthosiphon pisum (NP_001155610), Sa Sitobion avenae (Deduced). Additional file 6: Table S3. Differentially expressed genes after aphid tethered migration. RPS27a: ubiquitin-ribosomal S27a; IFM: indirect flight muscles; JH: juvenile hormone; TUNEL: Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay; DDRT-PCR: differential-display reverse transcription-PCR; SEM: scanning electron microscope; PCD: programmed cell death; RACE: rapid amplification of cDNA ends. IFM degeneration for energy re‑allocationf Supplementary information Supplementary information accompanie org/10.1186/s13104-019-4708-z. Supplementary information Supplementary information Supplementary information accompanies this paper at https://doi. org/10.1186/s13104-019-4708-z. Supplementary information Fat bodies occu- pied the corresponding thoracic locations in the wingless aphids. (E) Wing hair sensilla were also seen in the winged line. (F) It shows the wing hair sensilla of the adult wing aphid. Ubiquitin functions as a marker for aphid IFM degeneration Ubiquitin degrades proteins in eukaryotic cells. During cell apoptosis, short-lived proteins are subjected to ubiq- uitination, which triggers different degenerative pro- cesses [33, 34]. Here, we found that the S. avenae RPS27a was significantly differentially expressed pre-/post aphid migration. RPS27a showed a sharp increase before aphid migration and a sudden decrease after migration, which indicated that RPS27a is programed during IFM degen- eration. RPS27a may function as a trigger for apoptosis to regulate IFM degeneration [33–35] or RPS27a may degenerate waste proteins to facilitate energy realloca- tion [36]. Interestingly, from our qPCR results, RPS27a was primarily expressed in abdomen compared to head and thorax. During IFM degeneration, RPS27a may trig- ger a systematic energy reallocation during the shift from migration to reproduction; URS27a functions as a gen- eral protein degradation machinery throughout aphid body including abdomen, which is the major energy sink for reproduction. Additional file 2: Table S1. DDRT-PCR primers. Additional file 2: Table S1. DDRT-PCR primers. Additional file 3: Figure S2. Gel visualization of differentially expressed genes before and after aphid migration. 6% polyacrylamide gel electro- phoresis of PCR products from representative DDRT-PCR primer pairs. Arrowhead indicates genes that show different expressions before and after aphid migration. Additional file 3: Figure S2. Gel visualization of differentially expressed genes before and after aphid migration. 6% polyacrylamide gel electro- phoresis of PCR products from representative DDRT-PCR primer pairs. Arrowhead indicates genes that show different expressions before and after aphid migration. Additional file 4: Table S2. Primers used for the amplification of RPS27a sequences. Additional file 4: Table S2. Primers used for the amplification of RPS27a sequences. Consent for publication Consent for publication Consent for publication Not applicable. Abbreviations RPS27a: ubiquitin-ribosomal S27a; IFM: indirect flight muscles; JH: juvenile hormone; TUNEL: Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay; DDRT-PCR: differential-display reverse transcription-PCR; SEM: scanning electron microscope; PCD: programmed cell death; RACE: rapid amplification of cDNA ends. The timing of migration and IFM degeneration deter- mines whether insects can locate preferable host plants [37]. We investigated the IFM degeneration using teth- ered flight system on lab aphid population. However, the timing of aphid migration and IFM degeneration in nature remains elusive. Acknowledgements Not applicable. Feng et al. BMC Res Notes (2019) 12:672 Page 6 of 7 Feng et al. BMC Res Notes (2019) 12:672 Author details 20. Zera AJ, Larsen A. The metabolic basis of life history variation: genetic and phenotypic differences in lipid reserves among life history morphs of the wing-polymorphic cricket, Gryllus firmus. J Insect Physiol. 2001;47(10):1147–60. 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Insti- tute of Plant Protection, Chinese Academy of Agricultural Science, NO. 2 Yuanmingyuan Xilu, Haidian District, Beijing 100193, China. 2 College of Plant Science, Jilin University, No. 5333 Xi’an Road, Changchun 130062, Jilin, China. 3 Chongqing Academy of Agricultural Sciences, Baishiyi, Jiulongpo District, Chongqing 401329, China. 4 Present Address: Boyce Thompson Institute, 533 Tower Road, Ithaca, NY 14853, USA. 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Insti- tute of Plant Protection, Chinese Academy of Agricultural Science, NO. 2 Yuanmingyuan Xilu, Haidian District, Beijing 100193, China. 2 College of Plant Science, Jilin University, No. 5333 Xi’an Road, Changchun 130062, Jilin, China. 3 Chongqing Academy of Agricultural Sciences, Baishiyi, Jiulongpo District, 21. Zhao Z, Zera AJ. A morph-specific daily cycle in the rate of JH biosynthe- sis underlies a morph-specific daily cycle in the hemolymph JH titer in a wing-polymorphic cricket. J Insect Physiol. 2004;50(10):965–73. Chongqing 401329, China. 4 Present Address: Boyce Thompson Institute, 533 Tower Road, Ithaca, NY 14853, USA. y y 22. Zhao ZW, Zera AJ. The hemolymph JH titer exhibits a large-amplitude, morph-dependent, diurnal cycle in the wing-polymorphic cricket, Gryllus firmus. J Insect Physiol. 2004;50(1):93–102. Received: 8 August 2019 Accepted: 4 October 2019 Received: 8 August 2019 Accepted: 4 October 2019 23. Zera AJ. Intermediary metabolism and life history trade-offs: lipid metabolism in lines of the wing-polymorphic cricket, Gryllus firmus, selected for flight capability vs. early age reproduction. Integr Comp Biol. 2005;45(3):511–24. Funding This work was supported by the National Key R&D Program of China (Grant No. 2017YFD0201700), National Special Fund for Scientific Research on Public Causes (Grant No. 200803002) and the Public Welfare Project from Ministry of Agriculture of the People’s Republic of China (Grant No. 201103022). Funding agencies had no role in the design of the study and collection, analysis, and interpretation of the data, or writing of the manuscript. 14. Cheng D, Tian Z, Li H, Sun J, Chen J. Influence of temperature and humidity on the flight capacity of Sitobion avenae. Acta Entomol Sin. 2002;45(1):80–5. 15. Aubrey BJ, Kelly GL, Janic A, Herold MJ, Strasser A. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppres- sion? Cell Death Differ. 2018;25(1):104–13. 16. Kuwahara T, Inoue K, D’Agati VD, Fujimoto T, Eguchi T, Saha S, et al. LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts. Sci Rep. 2016;6:29945. Authors’ contributions 11. Shiga S, Yasuyama K, Okamura N, Yamaguchi T. Neural- and endocrine control of flight muscle degeneration in the adult cricket, Gryllus bimacu- latus. J Insect Physiol. 2002;48(1):15–24. HF, XG, KL and YC conceived of the study, HF, XG, HS, JX, and JY designed the experiments. HF, XG, HS, and SZ performed the experiments and data analysis. HF, XG, and KL wrote the manuscript. All authors contributed to preparation of the final version of the manuscript. All authors read and approved the final manuscript. 12. Hu XS, Liu YJ, Wang YH, Wang Z, Yu XL, Wang B, et al. Resistance of wheat accessions to the English grain aphid Sitobion avenae. PLoS ONE. 2016;11(6):e0156158. 13. Fujita SC, Inoue H, Yoshioka T, Hotta Y. Quantitative tissue-isolation from Drosophila freeze-dried in acetone. Biochem J. 1987;243(1):97–104. 13. Fujita SC, Inoue H, Yoshioka T, Hotta Y. Quantitative tissue-isolation from Drosophila freeze-dried in acetone. Biochem J. 1987;243(1):97–104. 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Oogenesis-flight syndrome in crickets: age-dependent egg production, flight performance, and biochemical composition of the flight muscles in adult female Gryllus bimaculatus. J Insect Physiol. 2007;53(8):819–32. 3. Johnson B. Studies on the degeneration of the flight muscles of alate aphids-II: histology and control of muscle breakdown. J Insect Physiol. 1959;3(4):367–72. 27. Tanaka S. Effects of wing-pad removal and corpus allatum implanta- tion on development of wings, flight muscles, and related structures in the striped ground cricket, Allonemobius-fasciatus. Physiol Entomol. 1985;10(4):453–62. 4. Johnson B. An electron microscopic study of flight muscle breakdown in an aphid Megoura viciae. Tissue Cell. 1980;12(3):529–38. 5. Kobayashi M, Ishikawa H. Breakdown of indirect flight muscles of alate aphids (Acyrthosiphon-Pisum) in relation to their flight, feeding and reproductive-behavior. J Insect Physiol. 1993;39(7):549–54. 28. Tanaka S. De-alation, flight-muscle histolysis, and oocyte development in the striped ground cricket, Allonemobius-Fasciatus. Physiol Entomol. 1986;11(4):453–8. 6. Ishikawa A, Miura T. Differential regulations of wing and ovarian develop- ment and heterochronic changes of embryogenesis between morphs in wing polyphenism of the vetch aphid. Evol Dev. 2009;11(6):680–8. 29. Nair CRM, Prabhu VKK. Entry of proteins from degenerating flight muscles into oocytes in Dysdercus-Cingulatus (Heteroptera, Pyrrhocoridae). J Insect Physiol. 1985;31(5):383–8. 7. Kobayashi M, Ishikawa H. Mechanisms of histolysis in indirect flight muscles of alate aphid (Acyrthosiphon-Pisum). J Insect Physiol. 1994;40(1):33–8. y 30. Stjernholm F, Karlsson B. Flight muscle breakdown in the green-veined white butterfly, Pieris napi (Lepidoptera: Pieridae). Eur J Entomol. 2008;105(1):87–91. 8. Kobayashi M, Ishikawa H. Involvement of juvenile-hormone and ubiquitin-dependent proteolysis in-flight muscle breakdown of alate aphid (Acyrthosiphon-Pisum). J Insect Physiol. 1994;40(2):107–11. 8. Kobayashi M, Ishikawa H. Involvement of juvenile-hormone and ubiquitin-dependent proteolysis in-flight muscle breakdown of alate aphid (Acyrthosiphon-Pisum). Competing interests 19. Piccirillo R, Demontis F, Perrimon N, Goldberg AL. Mechanisms of muscle growth and atrophy in mammals and Drosophila. Dev Dyn. 2014;243(2):201–15. p g The authors declare that they have no competing interests. Availability of data and materials integrity in diverse cellular contexts. Sci Rep. 2016;6:29945. All data generated or analyzed during this study are included in this published article and its additional files. All data generated or analyzed during this study are included in this published article and its additional files. 17. Barkefors I, Fuchs PF, Heldin J, Bergstrom T, Forsberg-Nilsson K, Kreuger J. Exocyst complex component 3-like 2 (EXOC3L2) associates with the exocyst complex and mediates directional migration of endothelial cells. J Biol Chem. 2011;286(27):24189–99. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate 18. Aguila S, Izaguirre G, Martinez-Martinez I, Vicente V, Olson ST, Cor- ral J. Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities. J Biol Chem. 2017;292(40):16513–20. References J Insect Physiol. 1994;40(2):107–11. 31. Bhakthan NM, Borden JH, Nair KK. Fine structure of degenerating and regenerating flight muscles in a bark Beetle, Ips-Confusus. I. Degeneration. J Cell Sci. 1970;6(3):807–19. y y 9. Selma-Soriano E, Artero R, Llamusi B. Optical cross-sectional muscle area determination of Drosophila Melanogaster adult indirect flight muscles. J Vis Exp. 2018;133:e56179. 32. Bhakthan NM, Nair KK, Borden JH. Fine structure of degenerating and regenerating flight muscles in a bark beetle, Ips-Confusus. II. Regenera- tion. Can J Zool. 1971;49(1):85–9. 10. Oliver RH, Albury AN, Mousseau TA. Programmed cell death in flight muscle histolysis of the house cricket. J Insect Physiol. 2007;53(1):30–9. 10. Oliver RH, Albury AN, Mousseau TA. Programmed cell death in flight muscle histolysis of the house cricket. J Insect Physiol. 2007;53(1):30–9. Feng et al. BMC Res Notes (2019) 12:672 Page 7 of 7 37. Feng H, Sun H, Yin J, Li K, Xi J, Cao Y. Progress of molecular biology researches in apoptosis of insect flight muscles. Chin J Appl Entomol. 2011;48(3):701–9. 33. Lee JC, Peter ME. Regulation of apoptosis by ubiquitination. Immunol Rev. 2003;193(1):39–47. 34. Gupta I, Singh K, Varshney NK, Khan S. Delineating crosstalk mechanisms of the ubiquitin proteasome system that regulate apoptosis. Front Cell Dev Biol. 2018;6:11. 36. Kano R, Kubota A, Nakamura Y, Watanabe S, Hasegawa A. Feline ubiquitin fusion protein genes. Vet Res Commun. 2001;25(8):615–22. 34. Gupta I, Singh K, Varshney NK, Khan S. Delineating crosstalk mechanisms of the ubiquitin proteasome system that regulate apoptosis. Front Cell Dev Biol. 2018;6:11. 35. Sun XX, DeVine T, Challagundla KB, Dai MS. Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to riboso- mal stress. J Biol Chem. 2011;286(26):22730–41. Feng et al. BMC Res Notes (2019) 12:672 33. Lee JC, Peter ME. Regulation of apoptosis by ubiquitination. Immunol Rev. 2003;193(1):39–47. 33. Lee JC, Peter ME. Regulation of apoptosis by ubiquitination. Immunol Rev. 2003;193(1):39–47. 34. Gupta I, Singh K, Varshney NK, Khan S. Delineating crosstalk mechanisms of the ubiquitin proteasome system that regulate apoptosis. Front Cell Dev Biol. 2018;6:11. 35. Sun XX, DeVine T, Challagundla KB, Dai MS. Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to riboso- mal stress. J Biol Chem. 2011;286(26):22730–41. 36. Kano R, Kubota A, Nakamura Y, Watanabe S, Hasegawa A. Feline ubiquitin fusion protein genes. Vet Res Commun. 2001;25(8):615–22. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? 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https://openalex.org/W2154225216	https://ijponline.biomedcentral.com/track/pdf/10.1186/1824-7288-40-S1-A13	English	null	Bleeding management in pediatric patients	The Italian Journal of Pediatrics/Italian journal of pediatrics	2,014	cc-by	1,005	Bleeding management in pediatric patients Paola Giordano, Giuseppe Lassandro From 70th Congress of the Italian Society of Pediatrics, Joint National Meeting SIP, SICuPP, SITIP Palermo, Italy. 11-14 June 2014 for patients with platelet dysfunction are: antifibrinolytics, desmopressin, platelet transfusions and recombinant factor VIIa [6,7]. Bleeding events still occurs in patients with hemophilia despite prophylactic factor replacement. Adequate management depends not only upon the hemo- philia subtype and site of bleeding, but also the episode severity, identifiable precipitants (e.g. trauma), the patients’ age and current place of residence, adequacy of venous access, presence of inhibitors, previous history of bleeding, and the time of onset in relation to prophylactic therapy. For all diseases other important initial considerations include the applicability of first aid measures (e.g ice, direct pressure, splinting) and appropriate analgesia [8]. Bleeding has always been an alarming clinical symptom in all human societies, and physicians have had varying degrees of success in diagnosing and treating bleeding patients [1]. Because bleeding is part of the human experi- ence, one the most challenging tasks for a physician is to discriminate between “normal” and “pathologic” bleeding. Hemorrhages or bleeds may occur in every tissue and organ. Every bleeding symptom may, however, vary greatly in terms of magnitude: for instance bleeds in the subcuta- neous tissues may present as small pinpoint lesions (pete- chiae) or large bruises, and epistaxis may range from some blood-streaked mucus to a massive hemorrhage [2]. Minor bleeding is a broad category encompassing a wide variety of symptoms that are, however, severe enough to interfere with the patient’ everyday life. Major bleeding defines those episodes that may cause permanent damage to the patient or threaten his or her life. Major bleeding is defined as bleeding in a critical area (intracranial, intrasp- inal, intraocular, retroperitoneal, intra-articular or pericar- dial, or intramuscular with compartment syndrome) resulting in an hemoglobin fall > 2 g/dl or requiring trans- fusion [3]. Bleeding in a child can be a diagnostic challenge because of the wide range of causes, but making a specific diagnosis is clinically important in order to provide appro- priate therapy. Bleeding disorders can be inherited or acquired, and include coagulation factor deficiencies or Von Willebrand diseases, platelet deficiencies and/or dys- functions, blood vessels alterations [4]. The evaluation of a child presenting with bleeding should include a compre- hensive medical and bleeding history, a complete family history, a detailed examination and selected laboratory tests. For immune thrombocytopenia, in newly diagnoses children, intravenous human immunoglobulin or corticos- teroids can be used [5]. MEETING ABSTRACT Open Access References 1. James P, Coller B: Phenotyping bleeding. Curr Opin Hematol 2012, 19:406-12. 1. James P, Coller B: Phenotyping bleeding. Curr Opin Hematol 2012, 19:406-12. 2. Rodeghiero F, Michel M, Gernsheimer T, Ruggeri M, Blanchette V, Bussel JB, Cines DB, Cooper N, Godeau B, Greinacher A, Imbach P, Khellaf M, Klaassen RJ, Kuhne T, Liebman H, Mazzucconi MG, Newland A, Pabinger I, Tosetto A, Stasi R: Standardization of bleeding assessment in immune thrombocytopenia: report from the International Working Group. Blood 2013, 121:2596-606. 2. Rodeghiero F, Michel M, Gernsheimer T, Ruggeri M, Blanchette V, Bussel JB, Cines DB, Cooper N, Godeau B, Greinacher A, Imbach P, Khellaf M, Klaassen RJ, Kuhne T, Liebman H, Mazzucconi MG, Newland A, Pabinger I, Tosetto A, Stasi R: Standardization of bleeding assessment in immune thrombocytopenia: report from the International Working Group. Blood 2013, 121:2596-606. 3. Schulman S, Kearon C: Definition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients. J Thromb Haemost 2005, 3:692-4. 4. Revel-Vilk S: Clinical and laboratory assessment of the bleeding pediatric patient. Semin Thromb Hemost 2011, 37:756-62. 5. Provan D, Stasi R, Newland AC, Blanchette VS: International consensus report on the investigations and management of primary immune thrombocytopenia. Blood 2010, 115:168-86. 6. Almeida AM, Khair K, Hann I, Liesner R: The use of recombinant factor VIIa in children with inherited platelet function disorders. Br J Haematol 2003, 121:477-81. 7. Giordano P, Lassandro G, Tesse R, Longo S, Valente F, Cappiello AR, Coppola A: Innovative use of recombinant activated factor VII during physical rehabilitation in an Italian child with Glanzmann’s thromboasthenia. Blood Transfus 2013, 11:143-7. thromboasthenia. Blood Transfus 2013, 11:143-7. 8. Giordano P, Franchini M, Lassandro G, Faienza MF, Valente R, Molinari AC: Issues in pediatric haemophilia care. Ital J Pediatr 2013, 39:24. 8. Giordano P, Franchini M, Lassandro G, Faienza MF, Valente R, Molinari AC: Issues in pediatric haemophilia care. Ital J Pediatr 2013, 39:24. doi:10.1186/1824-7288-40-S1-A13 Cite this article as: Giordano and Lassandro: Bleeding management in pediatric patients. Italian Journal of Pediatrics 2014 40(Suppl 1):A13. Cite this article as: Giordano and Lassandro: Bleeding management in pediatric patients. Italian Journal of Pediatrics 2014 40(Suppl 1):A13. © 2014 Giordano and Lassandro; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: paola.giordano@uniba.it Dipartimento di Scienze Biomediche e Oncologia Umana, Clinica Pediatrica F. Vecchio, Università degli Studi di Bari “Aldo Moro”, Bari, Italy © 2014 Giordano and Lassandro; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly 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. Bleeding management in pediatric patients Paola Giordano*, Giuseppe Lassandro From 70th Congress of the Italian Society of Pediatrics, Joint National Meeting SIP, SICuPP, SITIP Palermo, Italy. 11-14 June 2014 Individuals with platelet function defects should be managed by qualified specialist and pla- telet inhibitor medication should be avoid. First line drugs Published: 11 August 2014 Giordano and Lassandro Italian Journal of Pediatrics 2014, 40(Suppl 1):A13 http://www.ijponline.net/content/40/S1/A13 Giordano and Lassandro Italian Journal of Pediatrics 2014, 40(Suppl 1):A13 http://www.ijponline.net/content/40/S1/A13 References 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.
https://openalex.org/W4285732752	https://lexrussica.msal.ru/jour/article/download/2524/1236	Russian	null	Axiology of the Civil Law Principles in Modern Conditions	Lex Russica/Lex russica (Russkij zakon)	2,022	cc-by	7,413	© Волос А. А., 2022 * Волос Алексей Александрович, кандидат юридических наук, доцент, доцент департамента частного права факультета права Национального исследовательского университета «Высшая школа экономики» Б. Трехсвятительский пер., д. 3, г. Москва, Россия, 109028 volosalexey@yandex.ru ЧАСТНОЕ ПРАВО JUS PRIVATUM DOI: 10.17803/1729-5920.2022.188.7.009-018 А. А. Волос* Аксиология принципов гражданского права в современных условиях Аннотация. В исследовании рассмотрена проблема принципов гражданского права с концептуальных по- зиций автора о принципах-методах гражданского права и их аксиологии, а также в связи с современными изменениями социально-экономического и правового характера. Целью исследования стало установление аксиологического значения принципов гражданского права в современных условиях, в том числе с учетом экономико-правовых преобразований, связанных с пандемией коронавируса и цифровизацией общества. Методологическую основу исследования составили общенаучные и частнонаучные методы познания. Среди них большая роль отведена историко-правовому и сравнительно-правовому методу. Кроме того, активно анализировалась отечественная судебная практика с позиции поставленных в работе проблем. Автором обосновано выделение самостоятельной категории «аксиология принципов гражданского права», предложены ее характерные черты. Была раскрыта аксиология принципов в теоретическом и практическом аспекте. Сделан вывод о том, что аксиологическое значение принципов права является константным и устойчивым, не зависит от исторических или социально-экономических условий. В самых разных ситуациях, в том числе в период пандемии коронавируса или широкой цифровизации общества, принципы гражданского права сохраняют свою ценность и способны решать конкретные теоретиче- ские и практические задачи. Существенная роль при этом будет принадлежать педагогической функции принципов. По мнению автора, именно педагогическая функция права в настоящий момент реализуется в меньшей степени, чем остальные. Студентам, а затем и будущим юристам, далеко не всегда удается осуществлять толкование норм законодательства в контексте действия принципов гражданского права, основного смысла законодательства. Ключевые слова: принципы гражданского права; аксиология права; цифровое право; смарт-контракты; принцип добросовестности; гражданское право; цивилистика; цифровизация общества. Для цитирования: Волос А. А. Аксиология принципов гражданского права в современных условиях // Lex russica. — 2022. — Т. 75. — № 7. — С. 9–18. — DOI: 10.17803/1729-5920.2022.188.7.009-018. Ключевые слова: принципы гражданского права; аксиология права; цифровое право; смарт-контракты; принцип добросовестности; гражданское право; цивилистика; цифровизация общества. LEX RUSSICA the coronavirus pandemic and digitalization of society. The methodology of the study was based on general scientific and private scientific methods of cognition. Among them, a large role is assigned to the historical-legal and comparative-legal methods. In addition, the author analized domestic jurisprudence from the perspective of the problems posed in the paper. p p p p The author substantiates the definition of an independent category «axiology of principles of civil law» and describes its characteristic features. The author elucidated the axiology of principles in theoretical and practical aspects. It is concluded that the axiological meaning of the principles of law is constant and stable, does not depend on historical or socio-economic conditions. In a variety of situations, including the coronavirus pandemic or widespread digitalization of society, the principles of civil law retain their value and are able to solve specific theoretical and practical problems. An essential role in this case will belong to the pedagogical function of the principles. According to the author, it is the pedagogical function of law that is currently being implemented to a lesser extent than the rest functions. Students, and then future lawyers, are not always able to interpret the norms of legislation in the context of the principles of civil law and the basic meaning of legislation. K d i i l f i il l i l f l di it l l t t t i i l f d f ith i il l Keywords: principles of civil law; axiology of law; digital law; smart contracts; principle of good faith; civil law; civics; digitalization of society. Cite as: Volos AA. Aksiologiya printsipov grazhdanskogo prava v sovremennykh usloviyakh [Axiology of the Civil Law Principles in Modern Conditions]. Lex russica. 2022;75(7):9-18. DOI: 10.17803/1729-5920.2022.188.7.009- 018. (In Russ., abstract in Eng.). Принципы гражданского права следует из- учать не столько с позиции их понятия, содер- жания или системы, как делают многие авторы, сколько с позиции реального назначения и цен- ности основополагающих начал для законода- теля, суда, участников гражданских правоотно- шений. Подобный подход предложен автором настоящих строк несколько лет назад1, и с того момента накопился достаточный эмпирический и теоретический материал, который еще больше подтверждает выдвинутую гипотезу. В частно- сти, ситуация с COVID-19 предопределила теоре- тическую сложность обоснования исключений из действия принципов гражданского права, научная идея которых была описана с позиции аксиологии принципов гражданского права. Другая тенденция последних лет — цифрови- зация общества. 1 См.: Волос А. А. Принципы-методы гражданского права и их система. М., 2018. С. 114–138. Axiology of the Civil Law Principles in Modern Conditions Aleksey A. Volos, Cand. Sci. (Law), Associate Professor, Depa National Research University «Higher School of Economics» per. B. Trekhsvyatitelskiy, d. 3, Moscow, Russia, 109028 volosalexey@yandex.ru Abstract. The study examines the problem of the principles of civil law in the context of the author’s conceptual standings concerning the principles and methods of civil law and their axiology, as well as in connection with modern changes of socio-economic and legal nature. The aim of the study was to establish axiological significance of the principles of civil law in modern conditions, including economic and legal transformations associated with LEX RUSSICA Том 75 № 7 (188) июль 2022 9 Частное право JUS PRIVATUM Частное право JUS PRIVATUM LEX RUSSICA делает акцент на теоретическом аспекте, ука- зывая, что принципы пронизывают все граж- данско-правовые структурные подразделения, по своей сути являются первичными нормами, связаны генетическими и функциональными зависимостями и отличаются самобытностью7. Трудно спорить с приведенными суждения- ми, а также многими иными мнениями, выска- занными в науке. При этом, как легко заметить, рассуждения разновекторные. Чтобы исправить ситуацию, нужно системное преставление о принципах гражданского права. Здесь поможет сформированная авторская конструкция аксио- логии принципов гражданского права. В таком понимании аксиология содержит два аспекта: теоретический и практический. делает акцент на теоретическом аспекте, ука- зывая, что принципы пронизывают все граж- данско-правовые структурные подразделения, по своей сути являются первичными нормами, связаны генетическими и функциональными зависимостями и отличаются самобытностью7. характеристики принципов, выражающейся в их необходимости, полезности для человека, общества и государства, в возможности дости- жения с их помощью положительного резуль- тата. Значимость предложенной категории су- щественна. В научной литературе отсутствует системное понимание того, что́ есть значение принципов. Имеющиеся подходы не всегда обладают свойствами всесторонности и пол- ноты. Очень часто при выявлении назначения принципов авторы рассматривают этот вопрос однобоко: например, с позиции правопримени- тельного или сугубо теоретического значения. Более того, зачастую наблюдается разнопла- новое, разностороннее, а в иных случаях даже хаотичное, несистематизированное рассмотре- ние проблемы значимости принципов граждан- ского права. Все это может привести к ложной мысли о том, что значение принципов граждан- ского права неконкретно или неоднозначно. Трудно спорить с приведенными суждения- ми, а также многими иными мнениями, выска- занными в науке. При этом, как легко заметить, рассуждения разновекторные. Чтобы исправить ситуацию, нужно системное преставление о принципах гражданского права. Здесь поможет сформированная авторская конструкция аксио- логии принципов гражданского права. В таком понимании аксиология содержит два аспекта: теоретический и практический. Автор статьи полагает, что с теоретической точки зрения принципы гражданского пра- ва, являясь фундаментом отрасли граждан- ского права, выполняют различные функции: системоформирующую, герменевтическую, гносеологическую, коммуникативную, инди- видуалистическую, этическую. Практическая значимость принципов различна в зависимости от субъекта, который использует основные на- чала в своей деятельности (законодатель, суд, иной правоприменительный орган, субъекты гражданского оборота). На основе изложен- ного подхода возможен всесторонний взгляд на аксиологию принципов гражданского права. При этом значимость принципов подчерки- валась многими отечественными авторами еще с XIX в. Например, Г. Ф. Шершеневич писал, что с помощью основных начал «мы предугады- ваем частные правила на не предусмотренные законодателем случаи»2. Е. В. Васьковский рассматривал использование принципов как один из способов толкования и применения гражданских законов3. Кроме того, принципы активно применялись в законодательной дея- тельности, использовались, например, в про- цессе кодификации гражданского права4. 2 Шершеневич Г. Ф. Задачи и методы гражданского правоведения. Казань, 1898. С. 13. 3 См.: Васьковский Е. В. Цивилистическая методология. Учение о толковании и применении гражданских законов. Одесса, 1901. С. 153. 4 О кодификации гражданского права в России в XIX в. и о роли принципов гражданского права при ее осуществлении см., например: Пахман С. В. История кодификации гражданского права. СПб., 1876. Т. 2. 5 См.: Грибанов В. П. Осуществление и защита гражданских прав. М., 2000. С. 215. 6 Алексеев С. С. Структура советского права // Собрание сочинений : в 10 т. М., 2010. Т. 2 : Специальные вопросы правоведения. С. 79. 7 См.: Свердлык Г. А. Принципы советского гражданского права : автореф. дис. … д-ра юрид. наук. М., 1985. С. 21. 8 Dziedziak W. Axiological Basis for the Application of Law — a Perspective of the Equitable Law // Studia Iuridica Lublinensia. 2015. Vol. XXIV, 2. P. 68–69. 4 О кодификации гражданского права в России в XIX в. и о роли принципов гражданского права при ее осуществлении см., например: Пахман С. В. История кодификации гражданского права. СПб., 1876. Т. 2. 5 См.: Грибанов В. П. Осуществление и защита гражданских прав. М., 2000. С. 215. LEX RUSSICA В качестве примера можно привести правовое регулирование отношений, связанных с использованием технологии смарт- контракта. Была предложена концепция право- вого регулирования таких отношений, основой которой стали принципы гражданского права, их интерпретация через аксиологию. Значимость принципов гражданского права и необходимость их изучения неоспоримы, подчеркивается фактически всеми цивилиста- ми. При этом проблемы, связанные с понятием, значением, содержанием и системой прин- ципов гражданского права, рассматриваются самыми разными авторами. Показательно, что общего решения или единства хотя бы по некоторым аспектам темы не прослеживается. В этой сложной научной ситуации, когда найти решение проблем не представляется возмож- ным даже при наличии большого количество исследований, необходим новый методологи- ческой подход. Принципы гражданского права существуют не сами по себе: для человека, общества и государства они имеют реальную ценность, на выявление которой должна быть направлена работа современной цивилистики. Принципы выполняют конкретные теоретические и прак- тические функции. Однако возможен и обрат- ный взгляд на проблему. В принципах граждан- ского права изначально должны быть заложены определенные ценностные установки и на- правленность на решение фундаментальных и правоприменительных задач. Если же право- вой принцип в силу определенных причин не выполняет ту роль, которая ему присуща, он должен быть исключен из перечня принципов. Предложенный вывод важен прежде всего для законодателя, которому следует воздерживать- ся от непродуманного изменения количества и сущности принципов гражданского права. В настоящем исследовании рассмотрена проблема принципов гражданского права с учетом концептуальных позиций автора о прин- ципах-методах гражданского права и их аксио- логии, а также в связи с современными измене- ниями в обществе социально-экономического и правового характера. Аксиологию принципов гражданского пра- ва следует понимать в качестве определенной Том 75 № 7 (188) июль 2022 10 Волос А. А. Аксиология принципов гражданского права в современных условиях Волос А. А. Волос А. А. Аксиология принципов гражданского права в современных условиях Аксиология принципов гражданского права в современных условиях характеристики принципов, выражающейся в их необходимости, полезности для человека, общества и государства, в возможности дости- жения с их помощью положительного резуль- тата. Значимость предложенной категории су- щественна. В научной литературе отсутствует системное понимание того, что́ есть значение принципов. Имеющиеся подходы не всегда обладают свойствами всесторонности и пол- ноты. Очень часто при выявлении назначения принципов авторы рассматривают этот вопрос однобоко: например, с позиции правопримени- тельного или сугубо теоретического значения. Более того, зачастую наблюдается разнопла- новое, разностороннее, а в иных случаях даже хаотичное, несистематизированное рассмотре- ние проблемы значимости принципов граждан- ского права. Все это может привести к ложной мысли о том, что значение принципов граждан- ского права неконкретно или неоднозначно. 2 Шершеневич Г. Ф. Задачи и методы гражданского правоведения. Казань, 1898. С. 13. 3 См.: Васьковский Е. В. Цивилистическая методология. Учение о толковании и применении гражданских законов. Одесса, 1901. С. 153. 8 Dziedziak W. Axiological Basis for the Application of Law — a Perspective of the Equitable Law // Studia Iuridica Lublinensia. 2015. Vol. XXIV, 2. P. 68–69. законов. Одесса, 1901. С. 153. 4 О кодификации гражданского права в России в XIX в. и о роли принципов гражданского права при ее осуществлении см., например: Пахман С. В. История кодификации гражданского права. СПб., 1876. Т. 2. 5 См.: Грибанов В. П. Осуществление и защита гражданских прав. М., 2000. С. 215. 6 Алексеев С. С. Структура советского права // Собрание сочинений : в 10 т. М., 2010. Т. 2 : Специальные вопросы правоведения С 79 еневич Г. Ф. Задачи и методы гражданского правоведения. Казань, 898. С. 3. аськовский Е. В. Цивилистическая методология. Учение о толковании и применении гражданских ов. Одесса, 1901. С. 153. еневич Г. Ф. Задачи и методы гражданского правоведения. Казань, 1898. С. 13. 6 Алексеев С. С. Структура советского права // Собрание сочинений : в 10 т. М., 2010. Т. 2 : Специальные вопросы правоведения. С. 79. 9 Weilinger A. Privatrecht. Eine Einführung. Wien, 2016. S. 6–8. 10 Богданов Е. В., Богданова Е. Е., Богданова Д. Е. Принцип солидарности в гражданском праве России // Журнал российского права. 2016. № 11. С. 37. 11 Семякин М. Н. Принципы вещного права в контексте реформирования российского гражданского зако- нодательства // Российский юридический журнал. 2021. № 4. С. 118. 12 См.: Семякин М. Н. Указ. соч. С. 118–130. 13 Нам К. В. Принцип добросовестности как правовой принцип // Вестник экономического правосудия Российской Федерации. 2020. № 2. С. 100. LEX RUSSICA их прибыли», максимизация которой как раз и возможна в ситуации учета прав и законных интересов друг друга, содействия двух сторон, представления необходимой информации. Однако в любом случае приведенный пример подчеркивает значимость дискуссий в связи с аксиологическим значением принципов граж- данского права при понимании права в целом. вопросы уже были подробно раскрыты в трудах ученых самых разных периодов. Принципы предопределяют смысл граж- данского права и его понимание. Так, частное право основывается на принципе свободы, самоопределении субъекта. Этот принцип вы- ражается в конкретных формах: свободе дого- вора, свободе завещательных распоряжений, свободе вступления в брак, свободе выбора письменной или нотариальной формы сделок и т.п.9 Действие принципов, обеспечивающих свободу участников соответствующих отноше- ний, есть отличительная черта частного права в целом. Рассуждая о теоретическом значении прин- ципов гражданского права, сто́ит осветить два момента, которые обычно упускаются из вида. Во-первых, прежде чем говорить о правовом значении и об аксиологии принципов граждан- ского права, нужно доказать, что то или иное явление — именно принцип. Так, в одной из работ автор отмечает, что «в цивилистической литературе нередко не проводится четкого различия между понятиями “принципы вещ- ного права” и “признаки вещного права”»11. При этом описываются некоторые категории, которые названы принципами, но, по сути, та- ковыми не являются. Среди них принцип нор- мативности вещных прав и принцип закрытого перечня вещных прав, которые на самом деле есть признаки вещных прав, но не принципы12. Рассуждая о ценности отдельных начал и их системы, ученые неизбежно переходят в об- ласть аксиологии гражданского права в целом и отдельных его норм. Например, обоснована идея о принципе солидарности в граждан- ском праве, который «отражает интересы об- щества и личности как единой социосистемы, что позволяет формировать отношения между членами общества на основе солидарности, а не индивидуализма, когда во имя своей при- были, своих интересов игнорируются интересы как общества, так и партнеров по договору»10. Показательно, что авторы тезиса основательно раскрывают свою идею через отдельные поло- жения ГК РФ, что подчеркивает аксиологиче- ский аспект принципа. Для разрешения проблемы нужно точно выяснить отличительные признаки принципов. Например, принцип как норма высокого уров- ня абстракции раскрывается через отдельные нормы права. Признак закрытого перечня вещ- ных прав не обладает такой характеристикой, хотя, безусловно, он является важной чертой подотрасли вещного права, и дискуссии по его применимости/неприменимости в России акту- альны и значимы. Говоря непосредственно о принципе соли- дарности в гражданском праве, следует под- держать необходимость дополнительных дискуссий по нему. LEX RUSSICA Вопросы аксиологии права неоднократно были в центре внимания зарубежных цивили- стических исследований. Аксиология понятий и принципов должна быть необходимой частью анализа права. Так, когда речь идет об аксио- логических основах применения закона, нужно найти способ, который вел бы к наиболее спра- ведливому разрешению спорных ситуаций8. В. П. Грибанов указывал на особую регули- рующую роль принципов гражданского права, которая заключается в определении основного содержания, характера толкования и приме- нения правовых норм отрасли или института права5. О том, что принципы «определяют ли- нию судебной и иной юридической практики», писал С. С. Алексеев6. Напротив, Г. А. Свердлык в докторской диссертации в большей степени В последние годы дискуссии по поводу тео- ретической роли принципов гражданского пра- ва только расширились, хотя, казалось бы, эти 6 Алексеев С. С. Структура советского права // Собрание сочинений : в 10 т. М., 2010. Т. 2 : Специальные вопросы правоведения. С. 79. LEX RUSSICA Том 75 № 7 (188) июль 2022 11 Частное право JUS PRIVATUM Частное право JUS PRIVATUM 14 Брановицкий К. Л., Ренц И. Г., Ярков В. В. Судебное правотворчество в условиях пандемии коронавиру- са: нонсенс или необходимость? // Закон. 2020. № 5. С. 113. 15 Суворов Е. Д. Применение принципов договорного права к договорным отношениям, осложненным цифровым элементом // Lex russica. 2022. Т. 75. № 1. С. 109. 16 Суворов Е. Д. Указ. соч. С. 109. 17 См.: Иванов А. А. Цифровая этика и право // Закон. 2021. № 4. С. 71. LEX RUSSICA Приводимые авторами при- меры из Общей и Особенной части ГК РФ сви- детельствуют скорее не о солидарности членов общества или о желании ее достичь, а о том, что законодатель стремится найти разумный баланс интересов сторон конкретного право- отношения. Например, цель п. 3 ст. 307 ГК РФ видится не в обеспечении солидарности или формировании единых интересов общества. Положения данного пункта вполне эффективно могут гарантировать интересы сторон «во имя Во-вторых, каждый принцип гражданского права может иметь свое особое, отличное от других начал, теоретическое предназначение. Так, принцип добросовестности «призван кор- ректировать издержки нормативной системы правового регулирования, когда общая пози- тивная норма в отдельной конкретной ситуа- ции не соответствует тому результату, который предполагался правом»13. Здесь не наблюда- Том 75 № 7 (188) июль 2022 12 Волос А. А. Аксиология принципов гражданского права в современных условиях Волос А. А. Волос А. А. Аксиология принципов гражданского права в современных условиях нению теории субъектов права. Токенизация также пока не привела к существенному изме- нению представлений об объектах гражданских прав, ведь их перечень традиционно неисчер- пывающий и может быть дополнен в связи с изменением социально-экономических усло- вий, что и произошло, например, пару веков назад с таким объектом, как интеллектуальная собственность. ется противоречия. Каждый принцип граждан- ского права обладает как общей, так и специ- альной аксиологической характеристикой. Теоретическая роль принципов права стала объектом дискуссий в связи с современными условиями и изменениями. Началось всё с процессуальных вопросов, которые возникли в ситуации тех ограничений, с которыми Рос- сия живет уже с начала 2020 г. Так, «исходя из вызванного пандемией ограничения личного процессуального общения, но преследуя цель разрешения дела, суды применили аналогию права, создав новые правила, не противореча- щие, а, наоборот, соответствующие в полной мере основным принципам гражданского про- цессуального права»14. Тот факт, что смарт-контракты автором выне- сены в качестве примера пересмотра категории «исполнение обязательств», также иллюстри- рует спорность суждений, поскольку далеко не факт, что смарт-контракт предполагает особен- ности именно исполнения обязательств. Мно- гие ученые и специалисты видят смарт-контракт в качестве инструмента, предполагающего осо- бенности в заключении договора и гарантирую- щего неизменность его условий. Вместе с тем главная роль принципов граж- данского права стала раскрываться при обсуж- дении проблем цифровизации и права. И здесь всё чаще появляются идеи о том, что «в усло- виях недостатка нормативного регулирования соответствующие принципы права являются востребованными источниками права для разрешения потенциальных конфликтов инте- ресов»15. LEX RUSSICA цессами цифровизации общества и пандемии коронавируса, встает вопрос о том, насколько общепризнанные принципы актуальны и при- менимы и нет ли необходимости трансформа- ции их системы и содержания. О практической роли принципов гражданского права стали в по- следнее время упоминать всё чаще различные ученые, в том числе те, кто имел опыт работы в судах (в частности, А. А. Иванов, С. В. Сарбаш). Действительно, сейчас вряд ли многие рискнут утверждать, что принципы гражданского права имеют лишь сугубо теоретическую ценность, не связанную с практикой и реальной жизнью. Наоборот, авторы пытаются систематизировать различные дела, в которых суд разрешил спор на основании принципов21. рубежом. Встречаются диаметрально противо- положные мнения. Например, о том, что ком- пьютеризация в полной мере соответствует не только принципам права, но и сложившимся в конкретных государствах традициям и обы- чаям. В частности, такое утверждение можно встретить среди правоведов, изучающих му- сульманское право: «Отличительные особен- ности смарт-контракта защищают права обоих сторон договора, их интересы, обеспечивают справедливое перераспределение, основан- ное на доверии, добросовестности, равен- стве сторон, то есть на ключевых принципах шариата»18. Другие авторы (из Великобрита- нии и Нидерландов) обращают внимание, что компьютер будет «беспощаден» к применению принципов равенства и добросовестности19. Есть и более компромиссный вариант: действие принципа добросовестности продолжается и в случае использования цифровых технологий, правда в ограниченном виде20. Однако куда более важными стали выво- ды о процессе совершенствования практики, связанной с применением судами принципов гражданского права, о конкретных проблемах именно по данному вопросу. Таким образом, особенность применения судами принципов постепенно становится отдельным вопросом научных исследований. Последнее видится ближе к истине. Тот факт, что компьютер будет «равнодушен» или «бес- пощаден» к принципам права, очевиден, по крайней мере на данном этапе развития техно- логий. Вместе с тем это вовсе не означает, что такого же мнения будет придерживаться право- применитель. В противном случае возникло бы нарушение формальных правил ст. 1 ГК РФ. При всей сложности судебной защиты сторон суды должны реагировать на грубейшие наруше- ния сторон при использовании технологий вне зависимости от правильности или неправиль- ности технических программ. Главной задачей в настоящий момент видится разработка гаран- тий по судебной и иной защите участников ци- фровых отношений. Так, С. В. Сарбаш выделил «девиантные практики использования принципов при моти- вировке судебного решения», среди которых неконкретная отсылка, избыточность ссылки на принципы, ссылка не на те принципы, которые релевантны, неумение или нежелание подроб- но отражать в тексте судебного акта рассужде- ния «от принципов», игнорирование судами ссылки сторон на принципы права, необосно- ванное ограничение принципов22. 18 Alam N., Gupta L., Zameni A. Fintech and Islamic Finance: Digitalization, Development and Disruption. Palgrave Macmillan, 2019. P. 129. 19 Durovic M., Janssen А. The Formation of Smart Contracts and Beyond: Shaking the Fundamentals of Contract Law? 2018. September. P. 23. 20 Herian R. Legal Recognition of Blockchain Registries and Smart Contracts. Paris, 2018. P. 22. 21 Попытку систематизации по отдельным вопросам практики предложил С. В. Сарбаш, см.: Основные положения гражданского права : постатейный комментарий к статьям 1–16.1 Гражданского кодекса Российской Федерации [Электронное издание. Редакция 1.0] / А. В. Асосков, В. В. Байбак, Р. С. Бевзенко [и др.] ; отв. ред. А. Г. Карапетов. М. : М-Логос, 2020. С. 109 и сл. 22 См.: Основные положения гражданского права: постатейный комментарий к статьям 1–16.1 Граждан- ского кодекса Российской Федерации. С. 116 и сл. LEX RUSSICA Есть мнение, что «в условиях, когда пересматриваются основные категории граж- данского права — субъекты (идентификация личности), объекты (токенизация), сделки (спо- собы волеизъявления в цифровой экономике), исполнение обязательств (смарт-контракты), основное, что у права останется неизмен- ным, — это его принципы»16. Таким образом, следует поддержать вывод о неизменности принципов гражданского пра- ва. Однако именно то, что они «неизменны», позволяет отказаться от фундаментального пересмотра основных категорий гражданского права (субъект, объект и др.). С учетом толко- вания принципов права и вновь возникающих социально-экономических условий следует говорить лишь о трансформации содержания самих принципов и этих категорий. Указанной сейчас трансформации подвер- гаются многие категории, даже этика права. Среди особенностей развития этики примени- тельно к цифровой среде выделено «различное влияние этики на нормы периферии и ядра». Принципы отнесены к нормам высокого уровня абстракции, «которые если и будут машинизи- рованы, то в последнюю очередь»17. Означает ли это, что принципы совершенно не испыты- вают на себе влияние цифровизации? Конечно, нет, но принципы, безусловно, имеют «особый статус» в контексте социально-экономических преобразований по сравнению с иными нор- мами права. Как видим, автор уверен в том, что прин- ципы права не только не будут вступать в про- тиворечие с современными процессами, но и будут востребованы при регулировании вновь возникающих отношений. Вместе с тем сложно в полной мере согласиться с предложенными выводами о том, что сейчас «пересматриваются основные категории гражданского права». Дей- ствительно, происходит определенная транс- формация содержания отдельных понятий, а также признание технической вариативности при осуществлении прав и исполнении обя- занностей. Например, появляются новые воз- можности по идентификации личности, но они нереляционны (по крайней мере пока) изме- Говоря о теоретической роли принципов гражданского права в период цифровизации общества, нельзя не обратить внимание на дискуссии, происходящие по этому вопросу за LEX RUSSICA 13 Том 75 № 7 (188) июль 2022 Частное право JUS PRIVATUM Частное право JUS PRIVATUM LEX RUSSICA Подчеркивая важность подобных выводов, следует сказать, что большая часть проблем при использовании принципов гражданского права вызвана неумением грамотно их приме- нять, толковать, выяснять суть. Для конкретной отсылки или ссылки на «правильный принцип» суду вряд ли достаточно будет буквального толкования законодательства, это более слож- Не менее важен и практический аспект аксиологии принципов гражданского права. В ситуации существенных изменений социаль- но-экономических условий, ускоренных про- Том 75 № 7 (188) июль 2022 14 Волос А. А. Аксиология принципов гражданского права в современных условиях Волос А. А. Волос А. А. Аксиология принципов гражданского права в современных условиях ная задача, требующая погружения в историю, доктрину, судебную практику по различным делам. Вместе с тем по ряду аспектов можно и поспорить с мнением С. В. Сарбаша. Так, слож- но согласиться с тем, что «избыточность ссылки на принципы» есть девиантная практика. Уси- ление аргументации по делу — важная часть решения суда, которая показывает, насколько отдельные примененные по делу нормы права соответствуют всей системе права, основанной на принципах. Кроме того, противоречивость права приводит к тому, что часто для правиль- ного разрешения дела одного (специального) аргумента недостаточно. Следует использовать и принципы. ная задача, требующая погружения в историю, доктрину, судебную практику по различным делам. Вместе с тем по ряду аспектов можно и поспорить с мнением С. В. Сарбаша. Так, слож- но согласиться с тем, что «избыточность ссылки на принципы» есть девиантная практика. Уси- ление аргументации по делу — важная часть решения суда, которая показывает, насколько отдельные примененные по делу нормы права соответствуют всей системе права, основанной на принципах. Кроме того, противоречивость права приводит к тому, что часто для правиль- ного разрешения дела одного (специального) аргумента недостаточно. Следует использовать и принципы. условиях ограничений, вызванных пандемией COVID-19. Сам факт ограничения прав в связи со сложившейся эпидемиологической обста- новкой и порядок установления исключений из действия принципов гражданского права, которые реально и широко применялись, еще должен стать объектом отдельных теоретиче- ских дискуссий. Однако несомненно, что прин- ципы гражданского права использовались для разрешения ряда ситуаций. условиях ограничений, вызванных пандемией COVID-19. Сам факт ограничения прав в связи со сложившейся эпидемиологической обста- новкой и порядок установления исключений из действия принципов гражданского права, которые реально и широко применялись, еще должен стать объектом отдельных теоретиче- ских дискуссий. Однако несомненно, что прин- ципы гражданского права использовались для разрешения ряда ситуаций. Так, возник вопрос, распространяется ли мо- раторий на начисление неустоек на собствен- ников и пользователей нежилых помещений в многоквартирном доме. LEX RUSSICA Учитывая, что понятие платы за жилое помещение и коммунальные услуги равным образом относится к собствен- никам и пользователям как жилых, так и нежи- лых помещений в многоквартирных домах, на поставленный вопрос Верховный Суд РФ дал положительный ответ24. Думается, что иное толкование норм законодательства привело бы к нарушению принципа равенства участников жилищных отношений. Надо сказать, что принципы гражданского права продолжают быть эффективным ин- струментом разрешения ситуаций, когда закон не дает конкретного решения кейса. Так, в недавнем Обзоре судебной практики Верхов- ный Суд РФ был вынужден признать, что закон о банкротстве прямо не регулирует вопрос о возможности последующего изменения кре- дитором своего выбора способа распоряжения требованием к субсидиарным ответчикам и это обстоятельство не позволило судам произве- сти процессуальную замену должника на его кредитора. Однако отсутствие прямого право- вого регулирования может быть восполнено общими нормами-принципами гражданского права, поскольку правоотношения по процес- суальному правопреемству производны от их материального содержания, а в данном случае материальные правоотношения между креди- тором и должником по имущественным требо- ваниям относятся к сфере действия граждан- ского законодательства23. Сегодня, как показывают выступления выс- ших государственных лиц государства и направ- ления большинства научных исследований, основным трендом является цифровизация общества, в том числе с правовых позиций. Например, после выступления В. В. Путина на- чался новый виток дискуссий по поводу право- вого режима криптовалют25. Однако судебной практики, которая могла бы стать эмпирической базой для исследований и образцом для участ- ников оборота, практически нет. Вместе с тем суды нередко пытаются обосновать допусти- мость рассмотрения криптовалют в качестве объекта гражданских прав, хотя бы потому, что иное не предусмотрено ГК РФ. Вместе с тем судебная практика не может быть в стороне от новых социально-экономи- ческих процессов. И здесь роль принципов гражданского права и правильного их толкова- ния только возрастает. Например, в довольно сложной ситуации оказался Верховный Суд РФ при решении вопроса о том, как должно при- меняться гражданское законодательство в Так, суд кассационной инстанции отменил решения нижестоящих судов о том, что отно- шения по поводу криптовалюты не могут быть объектом гражданско-правового регулирова- ния. Кассация отметила, что на «момент воз- никновения спорных правоотношений цифро- вая валюта де-факто существовала, имела 23 См.: Обзор судебной практики Верховного Суда РФ № 2 (2021), утв. Президиумом Верховного Суда РФ 30.06.2021 // Бюллетень Верховного Суда РФ. 2021. № 10. 24 См.: Обзор по отдельным вопросам судебной практики, связанным с применением законодательства и мер по противодействию распространению на территории Российской Федерации новой коронавирус- ной инфекции (COVID-19) № 3, утв. 23 См.: Обзор судебной практики Верховного Суда РФ № 2 (2021), утв. Президиумом Верховного Суда РФ 30.06.2021 // Бюллетень Верховного Суда РФ. 2021. № 10. 24 См.: Обзор по отдельным вопросам судебной практики, связанным с применением законодательства и мер по противодействию распространению на территории Российской Федерации новой коронавирус- ной инфекции (COVID-19) № 3, утв. Президиумом Верховного Суда РФ 17.02.2021 // Бюллетень Верхов- ного Суда РФ. 2021. № 4. 25 См., например: Путин отметил конкурентные преимущества России в майнинге криптовалют // URL: https://ria.ru/20220126/mayning-1769647271.html (дата обращения: 29.01.2022). 25 См., например: Путин отметил конкурентные преимущества России в майнинге криптовалют // URL: https://ria.ru/20220126/mayning-1769647271.html (дата обращения: 29.01.2022). 23 См.: Обзор судебной практики Верховного Суда РФ № 2 (2021), утв. Президиумом Верховного Суда РФ 30.06.2021 // Бюллетень Верховного Суда РФ. 2021. № 10. 24 См.: Обзор по отдельным вопросам судебной практики, связанным с применением законодательства и мер по противодействию распространению на территории Российской Федерации новой коронавирус- ной инфекции (COVID-19) № 3, утв. Президиумом Верховного Суда РФ 17.02.2021 // Бюллетень Верхов- ного Суда РФ. 2021. № 4. мер по противодействию распространению на территории Российской Федерации новой коронавирус- ной инфекции (COVID-19) № 3, утв. Президиумом Верховного Суда РФ 17.02.2021 // Бюллетень Верхов- ного Суда РФ. 2021. № 4. 25 См., например: Путин отметил конкурентные преимущества России в майнинге криптовалют // URL: https://ria.ru/20220126/mayning-1769647271.html (дата обращения: 29.01.2022). LEX RUSSICA некую экономическую ценность, с ней могли совершаться операции по покупке, продаже, обмену, в том числе на вещи, лицами, имею- щими материальный интерес в таких операци- ях». Кроме того, сделан акцент на диспозитив- ности гражданско-правового регулирования, открытости перечня объектов по ст. 128 ГК РФ и возможности применения аналогии закона и аналогии права26. До этого Верховный Суд РФ фактически подтвердил, что отношения по по- воду криптовалюты — законные и имуществен- ные. По одному из дел сделан такой вывод: пе- редав свое имущество (криптовалюту) взамен полученных денежных средств, гражданин пре- следовал определенную экономическую цель, заключив сделки по продаже криптовалюты. Таким образом, имелись правовые основания для получения денежных средств27. есть мнение, что они не формируют перспек- тиву высоких темпов их использования. Ввиду их важной роли в качестве стандартов обобще- ния они используются, только если не применя- ются другие источники28. Однако и здесь автор вынужден признать, что правовые принципы являются для судей единственным доступным источником права при рассмотрении отноше- ний, не регулируемых каким-либо положитель- ным писаным законом29. В таких случаях не только в России, но и во многих странах мира лишь общий принцип может быть использован для разрешения дела по существу. некую экономическую ценность, с ней могли совершаться операции по покупке, продаже, обмену, в том числе на вещи, лицами, имею- щими материальный интерес в таких операци- ях». Кроме того, сделан акцент на диспозитив- ности гражданско-правового регулирования, открытости перечня объектов по ст. 128 ГК РФ и возможности применения аналогии закона и аналогии права26. До этого Верховный Суд РФ фактически подтвердил, что отношения по по- воду криптовалюты — законные и имуществен- ные. По одному из дел сделан такой вывод: пе- редав свое имущество (криптовалюту) взамен полученных денежных средств, гражданин пре- следовал определенную экономическую цель, заключив сделки по продаже криптовалюты. Таким образом, имелись правовые основания для получения денежных средств27. Обсуждая аксиологию принципов граждан- ского права, нельзя не сказать о юридическом образовании, которое сочетает в себе тео- ретический и практический аспект. Уместно будет привести мнение профессора права из Гамбурга, который выделил самостоятельную педагогическую функцию принципов граждан- ского права: они могут оказать значительную помощь студенту при изучении права, помо- гают придать закону более понятный вид с вы- соты птичьего полета сложной области права30. По данным двум делам суд не писал в решении, что оно основано на «принципах гражданско-правового регулирования». Воз- можно, с политико-правовой точки зрения в отсутствие окончательного решения по поводу запрета (регулирования, свободного обраще- ния) криптовалют в России такая аргументация формально была бы не слишком удачной. 26 См.: определение Седьмого кассационного суда общей юрисдикции от 30.06.2021 № 88-10336/2021 // Документ опубликован не был. Доступ из СПС «КонсультантПлюс». 27 Определение Судебной коллегии по гражданским делам Верховного Суда РФ от 02.02.2021 № 44-КГ20- 17-К7, 2-2886/2019 // Доступ из СПС «КонсультантПлюс». 28 Jordan D. Legal Principles, Legal Values and Legal Norms: are they the same or different? // Academicus. International Scientific Journal. 2010. July. P. 109–110. 29 Jordan D. Op. cit. P. 109–110. 30 Drobnig U General Principles of European Contract Law // International Sale of Goods: Dubrovnik Lectures LEX RUSSICA Президиумом Верховного Суда РФ 17.02.2021 // Бюллетень Верхов- ного Суда РФ. 2021. № 4. 25 См., например: Путин отметил конкурентные преимущества России в майнинге криптовалют // URL: https://ria.ru/20220126/mayning-1769647271.html (дата обращения: 29.01.2022). LEX RUSSICA 15 Том 75 № 7 (188) июль 2022 Частное право JUS PRIVATUM LEX RUSSICA 30 Drobnig U. General Principles of European Contract Law // International Sale of Goods: Dubrovnik Lectures, Oceana / P. Sarcevic & P. Volken eds. 1986. Ch. 9. P. 305–332. 29 Jordan D. Op. cit. P. 109–110. БИБЛИОГРАФИЯ 1. Алексеев С. С. Собрание сочинений : в 10 т. Т. 2 : Специальные вопросы правоведения. — М. : Статут, 2010. — 471 с. 1. Алексеев С. С. Собрание сочинений : в 10 т. Т. 2 : Специальные вопросы правоведения. — М. : Статут, 2010. — 471 с. 2. Богданов Е. В., Богданова Е. Е., Богданова Д. Е. Принцип солидарности в гражданском праве России // Журнал российского права. — 2016. — № 11. — С. 37–45. 3. Брановицкий К. Л., Ренц И. Г., Ярков В. В. Судебное правотворчество в условиях пандемии коронави нонсенс или необходимость? // Закон. — 2020. — № 5. — С. 107–117. 3. Брановицкий К. Л., Ренц И. Г., Ярков В. В. Судебное правотворчество в условиях пандемии корон нонсенс или необходимость? // Закон. — 2020. — № 5. — С. 107–117. 4. Васьковский Е. В. Цивилистическая методология. Учение о толковании и применении гражданских законов. — Одесса : Экон. тип., 1901. — 376 с. 4. Васьковский Е. В. Цивилистическая методология. Учение о толковании и применении гражданских законов. — Одесса : Экон. тип., 1901. — 376 с. 5. Волос А. А. Принципы-методы гражданского права и их система. — М. : Юстицинформ, 2018. — 258 с. 6 Г б В П О М С 2001 411 5. Волос А. А. Принципы-методы гражданского права и их система. — М. : Юстицинформ, 2018. — 258 с. 6. Грибанов В. П. Осуществление и защита гражданских прав. — М. : Статут, 2001. — 411 с. 7. Иванов А. А. Цифровая этика и право // Закон. — 2021. — № 4. — С. 67–73. 5. Волос А. А. Принципы-методы гражданского права и их система. — М. : Юстицинформ, 2018. — 258 с. 6. Грибанов В. П. Осуществление и защита гражданских прав. — М. : Статут, 2001. — 411 с. 7. Иванов А. А. Цифровая этика и право // Закон. — 2021. — № 4. — С. 67–73. 7. Иванов А. А. Цифровая этика и право // Закон. — 2021. — № 4. — С. 67–73. 8. Нам К. В. Принцип добросовестности как правовой принцип // Вестник экономического правос Российской Федерации. — 2020. — № 2. — С. 88–103. 9. Основные положения гражданского права: постатейный комментарий к статьям 1–16.1 Гражданского кодекса Российской Федерации [Электронное издание. Редакция 1.0] / А. В. Асосков, В. В. Байбак, Р. С. Бевзенко [и др.] ; отв. ред. А. Г. Карапетов. — М. : М-Логос, 2020. — 1469 с. 10. Пахман С. LEX RUSSICA Ина- че пришлось бы утверждать, что гражданский оборот криптовалют допустим в соответствии с принципами гражданского права. Вместе с тем суды в приведенных случаях фактически сделали такой вывод. Действительно, среди основных принципов гражданского права сво- бода договора, автономия воли сторон, кото- рые позволяют заключать сделку постольку, поскольку иное не указано в законе. Таким образом, на сегодня нет причин отрицать воз- можность гражданского оборота криптовалют на основании принципов гражданского права, смысла законодательства и, как следствие, ана- логии права. К сожалению, именно педагогическая функ- ция права в настоящий момент реализуется в меньшей степени, чем остальные. Студентам, а затем и юристам, далеко не всегда удается осу- ществлять толкование норм законодательства в контексте действия принципов гражданского права, основного смысла законодательства. Таким образом, проведенное исследование показало, что принципы гражданского права имеют собственную аксиологию, которая выра- жается в теоретическом и практическом аспек- тах. Каждый из этих аспектов не в полной мере раскрыт в действительности. Для решения ука- занных проблем следует начинать с педагогиче- ской функции принципов, глубокого их обсуж- дения и анализа, понимания права с позиции не его норм, а прежде всего принципов и смыс- ла. При этом аксиологическое значение прин- ципов является константным и устойчивым, не зависит от исторических или социально-эконо- Несмотря на все сказанное, в литературе иногда можно встретить некоторые сомнения в том, что принципы права могут быть непо- средственно использованы судом. Например, Том 75 № 7 (188) июль 2022 16 Волос А. А. Аксиология принципов гражданского права в современных условиях ципы гражданского права сохраняют свою цен- ность и способны решать конкретные теорети- ческие и практические задачи. мических условий. 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https://openalex.org/W4388692423	https://www.researchsquare.com/article/rs-3483783/latest.pdf	English	null	Preoperative reduced hand grip strength and oral frailty as a predictor of disability in the elderly following hepatobiliary-pancreatic surgery	Research Square (Research Square)	2,023	cc-by	7,590	Preoperative reduced hand grip strength and oral frailty as a predictor of disability in the elderly following hepatobiliary- pancreatic surgery Mariko Tsukagoshi (  marikot@gunma-u.ac.jp ) Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0003-3748-9615 Kenichiro Araki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takamichi Igarashi Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norihiro Ishii Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Shunsuke Kawai Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kei Hagiwara Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kouki Hoshino Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takaomi Seki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norifumi Harimoto Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0002-8085-2857 Ken Shirabe Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Research Article Preoperative reduced hand grip strength and oral frailty as a predictor of disability in the elderly following hepatobiliary- pancreatic surgery Mariko Tsukagoshi (  marikot@gunma-u.ac.jp ) Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0003-3748-9615 Kenichiro Araki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takamichi Igarashi Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norihiro Ishii Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Shunsuke Kawai Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kei Hagiwara Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kouki Hoshino Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takaomi Seki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norifumi Harimoto Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0002-8085-2857 Ken Shirabe Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Research Article Preoperative reduced hand grip strength and oral frailty as a predictor of disability in the elderly following hepatobiliary- pancreatic surgery Mariko Tsukagoshi (  marikot@gunma-u.ac.jp ) Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0003-3748-9615 Kenichiro Araki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takamichi Igarashi Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norihiro Ishii Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Shunsuke Kawai Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kei Hagiwara Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kouki Hoshino Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takaomi Seki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norifumi Harimoto Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0002-8085-2857 Ken Shirabe Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Research Article Preoperative reduced hand grip strength and oral frailty as a predictor of disability in the elderly following hepatobiliary- pancreatic surgery Page 1/16 predictor of disability in the elderly following hepatobiliary- pancreatic surgery Mariko Tsukagoshi (  marikot@gunma-u.ac.jp ) Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0003-3748-9615 Kenichiro Araki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takamichi Igarashi Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norihiro Ishii Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Shunsuke Kawai Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kei Hagiwara Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Kouki Hoshino Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takaomi Seki Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Norifumi Harimoto Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0002-8085-2857 Ken Shirabe Gunma University Graduate School of Medicine School of Medicine: Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu Research Article Keywords: disability, elderly, frailty, hand grip strength, surgery Posted Date: November 15th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3483783/v1 Methods We retrospectively analyzed data of 150 elderly patients (≥ 70 years) following hepatobiliary-pancreatic surgery for malignancies between June 2020 and June 2022. Disability was defined as a new need for long-term health care or an increase in the level of care within six months after surgery. We assessed frailty using the frailty checkup introduced by the Ministry of Health, Labor, and Welfare to check the state of frailty. Background This study aimed to investigate the usefulness of preoperative assessment of frailty in elderly patients using a self-check questionnaire and hand grip strength assessment on surgical outcomes and disability after hepatobiliary-pancreatic surgery. Conclusion Oral frailty and reduced handgrip strength may be useful screening tools for predicting postoperative disability in patients undergoing hepatobiliary-pancreatic surgery. Results The incidence of disability was significantly correlated with reduced grip strength (P = 0.001), difficulty eating hard foods (P = 0.004), and falling (P = 0.049). Multivariate analysis showed that difficulty eating hard foods (P = 0.016), oral frailty, and reduced hand grip strength (P = 0.007) were independent risk factors for the incidence of postoperative disability. Patients at risk of reduced hand grip strength and difficulty eating hard foods showed significantly lower albumin and zinc levels. Furthermore, patients with both risks were significantly associated with increased postoperative complications (P = 0.026), prolonged postoperative hospital stay (P = 0.015), increased hospital transfer (P < .001), and the incidence of disability (P < .001). Research Article Keywords: disability, elderly, frailty, hand grip strength, surgery License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/16 Introduction The continuous increase in the aging population is accompanied by an increase in the number of elderly patients eligible for hepatobiliary-pancreatic (HBP) surgery. According to the annual report on National Clinical Database in Japan, the ratio of age ≥ 70 years increased from 45.1–54.8% for liver surgery and 47.6–60.3% for pancreatic surgery in the last 10 years [1]. It is speculated that the proportion of elderly patients undergoing HBP surgery will continue to increase. In general, elderly patients have various histories, such as multiple comorbidities, multiple oral medications, and social and economic restrictions, in addition to declining physical, organ, and cognitive functions. Another characteristic is that individual differences are extremely large. Therefore, regarding the tolerability of surgical treatment in elderly patients, it is difficult to assess surgical indications based on age alone, and it is necessary to make a comprehensive judgment on an individual basis. Page 3/16 Frailty is considered highly prevalent in old age, conferring a high risk of adverse health outcomes, including falls, disability, hospitalization, and mortality [2]. Frailty is a state of increased vulnerability to adverse outcomes due to decreased reserves and resistance to stressors resulting from various functional changes associated with aging [3–4]. Fried et al. provided a potential standardized definition of frailty in which three or more of the following criteria were present: unintentional weight loss, exhaustion, weakness (low grip strength), slow walking speed, and low physical activity [4]. In surgery, frailty is considered a higher risk factor for unfavorable surgical outcomes [5–6]. Panayi et al. showed the modified frailty index (mFI) Page 3/16 Page 3/16 was a prognostic indicator that strongly correlates with the risk of postsurgical morbidity and mortality [5]. MacIsaac et al. reported that frailty was the only significant predictor of mortality or new disability in non-cardiac surgery, and the Clinical Frailty Scale (CFS) was as accurate as the mFI and easier to use [6]. The Kihon checklist, which is extensively used in Japan, has also been reported as a useful tool for frailty screening and identifying elderly individuals who are at risk of requiring support or care [7]. Tanaka et al. previously reported that frailty was defined by the Kihon checklist, age ≥ 76 years, and open surgery, which are independent risk factors for postoperative loss of independence following hepatic resection [8]. Thus, various indicators are used to screen for and assess frailty, some of which require time. Treatment and data collection The demographic and clinical characteristics and treatment-related details of all the patients were collected from their medical records. Surgical procedures were performed according to the institutional policies and cancer board recommendations. Postoperative complications within 30 days were recorded and scored, according to the Clavien–Dindo classification [9]. Disability was defined based on long-term healthcare support requirements. Long-term Care Insurance is a system in which the society as a whole support individuals and their families faced with situations where someone needs long-term care in Japan. When they require long-term care or support and qualify for such care or support, they can use the Long-term Care Insurance services. In this study, disability was defined as a new need for long-term support required within six months after surgery, or an increase in the level of care within six months after surgery. Two patients died within six months of surgery and were excluded from the disability analysis. Study design and participants We performed a retrospective study of 150 elderly patients (≥ 70 years) following HBP surgery for malignancies between June 2020 and June 2022 in the Department of Hepatobiliary and Pancreatic Surgery at Gunma University Hospital. This study was approved by the Ethics Committee of the hospital, and it met the institutional guidelines of the Declaration of Helsinki Introduction Meanwhile, the frailty checkup for elderly people was introduced in 2020 fiscal year in Japan. Frailty factors that affect surgical outcomes have not yet been established. Therefore, we aimed to investigate whether preoperative assessment of frailty using the frailty checkup and hand grip strength in elderly patients is useful for predicting surgical outcomes and disability after HBP surgery. Frailty assessment We used the "Late-Stage Elderly Questionnaire" newly introduced by the Ministry of Health, Labor, and Welfare to check the state of frailty [10]. This checklist is a self-administered questionnaire consisting of 15 items on health condition, mental health, eating habits, oral function, weight change, exercise and falling, cognitive function, smoking, social participation, and social support (Table 1). Muscle strength was assessed by handgrip strength. We considered only the maximum handgrip strength of at least two trials [11]. Cut-off values for handgrip strength were 28 kg for males and 18 kg for females, according to the revised Japanese version of the Cardiovascular Health Study criteria [12]. Page 4/16 Table 1 Table 1 “Late-Stage Elderly Questionnaire” introduced by the Ministry of Health, Labor, and Welfare to check the state of frailty Factors Questions Answer Weakness Reduced grip strength If hand grip strength is < 28 kg for men and 18 kg for women, this item is scored. No Yes Health condition What is your current state of health? Good/Somewhat good/Normal Not so good/Bad Mental health Are you satisfied with your daily life? Satisfied/Somewhat satisfied Somewhat dissatisfied/Dissatisfied Eating habits Do you eat three meals a day regularly? Yes No Oral function Difficulty to eat hard foods Has it become difficult to eat hard foods compared to six months ago? No Yes Choking Do you choke when drinking water or soups? No Yes Weight loss Have you lost more than 2–3 kg over a period of six months? No Yes Exercise and falling Slow walking speed Does it feel like you walk slower than before? No Yes Falling Have you experienced a fall over the last year? No Yes Lack of exercise Do you exercise, such as walking, at least once a week? Yes No Cognitive function Forgetfulness Has anyone around you mentioned that you have a tendency to forget things, such as repeating the same question? No Yes Losing track the date Do you ever lose track of the day of the month? No Yes Smoking Do you smoke? No/I quit Yes Social participation Going out Do you go out at least once a week? Yes No Social participation Do you usually interact with your family or friends? Yes No Social support Do you have someone nearby whom you can talk to if you are not feeling well? Frailty assessment Yes No Evaluation of inflammatory and nutritional factors We evaluated prognostic indicators based on inflammatory and nutritional factors, including neutrophil-to-lymphocyte ratio and prognostic nutritional index (PNI). The PNI was calculated as follows: 10 × serum albumin (g/dL) + 0.005 × total l h t t (/ 3) [13] Evaluation of inflammatory and nutritional factors We evaluated prognostic indicators based on inflammatory and nutritional factors, including neutrophil-to-lymphocyte ratio and prognostic nutritional index (PNI). The PNI was calculated as follows: 10 × serum albumin (g/dL) + 0.005 × total lymphocyte count (/mm3) [13]. Page 5/16 Page 5/16 Page 5/16 Categorical variables were assessed using chi-squared test or Fisher’s exact test, as appropriate. Cox proportional hazards model analysis was performed using univariate and multivariate analyses of prognostic factors. All statistical analyses were performed using JMP Pro 14 software (SAS Institute, Cary, NC, USA). A P-value of < 0.05 was considered statistically significant. We evaluated each factor on the frailty checklist and postoperative course (Table 3). The incidence of postoperative complications was significantly correlated with health (Q1; P = 0.006), mental health (Q2; P = 0.009), and weight loss (Q6; P = 0.016). Reduced grip strength (P < .001), health condition (Q1; P = 0.019), difficulty eating hard foods (Q4; P = 0.009), slow walking speed (Q7; P < .001), and losing track of the date (Q11; P = 0.019) were significantly associated with transfer to another hospital. The incidence of disability was significantly correlated with reduced grip strength (P = 0.001), difficulty eating hard foods (Q4; P = 0.004), and falling (Q8; P = 0.049). Clinical characteristics A total of 150 patients were included in the study. Eighty-three patients underwent hepatectomy, 64 underwent pancreatectomy, and 3 underwent concomitant hepatectomy and pancreatectomy. Characteristics of the patient are summarized in Table 2. Overall, 18.7% of the patients were octogenarians, and 6.0% had certification of long-term care or support preoperatively. Table 2 Patient characteristics Table 2 Patient characteristics All patients (n = 150) Age (years) 70–74 71 (47.3%) 75–79 51 (34.0%) ≥80 28 (18.7%) Sex Male 89 (59.3%) Female 61 (40.7%) Surgical procedure Hepatectomy 83 (55.3%) Pancreatectomy 64 (42.7%) Concomitant hepatectomy and pancreatectomy 3 (2.0%) Surgical technique Open 101 (67.3%) Laparoscopic 49 (32.7%) Primary cancer type Hepatobiliary 66 (44.0%) Colorectal 36 (24.0%) Duodenum 7 (4.7%) Pancreas 41 (27.3%) Preoperative Certification of Long-term Care or Support 9 (6.0%) between frailty factors and postoperative course Table 2 Page 6/16 We evaluated each factor on the frailty checklist and postoperative course (Table 3). The incidence of postoperative complications was significantly correlated with health (Q1; P = 0.006), mental health (Q2; P = 0.009), and weight loss (Q6; P = 0.016). Reduced grip strength (P < .001), health condition (Q1; P = 0.019), difficulty eating hard foods (Q4; P = 0.009), slow walking speed (Q7; P < .001), and losing track of the date (Q11; P = 0.019) were significantly associated with transfer to another hospital. The incidence of disability was significantly correlated with reduced grip strength (P = 0.001), difficulty eating hard foods (Q4; P = 0.004), and falling (Q8; P = 0.049). We evaluated each factor on the frailty checklist and postoperative course (Table 3). The incidence of postoperative complications was significantly correlated with health (Q1; P = 0.006), mental health (Q2; P = 0.009), and weight loss (Q6; P = 0.016). Reduced grip strength (P < .001), health condition (Q1; P = 0.019), difficulty eating hard foods (Q4; P = 0.009), slow walking speed (Q7; P < .001), and losing track of the date (Q11; P = 0.019) were significantly associated with transfer to another hospital. The incidence of disability was significantly correlated with reduced grip strength (P = 0.001), difficulty eating hard foods (Q4; P = 0.004), and falling (Q8; P = 0.049). Clinical characteristics Page 8/16 n = 150 Postoperative complications ≥ Grade III Clavien-Dindo classification Hospital transfer Disability No (n = 109) Yes (n = 41) P value No (n = 141) Yes (n = 9) P value No (n = 140) Yes (n = 8) P value No 64 (43%) 44 (40%) 20 (49%) 0.36 59 (42%) 5 (56%) 0.50 58 (41%) 5 (63%) 0.29 Q10. Has anyone around you mentioned that you have a tendency to forget things, such as repeating the same question? Yes 30 (20%) 20 (18%) 10 (24%) 0.49 26 (18%) 4 (44%) 0.079 26 (19%) 3 (38%) 0.19 Q11. Do you ever lose track of the day of the month? Yes 20 (13%) 13 (12%) 7 (17%) 0.43 16 (11%) 4 (44%) 0.019* 17 (12%) 2 (25%) 0.27 Q12. Do you smoke? Yes 17 (11%) 11 (10%) 6 (15%) 0.56 17 (12%) 0 0.60 17 (12%) 0 0.60 Q13. Do you go out at least once a week? No 12 (8%) 8 (7%) 4 (10%) 0.74 10 (7%) 2 (22%) 0.15 11 (8%) 1 (13%) 0.50 Q14. Do you usually interact with your family or friends? No 4 (3%) 3 (3%) 1 (2%) > .99 4 (3%) 0 > .99 4 (3%) 0 > .99 Q15. Do you have someone nearby whom you can talk to if you are not feeling well? No 3 (2%) 3 (3%) 0 0.56 3 (2%) 0 > .99 3 (2%) 0 > .99 * P value < 0.05 Frailty factors associated with postoperative disability Univariate and multivariate analyses were performed to analyze frailty factors associated with postoperative disability (Table 4). The univariate analysis revealed that reduced hand grip strength, not so good or bad health condition, difficulty eating hard foods, and a history of falls were significant factors for the incidence of disability. The multivariate analysis revealed that reduced hand grip strength (odds ratio [OR] = 12.97; 95% confidence interval [CI]: 2.03–82.76; P = 0.007) and difficulty eating hard foods (OR = 10.31; 95% CI: 1.56–68.33; P = 0.016) were independent risk factors for incidence of disability Q10. Has anyone around you mentioned that you have a tendency to forget things, such as repeating the same question? Frailty factors associated with postoperative disability Frailty factors associated with postoperative disability Univariate and multivariate analyses were performed to analyze frailty factors associated with postoperative disability (Table 4). Clinical characteristics Page 7/16 Table 3 Table 3 Correlation between frailty factors and postoperative course n = 150 Postoperative complications ≥ Grade III Clavien-Dindo classification Hospital transfer Disability No (n = 109) Yes (n = 41) P value No (n = 141) Yes (n = 9) P value No (n = 140) Yes (n = 8) P value Hand grip strength < 28 kg for males/<18 kg for female 31 (21%) 18 (17%) 13 (32%) 0.068 24 (17%) 7 (78%) < .001* 25 (18%) 6 (75%) 0.001* Q1. What is your current state of health? Not so good/Bad 31 (21%) 16 (15%) 15 (37%) 0.006* 26 (18%) 5 (56%) 0.019* 26 (19%) 4 (50%) 0.054 Q2. Are you satisfied with your daily life? Somewhat dissatisfied/Dissatisfied 21 (14%) 10 (9%) 11 (27%) 0.009* 18 (13%) 3 (33%) 0.11 18 (13%) 2 (25%) 0.30 Q3. Do you eat three meals a day regularly? No 15 (10%) 11 (10%) 4 (10%) > .99 15 (11%) 0 0.60 15 (11%) 0 > .99 Q4. Has it become difficult to eat hard foods compared to six months ago? Yes 38 (25%) 27 (25%) 11 (27%) 0.84 32 (23%) 6 (67%) 0.009* 32 (23%) 6 (75%) 0.004* Q5. Do you choke when drinking water or soups? Yes 25 (17%) 19 (17%) 6 (15%) 0.81 23 (16%) 2 (22%) 0.65 23 (16%) 1 (13%) > .99 Q6. Have you lost more than 2–3 kg over a period of six months? Yes 63 (42%) 39 (36%) 24 (59%) 0.016* 58 (41%) 5 (56%) 0.49 57 (41%) 6 (75%) 0.073 Q7. Does it feel like you walk slower than before? Yes 69 (46%) 51 (47%) 18 (44%) 0.86 60 (43%) 9 (100%) < .001* 63 (45%) 6 (75%) 0.15 Q8. Have you experienced a fall over the last year? Yes 17 (11%) 10 (9%) 7 (17%) 0.25 15 (11%) 2 (22%) 0.27 14 (10%) 3 (38%) 0.049* Q9. Do you exercise, such as walking, at least once a week? Q8. Have you experienced a fall over the last year? Correlation between risk score and incidence of dis We focused on reduced hand grip strength and difficulty eating hard foods as factors for predicting postoperative disability. A risk score of 0 (n = 89) was defined as neither, 1 (n = 51) was defined as either, and 2 (n = 10) was defined as both of the two factors. Postoperative disability significantly increased from 0% (0 of 89) with a risk score of 0 to 40% (4 of 10) with a risk score of 2 (P < 0.001; Fig. 1). Comparison of perioperative characteristics and postoperative course between the two groups classified by risk factors for disability Table 5 shows the perioperative characteristics and postoperative course of the two groups, classified according to risk factors for disability. The risk factors for disability were a score of 0 or 1 and a score of 2. Ten patients with a risk score of 2 were significantly correlated with older age and low albumin and zinc levels. There was a significant association between a risk score of 2 and prolonged postoperative hospital stay (15 vs. 31 days, P = 0.015), postoperative complications (25 vs. 60%, P = 0.026), hospital transfer (3 vs. 50%, P < .001), and incidence of disability (3 vs. 40%, P < .001). Clinical characteristics The univariate analysis revealed that reduced hand grip strength, not so good or bad health condition, difficulty eating hard foods, and a history of falls were significant factors for the incidence of disability. The multivariate analysis revealed that reduced hand grip strength (odds ratio [OR] = 12.97; 95% confidence interval [CI]: 2.03–82.76; P = 0.007) and difficulty eating hard foods (OR = 10.31; 95% CI: 1.56–68.33; P = 0.016) were independent risk factors for incidence of disability. Page 9/16 Table 4 Univariate and multivariate analyses to analyze frailty factors considered for postoperative disability Univariate analysis Multivariate analysis OR 95% CI P-value OR 95% CI P- value Hand grip strength < 28 kg for males/<18 kg for females 13.80 2.63–72.41 0.002* 12.97 2.03–82.76 0.007* Q1. What is your current state of health? Not so good/Bad 4.38 1.03–18.69 0.046* 3.45 0.60–19.78 0.17 Q2. Are you satisfied with your daily life? Somewhat dissatisfied/Dissatisfied 2.26 0.42–12.06 0.34 Q3. Do you eat three meals a day regularly? No NA NA NA Q4. Has it become difficult to eat hard foods compared to six months ago? Yes 10.13 1.95–52.63 0.006* 10.31 1.56–68.33 0.016* Q5. Do you choke when drinking water or soups? Yes 0.73 0.09–6.19 0.77 Q6. Have you lost more than 2 to 3 kg over a period of six months? Yes 4.37 0.85–22.42 0.077 Q7. Does it feel like you walk slower than before? Yes 3.67 0.72–18.80 0.12 Q8. Have you experienced a fall over the last year? Yes 5.40 1.16–25.04 0.031* 5.99 0.92–69.27 0.062 Q9. Do you exercise, such as walking, at least once a week? No 2.36 0.54–10.25 0.25 Q10. Has anyone around you mentioned that you have a tendency to forget things, such as repeating the same question? Yes 2.63 0.2–59–11.71 0.20 Q11. Do you ever lose track of the day of the month? Yes 2.41 0.45–12.92 0.30 Q12. Do you smoke? Yes NA NA NA Q13. Do you go out at least once a week? No 1.68 0.19–14.88 0.64 Q14. Do you usually interact with your family or friends? N NA NA NA Table 4 Univariate analysis Multivariate analysis Q15. Do you have someone nearby whom you can talk to if you are not feeling well? No NA NA NA Abbreviations: OR, odds ratio; CI, confidence interval; NA, not applicable. * P value < 0.05 Univariate analysis Multivariate analysis Correlation between risk score and incidence of disability Correlation between risk score and incidence of disability Comparison of perioperative characteristics and postoperative course between the two groups classified by risk factors for disability Table 5 shows the perioperative characteristics and postoperative course of the two groups, classified according to risk factors for disability. The risk factors for disability were a score of 0 or 1 and a score of 2. Ten patients with a risk score of 2 were significantly correlated with older age and low albumin and zinc levels. There was a significant association between a risk score of 2 and prolonged postoperative hospital stay (15 vs. 31 days, P = 0.015), postoperative complications (25 vs. 60%, P = 0.026), hospital transfer (3 vs. 50%, P < .001), and incidence of disability (3 vs. 40%, P < .001). Page 11/16 Page 11/16 Table 5 Table 5 Comparison of perioperative characteristics and postoperative course between the two groups classified by risk factors for disability Variables Risk factor P-value Score 0 or 1 (n = 140) Score 2 (n = 10) Age (years) 75 (70–89) 78 (72–88) 0.019* Male 85 (61%) 4 (40%) 0.32 Body mass index (kg/m2) 22.8 (16.5–33.0) 22.4 (17.1–27.6) 0.36 Parameters Albumin (g/dL) 4.1 (2.8–5.0) 3.8 (2.0–4.4) 0.012* Lymphocytes (/µL) 1510 (340–3410) 1720 (880–1960) 0.59 CRP (mg/dL) 0.09 (0.01–5.02) 0.21 (0.01–20.48) 0.58 Zn (µg/dL) 70 (44–133) 62 (33–78) 0.008* NLR 2.4 (1.0–23.1) 2.6 (1.4–7.4) 0.23 PNI 48.8 (33.1–62.1) 45.7 (25.3–53.5) 0.052 Operative procedures Pancreatectomy 59 (42%) 7 (70%) 0.11 Laparoscopic surgery 47 (34%) 2 (20%) 0.50 Postoperative hospitalization (days) 15 (7–62) 31 (9–43) 0.015* Complications ≥ Grade III Clavien-Dindo classification 35 (25%) 6 (60%) 0.026* Transfer to another hospital 4 (3%) 5 (50%) < .001* Disability 4 (3%) 4 (40%) < .001* Data are expressed as median (interquartile range), or number of patient (%). * P value < 0.05 Abbreviations: CRP, C-reactive protein; NLR, Neutrophil-to-lymphocyte ratio; PNI, Prognostic Nutritional Index; Zn, Zinc. Comparison of perioperative characteristics and postoperative course between the two groups classified by risk factors for disability Discussion Frailty defined by the CFS was found to be an independent prognostic factor in patients with colorectal liver metastasis [21], hepatocellular carcinoma [22], and perihilar cholangiocarcinoma [23] undergoing hepatectomy and in patients with pancreatic ductal adenocarcinoma undergoing pancreatic resection [24]. In April 2020, the Ministry of Health, Labor, and Welfare revised the health checkup questionnaire and introduced the "Late- Stage Elderly Questionnaire" to assess the state of frailty. This is the so-called "frailty checkup". In this study, we used this new questionnaire for frailty screening in patients undergoing HBP surgery and examined the relationship between 15 questionnaire items and hand grip strength during the postoperative course. This questionnaire is easily administered and can be answered by older adults. It is characterized by the inclusion of questions regarding a trivial decline in oral function, known as oral frailty. Among the oral frailty items in this study, difficulty eating hard foods was significantly associated with postoperative disability in patients undergoing HBP surgery. Recent studies reported that oral frailty is associated with physical frailty and sarcopenia. In the Kashiwa study, 16% of elderly individuals had oral frailty at baseline, which was significantly associated with 2.4-, 2.2-, 2.3-, and 2.2-fold increased risks of physical frailty, sarcopenia, disability, and mortality, respectively [25]. Iwasaki et al. demonstrated that poor oral function, as indicated by low maximum bite force, was associated with the development of frailty in community-dwelling elderly individuals [26]. The results of our study suggest that oral frailty may cause impairment of eating function, which may cause negative chain reaction, thereby leading to the deterioration of mental and physical functions after HBP surgery. The revised international consensus on sarcopenia suggests low muscle strength, which is identified using grip strength as a key parameter of sarcopenia [27]. Measurement of handgrip strength is a simple and non-invasive marker of muscle strength. Ishii et al. reported that comorbid physical frailty and low muscle mass have a significant impact on disability among community-dwelling Japanese older adults [28]. They also indicated that low muscle mass alone may not be associated with an increased risk of incident disability, and that low muscle mass was a risk factor for disability in older adults with physical frailty. In the present study, reduced hand grip strength and oral frailty were independent risk factors for disability after HBP surgery, and their coexistence was a high-risk factor. Discussion In this study, preoperative oral frailty and reduced handgrip strength were significantly associated with the incidence of disability after HBP surgery. Furthermore, both risks were significantly associated with increased postoperative complications, prolonged postoperative hospital stay, and increased hospital transfers. These findings suggest that preoperative frailty assessment using the frailty checkup and hand grip strength could be a novel and simple screening tool for predicting potential outcomes in patients undergoing HBP surgery. Frailty is one of the most problematic manifestations among the aging population. Elderly individuals with frailty are at risk of minor stress, which causes major health changes [14]. Surgery in frail elderly patients is predicted to be at an even higher risk, and sufficient consideration must be given to surgical indications. Two meta-analyses reported significantly higher rates of postoperative complications and mortality in frail patients [5, 15]. Panayi et al. reported that the risk of mortality was 4.19 times higher in frail patients. Although studies on frailty in elderly patients with HBP malignancies are limited, frailty has been reported to be associated with negative short- and long-term outcomes [16, 17]. In the present study, some items of the frailty questionnaire were associated with worse short-term outcomes after HBP surgery. Page 12/16 Page 12/16 In the surgical field, the frailty index or mFI, which evaluates risk as a continuous numerical value of the degree of frailty, has been widely used as a predictor of surgical outcomes [18]. High mFI has been reported to be an independent predictor of major postoperative complications and loss of independence in patients undergoing liver resection [19, 20]. The CFS is frequently used to assess frailty in the surgical field. Frailty defined by the CFS was found to be an independent prognostic factor in patients with colorectal liver metastasis [21], hepatocellular carcinoma [22], and perihilar cholangiocarcinoma [23] undergoing hepatectomy and in patients with pancreatic ductal adenocarcinoma undergoing pancreatic resection [24]. In the surgical field, the frailty index or mFI, which evaluates risk as a continuous numerical value of the degree of frailty, has been widely used as a predictor of surgical outcomes [18]. High mFI has been reported to be an independent predictor of major postoperative complications and loss of independence in patients undergoing liver resection [19, 20]. The CFS is frequently used to assess frailty in the surgical field. Discussion Preoperative evaluation of these two factors may be useful for decision-making when performing HBP surgery in elderly patients. In the present study, patients with both reduced hand grip strength and difficulty eating hard foods showed significantly lower albumin and zinc levels than other patients. Nishikawa et al. showed that serum zinc levels were well stratified, according to frailty status (i.e., frailty, pre-frailty, and robust) in patients with chronic liver diseases [29]. We previously reported that zinc deficiency is associated with hepatic resection complications in patients with hepatocellular carcinoma [30]. Serum zinc, a microelement, tends to decrease with advancing age [31], and its deficiency is thought to affect taste acuity [32]. Zinc deficiency is associated with reduced appetite through its interaction with leptin [33]. Zinc deficiency could be both a cause and effect of frailty through a decline in taste acuity and appetite. Oral frailty causes poor nutritional status and may lead to postoperative disability owing to the deterioration of frailty, such as loss of muscle mass and decreased physical function. Therefore, preoperative frailty assessment could inform an appropriate selection of elderly patients for invasive surgical procedures. This study has several limitations. First, this single-center, retrospective observational study had a small sample size, which limited the generalizability of the results. Therefore, nationwide multicenter studies are required to validate our findings. Second, the study had a selection bias; HBP surgery is often avoided in elderly patients because of high morbidity and mortality rates. Third, as the frailty check was self-administered, it is possible that the cognitive decline of the respondents affected the results. Finally, this study did not examine the effects of preoperative and postoperative chemotherapy. Chemotherapy may have influenced postoperative disability. However, the present study demonstrated that preoperative oral frailty and reduced handgrip strength significantly correlated with the incidence of disability after HBP surgery. Page 13/16 Page 13/16 In conclusion, we found that preoperative frailty checkups, especially for oral frailty, and the assessment of handgrip strength had a significant impact on disability after HBP surgery in elderly patients. Hence, frailty is a predictor of disability after HBP surgery and can be used to inform patients about potential outcomes. In conclusion, we found that preoperative frailty checkups, especially for oral frailty, and the assessment of handgrip strength had a significant impact on disability after HBP surgery in elderly patients. Discussion Hence, frailty is a predictor of disability after HBP surgery and can be used to inform patients about potential outcomes. References 1. Kajiwara Y, Takahashi A, Ueno H, et al (2023) Annual report on National Clinical Database 2020 for gastroenterological surgery in Japan. Ann Gastroenterol Surg 7:367–406 2. Rockwood K, Stadnyk K, MacKnight C, et al (1999) A brief clinical instrument to classify frailty in elderly people. Lancet 353:205–6 2. Rockwood K, Stadnyk K, MacKnight C, et al (1999) A brief clinical instrument to classify frailty in elderly people. Lancet 353:205–6 3. Campbell AJ, Buchner DM (1997) Unstable disability and the fluctuations of frailty. Age Ageing 26:315–8 3. Campbell AJ, Buchner DM (1997) Unstable disability and the fluctuations of frailty. Ag 3. Campbell AJ, Buchner DM (1997) Unstable disability and the fluctuations of frailty. Age Ageing 26:315–8 4. Fried LP, Tangen CM, Walston J, et al (2001) Frailty in older adults: evidence for a phenotype. J Gerontol A Biol Sci Med Sci 56:M146–56 4. Fried LP, Tangen CM, Walston J, et al (2001) Frailty in older adults: evidence for a phenotype. J Gerontol A Biol Sci Med Sci 56:M146–56 5. Panayi AC, Orkaby AR, Sakthivel D, et al (2019) Impact of frailty on outcomes in surgical patients: A systematic review and meta-analysis. Am J Surg 218:393–400 5. Panayi AC, Orkaby AR, Sakthivel D, et al (2019) Impact of frailty on outcomes in surgical patients: A systematic review and meta-analysis. Am J Surg 218:393–400 6. McIsaac DI, Taljaard M, Bryson GL, et al (2020) Frailty as a Predictor of Death or New Disability After Surgery: A Prospective Cohort Study. Ann Surg 271:283–9 6. McIsaac DI, Taljaard M, Bryson GL, et al (2020) Frailty as a Predictor of Death or New Disability After Surgery: A Prospective Cohort Study. Ann Surg 271:283–9 7. Satake S, Senda K, Hong YJ, et al (2016) Validity of the Kihon Checklist for assessing frailty status. Geriatr Gerontol Int 16:709–15 7. Satake S, Senda K, Hong YJ, et al (2016) Validity of the Kihon Checklist for assessing frailty status. Geriatr Gerontol Int 16:709–15 8. Tanaka S, Iida H, Ueno M, et al (2021) Preoperative Risk Assessment for Loss of Independence Following Hepatic Resection in Elderly Patients: A Prospective Multicenter Study. Ann Surg 274: e253–61 8. Tanaka S, Iida H, Ueno M, et al (2021) Preoperative Risk Assessment for Loss of Independence Following Hepatic Resection in Elderly Patients: A Prospective Multicenter Study. Ann Surg 274: e253–61 9. Acknowledgements: None. Acknowledgements: None. Funding Information: This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of interests: The authors declare no conflict of interest. The authors report no proprietary or commercial interests in any product mentioned or concept discussed in this article. Conflict of interests: The authors declare no conflict of interest. The authors report no proprietary or commercial interests in any product mentioned or concept discussed in this article. Ethics statements: The study was approved by the Ethics Committee of the study hospital and was conducted in accordance with the institutional guidelines and the Declaration of Helsinki. Patient consent was obtained using the opt-out method. Ethics statements: The study was approved by the Ethics Committee of the study hospital and was conducted in accordance with the institutional guidelines and the Declaration of Helsinki. Patient consent was obtained using the opt-out method. Author contributions: Study concepts: Mariko Tsukagoshi. Study design: Mariko Tsukagoshi. Data acquisition: Mariko Tsukagoshi, Takamichi Igarashi, Norihiro Ishii, Shunsuke Kawai, Kei Hagiwara, Kouki Hoshino, Takaomi Seki. Quality control of data and algorithms: Mariko Tsukagoshi, Norifumi Harimoto. Data analysis and interpretation: Mariko Tsukagoshi, Kenichiro Araki, Norifumi Harimoto, Ken Shirabe. Statistical analysis: Mariko Tsukagoshi. Manuscript preparation: Mariko Tsukagoshi. 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Letter to the editor: Normative data of handgrip strength in 26344 older adults - a pooled dataset from eight cohorts in Asia. J Nutr Health Aging 24:125–6 11. Auyeung TW, Arai H, Chen LK, et al (2020) Woo J. Letter to the editor: Normative data of handgrip strength in 26344 older adults - a pooled dataset from eight cohorts in Asia. J Nutr Health Aging 24:125–6 Page 14/16 Page 14/16 12. Satake S, Arai H (2020) The revised Japanese version of the Cardiovascular Health Study criteria (revised J-CHS criteria). Geriatr Gerontol Int 20:992–3 13. Chan AW, Chan SL, Wong GL, et al (2015) Prognostic Nutritional Index (PNI) Predicts Tumor Recurrence of Very Early/Early Stage Hepatocellular Carcinoma After Surgical Resection. Ann Surg Oncol 22:4138–48 14. Clegg A, Young J, Iliffe S, et al (2013) Frailty in elderly people. Lancet 381:752–62 15. Wagner D, DeMarco MM, Amini N, et al (2016) Role of frailty and sarcopenia in predicting outcomes among patients undergoing gastrointestinal surgery. World J Gastrointest Surg 8:27–40 16. Rostoft S, van Leeuwen B (2021) Frailty assessment tools and geriatric assessment in older patients with hepatobiliary and pancreatic malignancies. Eur J Surg Oncol 47:514–8 17. Mima K, Hayashi H, Nakagawa S (2021) Frailty is associated with poor prognosis after resection for pancreatic cancer. Int J Clin Oncol 26:1938–1946 18. McIsaac DI, Wong CA, Huang A, et al (2019) Derivation and Validation of a Generalizable Preoperative Frailty Index Using Population-based Health Administrative Data. Ann Surg 270:102–8 19. McKechnie T, Bao T, Fabbro M, et al (2021) Frailty as a Predictor of Postoperative Morbidity and Mortality Following Liver Resection. Am Surg 87: 648–54 20. References Jehan FS, Pandit V, Khreiss M, et al (2022) Frailty Predicts Loss of Independence After Liver Surgery. J Gastrointest Surg 26:2496–502 21. Tokuda K, Morine Y, Miyazaki K, et al (2021) Frailty Can Predict Prognosis After Hepatectomy in Patients With Colorectal Liver Metastasis. Anticancer Res 41:4637–44 22. Yamada S, Shimada M, Morine Y, et al (2021) Significance of Frailty in Prognosis After Hepatectomy for Elderly Patients with Hepatocellular Carcinoma. Ann Surg Oncol 28:439–46 23. Hosoda K, Shimizu A, Kubota K, et al (2022) Usefulness of frailty to predict short- and long-term outcomes in patients who have undergone major hepatectomy for perihilar cholangiocarcinoma. Ann Gastroenterol Surg 6:833–41 23. Hosoda K, Shimizu A, Kubota K, et al (2022) Usefulness of frailty to predict short- and long-term outcomes in patients who have undergone major hepatectomy for perihilar cholangiocarcinoma. Ann Gastroenterol Surg 6:833–41 24. Yamada S, Shimada M, Morine Y, et al (2021) Significance of frailty in prognosis after surgery in patients with pancreatic ductal adenocarcinoma. World J Surg Oncol 19(1):94 24. Yamada S, Shimada M, Morine Y, et al (2021) Significance of frailty in prognosis after surgery in patients with pancreatic ductal adenocarcinoma. World J Surg Oncol 19(1):94 25. Tanaka T, Takahashi K, Hirano H, et al (2018) Oral Frailty as a Risk Factor for Physical Frailty and Mortality in Community-Dwelling Elderly. J Gerontol A Biol Sci Med Sci 73:1661–7 25. Tanaka T, Takahashi K, Hirano H, et al (2018) Oral Frailty as a Risk Factor for Physical Frailty and Mortality in Community-Dwelling Elderly. J Gerontol A Biol Sci Med Sci 73:1661–7 26. Iwasaki M, Yoshihara A, Sato N, et al (2018) A 5-year longitudinal study of association of maximum bite force with development of frailty in community-dwelling older adults. J Oral Rehabil 45:17–24 26. Iwasaki M, Yoshihara A, Sato N, et al (2018) A 5-year longitudinal study of association of maximum bite force with development of frailty in community-dwelling older adults. J Oral Rehabil 45:17–24 27. Cruz-Jentoft AJ, Bahat G, Bauer J, et al (2019) Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing 48:16–31 27. Cruz-Jentoft AJ, Bahat G, Bauer J, et al (2019) Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing 48:16–31 28. Ishii H, Tsutsumimoto K, Doi T, et al (2020) Effects of comorbid physical frailty and low muscle mass on incident disability in community-dwelling older adults: A 24-month follow-up longitudinal study. Maturitas 139:57–63 28. References Ishii H, Tsutsumimoto K, Doi T, et al (2020) Effects of comorbid physical frailty and low muscle mass on incident disability in community-dwelling older adults: A 24-month follow-up longitudinal study. Maturitas 139:57–63 29. Nishikawa H, Yoh K, Enomoto H, et al (2020) Serum Zinc Level Is Associated with Frailty in Chronic Liver Diseases. J Clin Med 9:1570 29. Nishikawa H, Yoh K, Enomoto H, et al (2020) Serum Zinc Level Is Associated with Frailty in Chronic Liver Diseases. J Clin Med 9:1570 30. Harimoto N, Araki K, Muranushi R, et al (2022) Significance of zinc deficiency in patients with hepatocellular carcinoma undergoing hepatic resection. Hepatol Res 52:210–20 30. Harimoto N, Araki K, Muranushi R, et al (2022) Significance of zinc deficiency in patients with hepatocellular carcinoma undergoing hepatic resection. Hepatol Res 52:210–20 31. Maes M, DeVos N, Wauters A, et al (1999) Inflammatory markers in younger vs elderly normal volunteers and in patients with Alzheimer's disease. J Psychiatr Res 33:397–405 31. Maes M, DeVos N, Wauters A, et al (1999) Inflammatory markers in younger vs elderly normal volunteers and in patients with Alzheimer's disease. J Psychiatr Res 33:397–405 32. Stewart-Knox BJ, Simpson EE, Parr H, et al (2008) Taste acuity in response to zinc supplementation in older Europeans. Br J Nutr 99:129–36 32. Stewart-Knox BJ, Simpson EE, Parr H, et al (2008) Taste acuity in response to zinc supplementation in older Europeans. Br J Nutr 99:129–36 33. Konukoglu D, Turhan MS, Ercan M, et al (2004) Relationship between plasma leptin and zinc levels and the effect of insulin and oxidative stress on leptin levels in obese diabetic patients. J Nutr Biochem 15:757–60 33. Konukoglu D, Turhan MS, Ercan M, et al (2004) Relationship between plasma leptin and zinc levels and the effect of insulin and oxidative stress on leptin levels in obese diabetic patients. J Nutr Biochem 15:757–60 Page 15/16 Page 15/16 Figures Figure 1 Correlation between risk score and incidence of disability. A risk score of 0 (n = 89) was defined as neither, 1 (n = 51) was defined as either, and 2 (n = 10) was defined as both of reduced hand grip strength and difficulty eating hard foods. Postoperative disability significantly increased from 0% (0 of 89) with a risk score of 0 to 40% (4 of 10) with a risk score of 2 (P < 0.001). Figure 1 Correlation between risk score and incidence of disability. A risk score of 0 (n = 89) was defined as neither, 1 (n = 51) was defined as either, and 2 (n = 10) was defined as both of reduced hand grip strength and difficulty eating hard foods. Postoperative disability significantly increased from 0% (0 of 89) with a risk score of 0 to 40% (4 of 10) with a risk score of 2 (P < 0.001). Correlation between risk score and incidence of disability. A risk score of 0 (n = 89) was defined as neither, 1 (n = 51) was defined as either, and 2 (n = 10) was defined as both of reduced hand grip strength and difficulty eating hard foods. Postoperative disability significantly increased from 0% (0 of 89) with a risk score of 0 to 40% (4 of 10) with a risk score of 2 (P < 0.001). Page 16/16
https://openalex.org/W4321347530	https://www.j-las.lemkomindo.org/index.php/AFoSJ-LAS/article/download/335/499	Indonesian	null	Pengaruh Perputaran Piutang dan Perputaran Persediaan Terhadap Roa pada PT Bank Mandiri Tbk	All Fields of Science Journal Liaison Academia And Sosiety/All Fields of Science Journal Liaison Academia and Sosiety	2,022	cc-by	2,519	Abstract The purpose of this study was to find out whether there was an influence of accounts receivable turnover and inventory turnover to roa at PT. Bank Mandiri Tbk period 2011 to 2018. The method used in this study was a quantitative method with multiple linear regression analysis techniques. The data used were secondary data, hypothesis testing using the test of the coefficient of determination (R2), Simultaneous Test (F) and partial test (t) by calculation using the SPSS program. The results showed that partially Accounts Receivable Turnover has a significant effect on ROA. While Inventory Turnover has not a significant ROA effect on PT. Bank Mandiri Tbk, while simultaneously Receivable Turnover and Inventory Turnover has not a significant effect on ROA at PT. Bank Mandiri Tbk. Keywords: Accounts Receivable Turnover, Inventory Turnover, and ROA. AFoSJ-LAS, Vol.2, No.3, 01 Sept 2022 (hal: 56-62) AFoSJ-LAS, Vol.2, No.3, 01 Sept 2022 (hal: 56-62) e-ISSN.2776-2408 ; p-ISSN 2798-9267 Availabel Online: https://j-las.lemkomindo.org/index.php/AFoSJ-LAS/index Abstrak Tujuan dari penelitian ini adalah untuk mengetahui apakah ada pengaruh perputaran piutang dan perputaran persediaan terhadap roa pada PT. Bank Mandiri Tbk periode 2011 sampai 2018. Metode yang digunakan dalam penelitian ini adalah metode kuantitatif dengan teknik analisis regresi linier berganda. Data yang digunakan adalah data sekunder, pengujian hipotesis menggunakan uji koefisien determinasi (R2), Uji Simultan (F) dan uji parsial (t) dengan perhitungan menggunakan program SPSS. Hasil penelitian menunjukkan bahwa secara parsial Perputaran Piutang berpengaruh signifikan terhadap ROA. Sedangkan Inventory Turnover tidak berpengaruh signifikan terhadap ROA PT. Bank Mandiri Tbk, sedangkan secara simultan Perputaran Piutang dan Perputaran Persediaan tidak berpengaruh signifikan terhadap ROA pada PT. Bank Mandiri Tbk. Kata kunci: Perputaran Piutang, Perputaran Persediaan, dan ROA. 56 PENDAHULUAN Bank merupakan suatu organisasi yang memiliki tujuan memperoleh laba atau keuntungan, dari keuntungan tersebut digunakan untuk membiayai kegiatan perusahaan sehari-hari termasuk membayar gaji karyawan dan mengelola modal usaha. Untuk kelancaran dalam kegiatan operasional perusahaan tersebut dibutuhkan manajemen keuangan yang baik. Kita bisa menilai apakah keadaan keuangan perusahaan dalam keadaan yang stabil atau tidak dengan cara mengetahui kinerja keuangan perusahaan tersebut. Jika suatu perusahaan mengalami masalah dalam hal pendapatan atau manajemen keuangan nya, perusahaan harus mampu mengatasi nya segera jika tidak maka ini bisa berdampak pada kebangkrutan atau kelumpuhan total pada semua kegiatan perusahaan. Dari pendapatan yang diperoleh perusahaan dari kegiatan perusahaan ini bisa dimanfaatkan dengan efisien untuk memenuhi kebutuhan kegiatan perusahaan kedepannya. Dengan kata lain jika perusahaan mampu menghasilkan pendapatan yang stabil untuk menjaga kinerja keuangan perusahaannya. Profitabilitas mempunyai peranan penting dalam perusahaan sebagai cerminan masa depan apakah perusahaan mempunyai prospek yang baik di masa mendatang. Bagi perusahaan masalah profitabilitas sangat penting. Bagi pemimpin perusahaan profitabilitas digunakan untuk melihat seberapa besar kemajuan atau berhasil tidak perusahaan yang dipimpinnya. Untuk mengukur tingkat keuntungan suatu perusahaan, digunakan rasio profitablitisa yang dikenal juga dengan nama rasio rentabilitas. Rasio profitabilitas dalam penilitian ini menggunakan Return On Asset (ROA). Semakin besar ROA, berarti semakin efisien penggunaan aktiva perusahaan atau dengan kata lain dengan jumlah aktiva yang sama bisa dihasilkan laba yang lebih besar dan sebaliknya. Tabel Ikhtisar Keuangan PT Bank Mandiri Tbk N o Uraian 2013 2014 2015 2016 2017 1 ROA 3.66% 3.57% 3.15% 1.95% 2.72% 2 ROE 27.31% 25.81% 23.03% 11.12% 14.53% 3 NIM 5.68% 5.94% 5.90% 6.29% 5.63% 4 BOPO 62.41% 64.98% 69.67% 80.94% 71.78% 5 Rasio Laba (Rugi) terhadap Jumlah Aset 2.66% 2.57% 2.49% 1.42% 2.05% 6 Rasio Laba (Rugi) terhadap Jumlah Ekuitas 20.85% 19.96% 17.99% 9.07% 12.54% 7 Rasio Liabilitas terhadap Jumlah Aset 87.26% 87.14% 86.16% 84.31% 83.69% 8 Rasio Liabilitas terhadap Ekuitas 685.17% 677.79% 622.67% 537.32% 512.94% 9 Rasio Fee Based Income terhadap Total Pendapatan Operasional 23.48% 20.09% 22.26% 21.29% 23.29% Dinyatakan dalam (%) Penelitian ini dilakukan disalah satu perbankkan yang terdaftar di BEI yaitu PT. Bank Mandiri Tbk. Bank mandiri merupakan bank terbesar di Indonesia dalam hal aset, pinjaman, dan deposit. Bank ini berdiri pada tanggal 2 Oktober 1998 sebagai bagian dari program restrukturisasi perbankan yang dilaksanakan oleh Pemerintah Indonesia. y (%) Penelitian ini dilakukan disalah satu perbankkan yang terdaftar di BEI yaitu PT PENDAHULUAN Pada bulan Juli 1999, empat bank milik Pemerintah yaitu, Bank Bumi Daya (BBD), Bank Dagang Tabel Ikhtisar Keuangan PT Bank Mandiri Tbk p y g y Bank Mandiri Tbk. Bank mandiri merupakan bank terbesar di Indonesia dalam hal aset, pinjaman, dan deposit. Bank ini berdiri pada tanggal 2 Oktober 1998 sebagai bagian dari program restrukturisasi perbankan yang dilaksanakan oleh Pemerintah Indonesia. Pada bulan Juli 1999, empat bank milik Pemerintah yaitu, Bank Bumi Daya (BBD), Bank Dagang 57 Negara (BDN), Bank Ekspor Impor Indonesia (Bank Exim), dan Bank Pembangunan Indonesia (Bapindo), digabungkan ke dalam Bank Mandiri. Terlihat bahwa perbankan mandiri mengalami fluktuasi dalam pengelolahan aset (ROA) dari tahun 2011- 2018 adanya perubahan pendapatan yang didapat pada bank akan menyebabkan perubahan akan kinerja keungan perusahaan. Timbulnya piutang tak tertagih akan menyebabkan bank mengalami kerugian atau beban untuk perusahaan mengurangi pendapatan perusahaan, bahkan bisa menimbulkan kerugian jika jumlah piutang yang diterima kurang dari harga pokok barang yang dijual secara kredit dan mempengaruhi akan persediaan pada perusahaan persediaan pada bank merupa dana yang nantinya akan diberikan pada nasabah Persediaan terlalu rendah, akan berdampak dalam laporan posisi keuangan (neraca) yaitu jumlah persediaan, aset lancar, total aset, saldo laba akan menjadi dinyatakan terlalu rendah, dan modal kerja bersih serta saldo lancar akan menjadi lebih rendah pula dari seharusnya. Berdasarkan uraian diatas maka penulis tertarik untuk melakukan penelitian dengan judul “Pengaruh Perputaran Piutang dan Perpuatan Persediaan Terhadap ROA pada PT Bank Mandiri Tbk”. METODE PENELITIAN Peneliti tidak melakukan riset langsung ke lokasi tetapi peneliti hanya mengambil data yang diperlukan di website PT. Bank Mandiri, Tbk yang di laksanakan pada bulan April 2019 sampai Mei 2019. Data yang digunakan dalam penelitian ini adalah data sekunder. Sumber data yang digunakan yaitu data internal dan data eksternal, metode pengumpulan data yang digunakan adalah studi literatur dan metode dokumentasi. Sedangkan variabel yang digunakan dalam penelitian ini adalah variabel bebas yang terdiri dari perputaran piutang (X1) dan perputaran persediaan (X2) dan variabel terikat yaitu return on asset (Y). Metode analisis data yang digunakan dalam penelitian ini adalah metode deskriptif kuantitatif, sedangkan model analisis yang digunakan dalam penelitian ini adalah regresi linier berganda. Hasil Uji Regresi Linier Sederhana Hasil Uji Regresi Linier Sederhana Untuk mengetahui pengaruh Perputaran Piutang dan Perputaran Persediaan terhadap ROA pada PT. Bank Mandiri Tbk yang terdaftar di Bursa Efek Indonesia, maka digunakan uji Regresi Linier Berganda. g j g g Tabel 4.5. Hasil Pengujian Regresi Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 11,172 4,175 2,676 ,044 Perputaran Piutang ,140 ,052 ,868 2,702 ,043 Perputaran Persediaan -,069 ,073 -,303 -,944 ,389 a. Dependent Variable: ROA Berdasarkan hasil pengujian dibawah ini, maka diperoleh persamaan regresi sebagai berikut: Y = 11,172 + 0.140X1 +(-069)X2 Tabel 4.5. Hasil Pengujian Regresi 58 58 Pada model regresi diperoleh nilai konstanta ROA 11,17 artinya bahwa jika nilai variabel bebas (X1) bernilai 0,140 dan variabel (X2) nilai bernilai (0,069) variabel terikat (Y) il i b 11 17 K fi i i i b l b b b il i itif d tif ( ) ( ) ( ) (Y) nilainya sebesar 11,17. Koefisien regresi variabel bebas bernilai positif dan negatif. Berikut adalah tabel hasil pengujian regresi pada masing masing variabel: Pengujian Hipotesis g j p Koefisien Determinasi (R) Koefisien determinasi (R2) bertujuan untuk mengukur berapa besar kekmampuan model dalam menerangkan variabel terikat. Nilai koefisien determinasi (R2) dapat dilihat dari tabel dibawah : Tabel 4.6. Hasil Pengujian Koefisien Determinasi (R2) Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 ,773a ,598 ,437 3,16894 a. Predictors: (Constant), Perputaran Persediaan, Perputaran Piutang p g Nilai koefisien Determinasi (R2) yang diperoleh sebesar 0,59,8 atau 59,8 % menunjukan bahwa variabel Perputaran piutang dan Perputaran persediaan mampu menjelaskan variasi yang terjadi pada ROA pada PT. Bank Mandiri Tbk, sedangkan sisanya sebesar 0,402% atau 40,2% dijelaskan oleh variabel lain yang tidak diteliti dalam penilitian ini. p Uji Simultan (Uji F) Uji simultan (uji F) dilakukan untuk melihat perputaran piutang dan perputaran persediaan terhadap ROA, pengujian dilakukan dengan tingkat kepercayaan 95% atau tingkat kesalahan σ = 0.05 (5%), dengan kriteria jika Fhitung ≤ Ftabel maka diterima, H1 ditolak, artinya secara simultan penelitian ini tidak ada pengaruh, sedangkan jika Fhitung > Ftabel maka H0 ditolak, H1 diterima, artinya secara simultan penelitian terdapat pengaruh. Tabel 4.7 Hasil uji simultan (uji F) ANOVAa Model Sum of Squares Df Mean Square F Sig. 1 Regression 74,664 2 37,332 3,718 ,103b Residual 50,211 5 10,042 Total 124,875 7 a. Dependent Variable: ROA b. Predictors: (Constant), Perputaran Persediaan, Perputaran Piutang p b. Predictors: (Constant), Perputaran Persediaan, Perputaran Piutang Berdasarkan tabel diatas dapat terlihat pengaruh X1 dan X2 secara simultan terhadap Y adalah sebesar 0,1 > 0,05 dan nilai Fhitung 3,718 ≤ 5,41 Ftabel sehingga disimpulkan bahwa tidak terdapat pengaruh X1 dan X2 secara simultan terhadap Y. Dengan demikian variabel Perputaran Piutang dan Perputaran Persediaan simultan tidak berpengaruh positif dan tidak signifikan terhadap ROA pada PT. Bank Mandiri Tbk. Uji Parsial (Uji t) j ( j ) Uji parsial (Uji t) untuk melihat perputaran piutang dan perputaran persediaan secara parsial terhadap ROA, dengan kriteria jika thitung ≤ ttabel maka H0 diterima, H1 ditolak, artinya secara parsial penelitian ini tidak berpengaruh, sedangkan jika thitung > ttabel maka H0 ditolak, H1 diterima, artinya secara parsial penelitian ini terdapat pengaruh. 59 Hasil uji parsial pada penelitian ini terlihat pada tabel berikut: Tabel 4.8 Hasil uji parsial (uji t) Coefficientsa Model Unstandardized Coefficients Standardize d Coefficients t Sig. B Std. Error Beta 1 (Constant) 11,172 4,175 2,676 ,044 Perputaran Piutang ,140 ,052 ,868 2,702 ,043 Perputaran Persediaan -,069 ,073 -,303 -,944 ,389 a. Pengujian Hipotesis Dependent Variable: ROA Berdasarkan tabel tersebut di atas dapat terlihat bahwa : Berdasarkan tabel tersebut di atas dapat terlihat bahwa : p 1. Nilai thitung untuk variabel perputaran piutang = 2,702 dan ttabel = 2.570 dengan demikian pengaruh X1 dan Y sebesar 0,04 < 0,05 dan nilai thitung 2,702 ≤ 2,570 sehingga dapat disimpulkan bahwa H0 ditolak dan H1 diterima yang berarti ada pengaruh X1 dan Y, dengan demikian secara parsial variabel Perputaran Piutang berpengaruh dan signifikan terhadap ROA. 2. Nilai thitung untuk variabel perputaran persediaan = -9,44 dan ttabel = 2,702 dengan demikian pengaruh X2 dan Y sebesar 0,38 > 0,05 dan nilai thitung -9,44 ≤ ttabel 2,702 H2 ditolak dan H0 diterima yang berarti tidak ada pengaruh X2 dan Y, dengan demikian secara parsial variabel tidak ada berpengaruh dan tidak signifikan terhadap ROA. Hasil pengujian hipotesis pertama yaitu koefisien determinasi (R2) menunujukan bahwa variabel Perputaran Piutang dan Peputaran Persediaan mampu menjelaskan variasi yang terjadi pada ROA PT. Bank Mandiri Tbk yang terdaftar di bursa efek indonesia, hal ini dibuktikan dengan nilai koefisien determinasi (R2) yang diperoleh sebesar 0,59,8 atau 59,8 % menunjukan bahwa variabel Perputaran piutang dan Perputaran persediaan mampu menjelaskan variasi yang terjadi pada ROA pada PT. Bank Mandiri Tbk. Hasil pengujian hipotesis ketiga yaitu uji parsial (uji t) menunjukan bahwa pengaruh X1 dan Y sebesar 0,04 < 0,05 dan nilai thitung 2,702 ≤ 2,570 sehingga dapat disimpulkan bahwa H0 ditolak dan H1 diterima yang berarti ada pengaruh X1 dan Y, dengan demikian secara parsial variabel Perputaran Piutang berpengaruh dan signifikan terhadap ROA. Hasil penelitian sesuai dengan penelitian yang dilakukan oleh (Lestari, 2017) yang berjudul ”Pengaruh Perputaran kas, Perputaran Persediaan dan Perputaran Piutang terhadap Profitabilitas pada perusahaan manufaktur yang terdaftar di bursa efek indonesia” Perputaran persediaan tidak berpengaruh positif dan signifikan terhadap profitabilitas pada perusahaan manufaktur yang terdaftar di BEI. Perputaran Piutang berpengaruh positif dan signifikan terhadap profitabilitas pada perusahaan manufaktur yang terdaftar di BEI. Selanjutnya penelitian yang dilakukan oleh (Sompie, Murni, & Uhing, 2018) yang berjudul “Pengaruh Perputaran Modal Kerja, Piutang, Persediaan Terhadap Profitabilitas pada Perusahaan Kosmetik dan Keperluan Rumah Tangga di Bursa Efek Indonesia” Sejalan dengan hasil uji simultan yang menyatakan variabel perputaran modal kerja, piutang, 60 persediaan secara simultan tidak berpengaruh signifikan terhadap profitabilitas. Dan hasil uji parsial Rasio perputaran piutang berpengaruh signifikan terhadap profitabilitas pada perusahaan kosmetik dan keperluan rumah tangga di Bursa Efek Indonesia (BEI). KESIMPULAN Secara parsial Perputaran piutang berpengaruh signifikan terhadap ROA pada PT. Bank Mandiri Tbk, artinya semakin baik perusahaan dalam mengolah piutang maka perusahaan baik dalam mengolah aset sehingga mempengaruhi akan pendapatan perusahaan. Dan Perputaran Persediaan tidak berpengaruh signifikan g g Secara simultan Perputaran Piutang dan Perputaran Persediaan tida berpengaruh signifikan terhadap ROA pada PT. Bank Mandiri Tbk. Berdasarkan kesimpulan diatas, penulis memberikan beberapa saran yang mungkin akan bermanfaat, bahwa Pihak Perusahaan diharapkan memperhatikan Perputaran Piutang dan Perputaran Persediaan yang mempengaruhi profitabilitas perusahaan (ROA), sedangkan Peneliti selanjutnya disarankan memperluas jenis perusahaan, memperpanjang jangka waktu penelitian. Pengujian Hipotesis Namun tidak sejalan dengan hasil Rasio persediaan berpengaruh terhadap profitabilitas pada perusahaan kosmetik dan keperluan rumah tangga di Bursa Efek Indonesia (BEI). Hasil pengujian hipotesis kedua yaitu uji simultan (uji t) menunjukan bahwa secara simultan variabel Perputaran Piutang dan Perputaran Persediaan tidak berpengaruh terhadap ROA pada PT. Bank Mandiri Tbk yang terdaftar dibursa efek indonesia. Silaen, S. (2018). Metodologi Penelitian Sosial Untuk Penulisan Skripsi dan Tesis. Bogo Penerbit in media. gg ( ) Sugiyono. (2017). Metode Penilitian Kuantitatif,Kualitatif, dan R&D. Bandung: Alfabeta. Somple, A. G., Murni, S., & Uhing, Y. (2018). Pengaruh Perputaran Modal Kerja , Piutang , Persediaan Terhadap Profitabilitas pada perusahaan kosmetik dan keperluan rumah tangga di Bursa Efek Indonesia, 6(4), 1888–1897. Jakarta: Salemba empat. Silaen, S. (2018). Metodologi Penelitian Sosial Untuk Penulisan Skripsi dan Tesis. Bogor: Penerbit in media. Somple, A. G., Murni, S., & Uhing, Y. (2018). Pengaruh Perputaran Modal Kerja , Piutang , Persediaan Terhadap Profitabilitas pada perusahaan kosmetik dan keperluan rumah tangga di Bursa Efek Indonesia, 6(4), 1888–1897. Sugiyono. (2017). Metode Penilitian Kuantitatif,Kualitatif, dan R&D. Bandung: Alfabeta. Jakarta: Salemba empat. DAFTAR PUSTAKA Aish, S. (2018). Pengaruh Perputaran Kas dan Perputaran Piutang terhadap Return on assets pada perusahaan keramik yang terdaftar di bursa efek indonesia periode 2012-2017. Aish, S. (2018). Pengaruh Perputaran Kas dan Perputaran Piutang terhadap Return on assets pada perusahaan keramik yang terdaftar di bursa efek indonesia periode 2012-2017. Diana, A., & Setiawati, L. (2017). Akutansi Keuangan Menengah. Yogyakarta: C.V An Offset. Giri, Ferdinan, E. (2017). Akutansi Keuangan Menengah 1 Perspektif IFRS. Yogyakarta: Upp Stim Ykpn. Harahap, A. N. K. (2018). Pengaruh Perputaran Persediaan Dan Perputaran Piutang Terhadap Likuiditas Pada Perusahaan Otomotif Yang Terdaftar Di Bursa Efek Indonesia Priode 2010-2013. Jurnal Ilmiah Manajemen Dan Bisnis, 17(2), 116– 127. https://doi.org/10.30596/jimb.v17i2.1000 Hastuti, W. (2018). Pengaruh Perputaran Piutang dan Perputaran Persediaan terhadap Margin Laba Bersih pada perusahaan makanan dan minuman yang terdaftar dibursa efek indonesia, III(2), 224–234. Hery. (2015). Analisis Laporan Keuangan Pendekatan Rasio Keuangan. Yogyakarta: CAP (Center of Academic Publishing Service). ( g ) Hery. (2016). Analisis Laporan Keuangan-Integrated and Comprehensive. Jakarta: Grasindo. Hery. (2016). Analisis Laporan Keuangan-Integrated and Comprehensive. Jakarta: Grasindo Hery. (2016). Analisis Laporan Keuangan-Integrated and Comprehensive. Jakarta: Grasindo. Husnan, S., & Pudjiastuti, E. (2015). Dasar-Dasar Manajemen Keuangan. Yogyakarta: UPP AMP YKPN. Lestari, A. P. T. (2017). Pengaruh Perputaran kas, Perputaran Persediaan dan Perputaran Piutang terhadap Profitabilitas pada perusahaan manufaktur yang terdaftar di bursa efek indonesia. Muchson. (2015). Metode Riset Akutansi. Bogor: spasi media. Muchson. (2015). Metode Riset Akutansi. Bogor: spasi media. Musthafa, H. (2017). Manajemen Keuangan. Yogyakarta: C.V Andi Offset. j g g Revee, Warren, & Duchac. (2015). Pengantar Akuntansi (Adaptasi Indonesia) (25th ed 61 62 62
https://openalex.org/W2935769945	https://elar.urfu.ru/bitstream/10995/82840/1/10.18502_keg.v1i1.4407.pdf	English	null	The Formation of the Structure and Tribological Properties of Composite Bronzes Reinforced with Steel Dendrites	KnE engineering	2,019	cc-by	3,089	Conference Paper Conference Paper The Formation of the Structure and Tribological Properties of Composite Bronzes Reinforced with Steel Dendrites Zhilyakov A.Yu.1, Khristolyubov A.S.2, Potekhin B.A.2, and Ilyushin V.V.2 1Department of Heat Treatment and Physics of Metals, Institute of New Materials and Technologies, Ural Federal University named after the ﬁrst President of Russia B.N.Yeltsin, 620002, 19 Mira Street, Ekaterinburg, Russia 2Ural State Forest Engineering University, Yekaterinburg, 620100, 36 Siberian Road, Ekaterin- burg Russia 1Department of Heat Treatment and Physics of Metals, Institute of New Materials and Technologies, Ural Federal University named after the ﬁrst President of Russia B.N.Yeltsin, 620002, 19 Mira Street, Ekaterinburg, Russia 2 Abstract The possibility of creating of composite bronzes reinforced with steel dendrites from martensitic, austenitic and ferritic steels was considered. Compared to the BrO10 bronze widely used in the sliding friction units, bronze BRZHNA 12-7-1 has an increased complex of mechanical, technological and, especially, tribological properties. At comparable values of the friction coefﬁcient, the wear resistance of bronze BRZHNA 12-7-1 is much higher. In contrast to the classic BrO10 bronze, composite bronze can be obtained in a hot-deformed state, welded, welded on steel and cast iron. The level of mechanical properties: σ0.2 = 220 MPa; σ𝑢= 295 MPa; ψ = 38% is 1.5-3 times higher than that of BrO10. The possibility of creating an industrial version of composite bronze with a particularly high level of tribological properties was proved for the ﬁrst time at the laboratory level. Keywords: composite bronze, mechanical and technological properties, martensitic steel, austenitic steel, ferritic steel. Zhilyakov A.Yu. et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. How to cite this article: Zhilyakov A.Yu., Khristolyubov A.S., Potekhin B.A., and Ilyushin V.V., (2019), “The Formation of the Structure and Tribological Properties of Composite Bronzes Reinforced with Steel Dendrites” in XIX International scientiﬁc-technical conference “The Ural school-seminar of metal scientists-young researchers”, KnE Engineering, pages 176–183. DOI 10.18502/keg.v1i1.4407 Page 176 The Ural school-seminar of metal scientists-young researchers XIX International scientiﬁc-technical conference “The Ural school-seminar of metal scientists-young researchers” Volume 2019 The Ural school-seminar of metal scientists-young researchers XIX International scientiﬁc-technical conference “The Ural school-seminar of metal scientists-young researchers” Volume 2019 The Ural school-seminar of metal scientists-young researchers XIX International scientiﬁc-technical conference 1. Intorduction Tin bronzes, such as BrO10 gave a good account of itself in sliding friction units as anti- friction sliding alloys. However, it cannot be deformed either in hot or cold states due to the presence in it of intermetallic compounds like Cu31Sn8. Therefore the range of their rational use limits [1]. Welding, restoration surfacing is difﬁcult, since the intermetallic compounds are brittle at all temperatures of their existence and have an unfavorable acute-angular shape. Selection and Peer-review under the responsibility of The Ural school-seminar of metal scientists-young researchers Conference Committee. The technological plasticity of BrO10 bronze can be made satisfactory by dissolving and transferring coarse, brittle intermetallic compounds into α (Cu-Sn) solid solution. In this case, the rule of Charpie-Bochwara (for anti-friction alloys) is violated and bronze completely loses its service, that is, tribological properties. The Ural school-seminar of metal scientists-young researchers Our attempts to improve the morphology of intermetallic compounds using additional alloying of BrO10 by Ni and Co did not give positive results, and we replaced the Cu31Sn8 intermetallic compound with steel dendrites, the formation of which is known in the Cu – Fe alloys [2-6]. 2. Materials and Methods Experimental bronze ingots with a diameter of 65 mm and a height of 120 mm (weight about 2 kg) were prepared by melting pure charge materials in a Tamman furnace using alundum crucibles in a reducing atmosphere of carbon monoxide. Further, the rod ingots 7 mm in diameter were made by vacuum casting, the rate of their crystallization and cooling was about 700 ∘C / s [2]. In addition, the castings of the studied bronzes were melted down by a tungsten electrode in an argon environment to a depth of 5 mm. Table 1 shows the chemical composition of studied bronze. Table 1: The chemical composition of the studied bronze, [wt.%]. Table 1: The chemical composition of the studied bronze, [wt.%]. Bronze Fe Ni Co Cr Al Si BrZhNKA 9-4-1-1 8,6 3,9 1,1 - 0,8 - BrZhNA 12-7-1 12,9 6.0 - - 1,0 - BrZhNKA 23-8-3-1 22,9 82 2,8 - 1,0 - BrZhNKhA 12-9-3-1 13,1 9,7 - 2,3 1,1 - BrZhNKhK 12-7-5-1 13,4 7,2 - 5,8 - 1.0 Note: 1) Cu - the rest; 2) the amount of impurities does not exceed 0.30%, S and P ≤0.02% A local chemical analysis was performed using Jeol JSM 6490 LV scanning electron microscope with an attachment for an energy dispersive X-ray microanalysis (Oxford Inca Dry Cool) with a resolution of 133 eV. A total chemical analysis of the alloy was performed on an area of 1 mm2 with the averaging of the results of three measurements taken at different regions. The heat treatment was performed in a SNOL 8.2/1100 chamber furnace, the deviations from the speciﬁed temperature did not exceed ±5 ∘С. The number of dendrites by their relative area on the surface of the metallographic section (sample) was estimated according to the method [7]. The microhardness of the structural constituents of cast and heat treated alloys were measured using PMT-3M and 402MVD devices under a load of 0.5 N. The mechanical characteristics of the alloy were determined using standard (ﬁvefold) tensile specimens with a diameter of the gage part of 5 mm at room temperature using an Instron 3382 testing complex. DOI 10.18502/keg.v1i1.4407 Page 177 Page 177 The Ural school-seminar of metal scientists-young researchers The coefﬁcient of friction and the rate of wear were determined using a special setup mounted on the base of a lathe tool with a numerical program control. 2. Materials and Methods The tests were performed using the disk–ﬁnger scheme with a continuous computer ﬁxation of the testing parameters (pressure, rate of sliding, temperature). In each experiment, three specimens with dimensions of 6 × 6 × 12 mm were used; a disk made of steel ShKh15 (45 HRC) was used as a counterbody. The test procedure has been described in [8]. 3. Results and Discussion The low solubility of Fe and Co in Cu in the solid state: 1.92 and 3.5 at.% at 950 ∘C and less than 0.5 at.% at 600 ∘С, respectively, determines the formation of dendrites in the process of crystallization, the basis of which are Fe, Ni and Co. The crystallization of iron dendrites begins at temperatures of about 1250 ∘C, at which temperature the solubility of Cu in γFe is up to 10 at.% [9]. The size of dendrites in casting ingots weighing 3 kg is 15-20 microns (Fig. 1, a). With a high rate of crystallization and cooling of the melt (700 ∘C / s), their size decreases by an order of magnitude, while steel dendrites, unlike intermetallics such as Cu31Sn8, have a favorable shape, which, as a rule, affects mechanical and technological properties. First of all the quantity (volume) of dendrites depends on the content of iron in bronze, and the additional alloying of alloys by Ni, Co, Al, and others forms carbon-free steels of different classes. Figure 2a shows the linear dependence of the number of dendrites on the total content of Fe, Ni, Co. in bronze. The composition of dendrites is presented in the diagram (Fig. 2, b), from which it follows that the amount of Fe in them is almost always the same and is 58–62%, the copper content is also stable and is 19–23%, and the content of Ni and Co is proportional to their number in the charge. During the bronzes cooling after crystallization zones enriched in Cu, Ni, and Co are formed due to a decrease in the copper solubility in iron dendrites [10–13]. These zones (inclusions) are close in chemical composition to the matrix and are in the form of plates that coagulate after heating to 450 ∘C and holding at this temperature for 2 hours (Fig. 3, a, b). In other cases, these precipitates are shells around the grains that create the dendrite; a shell structure is formed (Fig. 3, c) [14–15]. The transverse size of all these inclusions is <0.2 μm. Whereas these areas are a substitution solid solution of Ni, Al in copper and are close in composition to the matrix. 3. Results and Discussion DOI 10.18502/keg.v1i1.4407 Page 178 The Ural school-seminar of metal scientists-young researchers Figure 1: Morphological features of the structure of composite bronze BRZHNA 12-7-1, depending on the technology of its manufacture: a) casting 3 kg; b) vacuum casting; c) arc remelting in argon; d) casting bronze BrO10. Figure 1: Morphological features of the structure of composite bronze BRZHNA 12-7-1, depending on the technology of its manufacture: a) casting 3 kg; b) vacuum casting; c) arc remelting in argon; d) casting bronze BrO10. a) b) Figure 2: The dependence of the number of dendrites (S𝑑) - a) andtheir chemical composition in bronze - b). Figure 2: The dependence of the number of dendrites (S𝑑) - a) andtheir chemical composition in bronze - b). For example, in bronze BRZhNA 12-7-1, the composition of the “steel” part of the dendrite is H15U1 steel, and the matrix and inclusions in the dendrites are bronze BrZhN 3-3 [16]. DOI 10.18502/keg.v1i1.4407 Page 179 The Ural school-seminar of metal scientists-young researchers a) b) c) Figure 3: The internal structure of the dendrite in bronze BRZHNKA 9-4-1-1 (a - casting, b- after aging 450 ∘ C for 2 hours) and BrZhNKA 23-8-3-1 (c - casting). b) a) b) a) c) a) b) c) a) c) Fi 3 Th i t l t t f th d d it i b BRZHNKA 9 4 1 1 ( c) Figure 3: The internal structure of the dendrite in bronze BRZHNKA 9-4-1-1 (a - casting, b- after aging 450 ∘ C for 2 hours) and BrZhNKA 23-8-3-1 (c - casting). The experimental data presented in Table 2 show that the hardness of the base surface (steel dendrites, intermetallic in BrO10) does not affect the coefﬁcient of friction and is in the range of 0.013–0.018, and the wear resistance varies widely from 0.001 to 0.025 μm / km. Whereas the wear resistance determines the reliability and durability of friction-slip units. In our opinion, the reason for high wear resistance is that steel dendrites (Fig. 3) have soft inclusions, and there are dispersed solid steel inclusions in the matrix [20, 22]. 3. Results and Discussion In the process of tribological testing, the hydraulic effect of the lubricant forms an oil-intensive microrelief on the friction-slip surface, which provides very high wear DOI 10.18502/keg.v1i1.4407 Page 180 The Ural school-seminar of metal scientists-young researchers able 2: The dependence of friction coefﬁcient (f) and wear rate (I) of the studied bronzes on the dendrite tructure and hardness. Table 2: The dependence of friction coefﬁcient (f) and wear rate (I) of the studied bronzes on the dendrite structure and hardness. Table 2: The dependence of friction coefﬁcient (f) and wear rate (I) of the studied bronzes on the dendrite structure and hardness. able 2: The dependence of friction coefﬁcient (f) and wear rate (I) of the studied bronzes on the dendrit ructure and hardness. № The composition of bronze, castings Dendrite base (base surface) HV50 f𝑚𝑝 I, [µm/km] 1 BrО10 Cu31Sn8 401 0,016 0,025 2 BrZhNA 12-7-1 aging martensite 338 0,018 0,026 3 BrZhNKhA 12-9-3-1 martensite+ austenite 200 0,015 0,010 4 BrZhNKhК 12-7-5-1 stainless austenite 189 0,013 0,006 5 BrZhNA 12-7-1 vacuum casting martensite - 0,017 0,002 6 BrZhNA 12-7-1 arc remelting martensite - 0,017 0,002 7 BrZhNKhК 12-7-5-1 vacuum casting stainless ferrite - 0,016 0,001 Note: 1) positions 1-4 - castings weighing 3 kg, positions 5-7 - rods 7 mm in diameter, length 600 mm, obtained by vacuum casting; 2) the microhardness of dendrites in bronze (positions 5-7) is not determined due to their high dispersion, but, in our opinion, cannot be higher than 200 HV50; 3) the microhardness of the matrix for different bronzes is in the range of 100-120 HV50; 4) the friction coefﬁcient is determined at P = 3 MPa and V = 3.3 m / s. Note: 1) positions 1-4 - castings weighing 3 kg, positions 5-7 - rods 7 mm in diameter, length 600 mm, obtained by vacuum casting; 2) the microhardness of dendrites in bronze (positions 5-7) is not determined due to their high dispersion, but, in our opinion, cannot be higher than 200 HV50; 3) the microhardness of the matrix for different bronzes is in the range of 100-120 HV50; 4) the friction coefﬁcient is determined at P = 3 MPa and V = 3.3 m / s. 3. Results and Discussion Note: 1) positions 1-4 - castings weighing 3 kg, positions 5-7 - rods 7 mm in diameter, length 600 mm, obtained by vacuum casting; 2) the microhardness of dendrites in bronze (positions 5-7) is not determined due to their high dispersion, but, in our opinion, cannot be higher than 200 HV50; 3) the microhardness of the matrix for different bronzes is in the range of 100-120 HV50; 4) the friction coefﬁcient is determined at P = 3 MPa and V = 3.3 m / s. resistance of composite bronzes, especially in the dispersed state (positions 5-6, Table 2). Composite bronze BrZhNKhK 12-7-5-1 has a particularly high wear resistance (position 7, table 2) in which the dendrites are stainless steel with the composition Kh23N15S1, “coated” with solid oxides of the (Fe,Cr)2O3 type. The mechanical properties of composite bronze BrZhNA 12-7-1 are signiﬁcantly higher than those of BrO10 bronze, especially in terms of plastic properties, even in casting without any heat treatment (position 2, Table 3). Table 3: Mechanical properties of composite bronze BrZhNA 12-7-1 after various treatments. № Bronze composition Heat treatment mode Mechanical properties σ0,2, [MPa] σ𝑢, [MPa] ψ, [%] δ, [%] δ𝑡, [%] 1 BrО10 - 170 215 10-14 3-10 < 1 2 BrZhNA 12-7-1 - 170 364 42,8 38,2 20,3 3 BrZhNA 12-7-1 quenching 950 ∘С, 30 min, water 147 300 69,5 66,9 34,2 4 BrZhNA 12-7-1 quenching + aging 450 ∘С, 2 h 149 301 39,4 51,4 39,3 5 BrZhNA 12-7-1 - 220 295 38,5 16,0 6,4 Note: Positions 1–4 ingots weighing 3 kg, diameter 50 mm; Position 5 - rod 7 mm in diameter, obtained by vacuum casting. Table 3: Mechanical properties of composite bronze BrZhNA 12-7-1 after various treatments. Note: Positions 1–4 ingots weighing 3 kg, diameter 50 mm; Position 5 - rod 7 mm in diameter, obtained by vacuum casting. In addition, unlike BrO10 bronze, widely used in mechanical engineering, composite bronze is well surfaced on steel and cast iron, more environmentally friendly (no tin) [22]. DOI 10.18502/keg.v1i1.4407 Page 181 The Ural school-seminar of metal scientists-young researchers 4. Conclusions At the laboratory level the studies substantiate the reasonability of creating an industrial version of composite bronze with particularly high wear resistance under conditions of sliding friction. The increased level of mechanical and technological properties makes it possible to use composite bronzes reinforced with dendrites from steels of different classes, both in cast and hot-deformed states, that is, to expand the range of their use in comparison with the “prototype” BrO10 bronze. BRZHNA 12-7-1 bronze was shown to be the best version of such bronze. References [1] B.N. Arzamasov Construction materials: a Handbook. Mechanical Engineering, Moscow, 1990. [2] Yu.S. Avraamov, A.D. Shlyapin Alloys based on systems with limited solubility in the liquid state. Intercontact science, Moscow, 2002 [3] C.P. Wang, X.J. Liu, I. Ohnuma, R. Kainuma, K. Ishida Thermodynamic database of the phase diagrams in Cu–Fe base ternary systems. J. Phase Equilib. Diffus. 25 (2004) 320–328. [4] M. Baricco, E. Bosco, G. Acconciaioco, P. Rizzi, M. Coisson Rapid solidiﬁcation of Cu– Fe–Ni alloys. Mater. Sci. Eng. A. 375–377 (2004) 1019–1023. [5] Y.Y. Chuang, R. Schmid, and Y.A. Chang Calculation of the equilibrium phase diagrams and the spinodally decomposed structures of the Fe–Cu–Ni system. Acta Mater. 8 (1985) 1369–1380. [6] K.P. Gupta The Cu–Fe–Ni (Copper–Iron–Nickel) system. Phase Diagram of Ternary Nickel Alloys. 1 (1990) 290–315. [7] S.A. Saltykov Stereometric metallography. Metallurgy, Moscow, 1970. [8] B.A. Potekhin, V.V. Ilyushin, A.S. Khristolyubov Special properties of babbitt B83 obtained by the turbulent casting method. Casting and metallurgy. 57 (2010) 78-81. [9] N.P. Lyakishev State diagrams of double metal systems: A Handbook. Mashinostroe- nie, Moscow, 1997 [10] G.W. Qin, G. Zhao, M. Jiang, H.X. Li, S.M. Hao. The isothermal sections of the Cu–Ni– Fe ternary system at 600, 800, 1000, and 1050∘C. Z. Metallkd. 5 (2000) 379–382. [11] V.M. Lopez, N. Sano, T. Sakurai, and K. Hirano. A study of phase decomposition in Cu–Ni–Fe alloys. Acta Metall. Mat. 1 (1993) 265–271. DOI 10.18502/keg.v1i1.4407 Page 182 Page 182 The Ural school-seminar of metal scientists-young researchers [12] K.J. Ronka, A.A. Kodentsov, P.J.J. Van Loon, J.K. Kivilahti, F.J.J. Van Loo. Thermo- dynamic and kinetic study of diffusion paths in the system Cu–Fe–Ni. Metall. Mater. Trans. A. 27 (1996) 2229–2238. [13] . U. Ugaste, A.A. Kodentsov, and F.J.J. Van Loo. Interdiffusion and Kirkendall-effect in the Fe–Ni–Cu system. Sol. St. Phenomena. 72 (2000) 117–122. [14] B. Potekhin, A. Hernández, A. Khristolyubov, V. Ilushin. Formación de la estructura y propiedades de los bronces Fe-Ni-Al. CIM 2011 – VI Congreso Internacional del Materiales. 27-30 Noviembre de 2011, Bogotá D.C., Colombia. [15] B.A. Potekhin, V.V. Ilyushin, A.S. Khristolyubov, A.Yu. Zhilyakov, A. Hernandez. The possibility of creating a composite bronze alloy - martensitic-aging steel. MITOM. 5 (2013) 6-10. [16] B.A. Potekhin, A.S. Khristolyubov, A.Yu. Zhilyakov, V.V. Ilyushin. Features of the formation of the structure of composite bronzes reinforced with steel dendrites. Voprosy Materialovedenia. 76 (2013) 43-49. [17] Ya.M. Potak. High-strength steels. Metallurgy, Moscow, 1972. [18] Ya.M. Potak, and E.A. Sagalevich. References Structural Diagram of Deformable Stainless Steels. MITOM. 9 (1971) 12–16. [19] E.A. Matsin. Charpy rule and antifriction alloy surface microrelief. Proceedings of the 2nd All-Union Conference on Friction and Wear in Machines. Academy of Sciences of the USSR, Moscow. 3 (1948) 222-229. [20] B.A. Potekhin, V.V. Ilyushin, A.S. Khristolyubov, and A.Yu. Zhilyakov. Formation of structure and properties of composite bronzes reinforced by steel dendrites. Phys. Met. Metallogr. 115 (2014) 413–419. [22] B.A. Potekhin, A.S. Khristolyubov, A.A. Hernandez Fereira. New class of composite bronze, armed with steel dendrites for antifriction technique. XXIV International scientiﬁc conference Trans & Motauto 16, Varna Bulgaria, June 2016. [22] V.I. Shumyakov, B.A. Potekhin, Yu.S. Korobov, A.S. Khristolyubov, V.V. Ilyushin, S.P. Kochugov, A.N. Balin, and A.A. Vishnevskii, RF Patent 170923, (2017). DOI 10.18502/keg.v1i1.4407 Page 183 Page 183
https://openalex.org/W1844715769	https://www.nature.com/articles/ncomms10102.pdf	English	null	Phase diagram for the transition from photonic crystals to dielectric metamaterials	Nature communications	2,015	cc-by	7,429	ARTICLE Received 20 May 2015 \| Accepted 31 Oct 2015 \| Published 2 Dec 2015 Received 20 May 2015 \| Accepted 31 Oct 2015 \| Published 2 Dec 2015 1 Ioffe Institute, St Petersburg 194021, Russia. 2 Department of Nanophotonics and Metamaterials, ITMO University, St Petersburg 197101, Russia. 3 Nonlinear Physics Center and the ARC Center of Excellence CUDOS, Australian National University, Canberra Australian Capital Territory 0200, Australia. Correspondence and requests for materials should be addressed to M.V.R. (email: m.rybin@mail.ioffe.ru) or to Y.S.K. (email: ysk@internode.on.net). URE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications Results A i An interplay between Bragg and Mie resonances. To analyse the PhC–MM phase transition, we investigate theoretically electro- magnetic properties of the dielectric periodic medium depending on the dielectric contrast, ﬁlling ratio and incident wave polar- ization. As an illustrative example, we consider here 2D square lattices of parallel dielectric circular rods inﬁnitely long in the z direction with the radius r and real frequency independent permittivity e. The rods are embedded in air (eair ¼ 1) and the arrays can be characterized by the ﬁlling ratio r/a. We have derived three key sets of spectroscopic data, namely: (i) spectra of the Mie scattering by an isolated rod; (ii) photonic band structure of an inﬁnite 2D square lattice composed of rods; and (iii) transmittance of a 2D square lattice of 10 layers in length. All data sets have been calculated for a wide range of the rod permittivity 1 e 100 with the step of De ¼ 1 (e-scenario) and in the range of ﬁlling ratio 0 r=a 0:5 with the step of 0.01 (r/a-scenario). In the r/a-scenario cylinder radius is supposed to be as a ﬁxed parameter. Note that at r/a ¼ 0.5 all rods touch each other and at r/a40.5 the rods penetrate into each other and the structure appears as inverted one of air holes in a dielectric matrix. First, we identify two different scenarios for the transition of a two-dimensional (2D) square lattice of dielectric rods from PhC to MM (Fig. 1). The ﬁrst scenario that has never been discussed previously occurs when the lattice constant a decreases in comparison with the ﬁxed radius of rods r and dielectric permittivity e. This transformation leads to an increase of the ﬁlling ratio r/a, and it makes possible the homogenization18 of the periodic dielectric structure with the negative effective permeability (mo0) at higher values of e (ref. 19–23). The second scenario occurs when the dielectric permittivity of rods e varies in a ﬁxed square lattice. This transformation leads to an increase of the Mie scattering wavelength, and it can also provide the conditions for the homogenization approach with negative m. While photonic structures with varying e were studied earlier theoretically24,25, the transition between PhCs and MMs First, we consider the Mie scattering by an isolated rod. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 a a Metamaterial Metamaterial Photonic crystal Mie Br < Mie Br Br Br Br > Mie Br < Mie Mie Mie ′ Decreasing lattice constant a′<a a′ r/a-scenario -scenario Increasing rod permittivity ′> a b c Figure 1 \| Transition from photonic crystals to metamaterials. Schematic illustrates two possible transformations of the PhC (b) to MM (a,c) through the competition between the Bragg and Mie scattering wavelengths. To satisfy the classical homogenization condition in the low-frequency long- wavelength limit (lBraggolMie), one may decrease the lattice constant and increase the ﬁlling ratio r/a (r/a-scenario) or, alternatively, increase the rod permittivity e (e-scenario). a a Metamaterial Metamaterial Photonic crystal Mie Br < Mie Br Br Br Br > Mie Br < Mie Mie Mie ′ Decreasing lattice constant a′<a a′ r/a-scenario -scenario Increasing rod permittivity ′> a b c T T he concepts of phase transitions and phase diagrams are among the most fundamental concepts in physics. Here we reveal and analyse a rather unusual type of phase transitions between two classes of artiﬁcial electromagnetic structures: photonic crystals (PhCs)1,2 and metamaterials (MMs)3,4. Rapidly growing attention to the study of PhCs and MMs is due to their unique electromagnetic properties and the exciting new physics these structures may offer for novel applications in photonics5–9. The resonant properties of PhCs are driven by the familiar Bragg scattering from a periodic structure when the lattice constant becomes comparable with the wavelength of light. MMs are regarded as effective media with averaged parameters which in turn can be engineered by changing the properties of their subwavelength elements modifying the propagation of electromagnetic waves with the wavelengths much larger than the lattice constant. a Metamaterial Metamaterial Photonic crystal Decreasing lattice constant a′<a Increasing rod permittivity ′> Metamaterial Figure 1 \| Transition from photonic crystals to metamaterials. Schematic illustrates two possible transformations of the PhC (b) to MM (a,c) through the competition between the Bragg and Mie scattering wavelengths. To satisfy the classical homogenization condition in the low-frequency long- wavelength limit (lBraggolMie), one may decrease the lattice constant and increase the ﬁlling ratio r/a (r/a-scenario) or, alternatively, increase the rod permittivity e (e-scenario). Figure 1 \| Transition from photonic crystals to metamaterials. Schematic illustrates two possible transformations of the PhC (b) to MM (a,c) through the competition between the Bragg and Mie scattering wavelengths. Phase diagram for the transition from photonic crystals to dielectric metamaterials Mikhail V. Rybin1,2, Dmitry S. Filonov2, Kirill B. Samusev1,2, Pavel A. Belov2, Yuri S. Kivshar2,3 & Mikhail F. Limonov1,2 V. Rybin1,2, Dmitry S. Filonov2, Kirill B. Samusev1,2, Pavel A. Belov2, Yuri S. Kivshar2,3 il F Li 1 2 Mikhail V. Rybin1,2, Dmitry S. Filonov2, Kirill B. Samusev1,2, Pavel A. Belov2, Yuri S. Kivshar2,3 & Mikhail F. Limonov1,2 Photonic crystals and dielectric metamaterials represent two different classes of artiﬁcial media but are often composed of similar structural elements. The question is how to distinguish these two types of periodic structures when their parameters, such as permittivity and lattice constant, vary continuously. Here we discuss transition between photonic crystals and dielectric metamaterials and introduce the concept of a phase diagram, based on the physics of Mie and Bragg resonances. We show that a periodic photonic structure transforms into a metamaterial when the Mie gap opens up below the lowest Bragg bandgap where the homogenization approach can be justiﬁed and the effective permeability becomes negative. Our theoretical approach is conﬁrmed by microwave experiments for a metacrystal composed of tubes ﬁlled with heated water. This analysis yields deep insight into the prop- erties of periodic structures, and provides a useful tool for designing different classes of electromagnetic materials with variable parameters. 1 Ioffe Institute, St Petersburg 194021, Russia. 2 Department of Nanophotonics and Metamaterials, ITMO University, St Petersburg 197101, Russia. 3 Nonlinear Physics Center and the ARC Center of Excellence CUDOS, Australian National University, Canberra Australian Capital Territory 0200, Australia. Correspondence and requests for materials should be addressed to M.V.R. (email: m.rybin@mail.ioffe.ru) or to Y.S.K. (email: ysk@internode.on.net). 1 NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 The transition we describe here can be mapped onto a phase diagram; however, it is quite different from phase transitions well known in other ﬁelds such as thermodynamics. The thermodynamical phase transitions appear as transformations of a system from one state or ‘phase’ to another with changing external variables such as pressure, temperature, chemical potential and so on. Here we reveal and analyse a rather unusual type of phase transitions between two regimes of light propagation, that is, the regime where the propagation of light is mainly inﬂuenced by the interference between multiple Bragg scattered (PhC ‘phase’), and the regime where the propagation of light is mainly determined by the properties of each single element of the materials (MM ‘phase’). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 To satisfy the classical homogenization condition in the low-frequency long- wavelength limit (lBraggolMie), one may decrease the lattice constant and increase the ﬁlling ratio r/a (r/a-scenario) or, alternatively, increase the rod permittivity e (e-scenario). g g Many properties of conventional MMs are usually deﬁned by the optically induced magnetic response of metallic wires or split- ring resonators, which has been demonstrated experimentally in many studies for both microwave and near-infrared spectral regions10,11. However, the scaling of such MMs to higher frequencies encounters many problems, including strong absorption of electromagnetic radiation in metals. Recently suggested electromagnetic periodic structures composed of high-permittivity dielectric elements12–14 can avoid these problems, while still delivering an effective magnetic response. The main physics underlying the properties of such all-dielectric MMs is based on the resonant modes of the Mie scattering15 supported by dielectric particles16,17. Mie resonances provide a mechanism for the generation of an optically induced resonant magnetic response based on the displacement current, and they offer a simpler and more versatile route towards the fabrication of isotropic MMs and metasurfaces operating at optical frequencies. has never been analysed previously to our best knowledge, especially in terms of the phase diagram, as discussed below. We notice that the aim of both scenarios is the same: to tune the wavelength of the Mie mode lMie higher than the Bragg wavelength lBr that can be achieved by either decreasing the Bragg wavelength or by increasing the Mie wavelength. Decreas- ing the Bragg wavelength means decreasing the lattice constant (building block size) a, which will be smaller than the operating wavelength for the MM behaviour, lMie. Here we integrate both scenarios and introduce a novel concept of a phase transition between all-dielectric PhC and MM and suggest the corresponding phase diagram. The phase diagram PhC–MM is represented on the plane ‘ﬁlling ratio versus dielectric permittivity’ for an example of 2D structure composed of dielectric circular rods. Our theory is conﬁrmed by very careful experimental design and analysis. p p g p q PhCs and all-dielectric MMs can be composed of the similar structural elements arranged in the similar geometries. Such structures offer a unique opportunity to study a transition from PhCs to MMs when their parameters, such as dielectric permittivity and lattice period, vary continuously. NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications Results A i The calculated data allow us to analyse precisely the evolution of the low-frequency region of the photonic band structure formed by a complicated mixture of the dispersion curves originating from Mie and Bragg resonances and propagating modes. Since our goal is to discuss the PhC–MM transition, we concentrate on the TE polarization where the lowest TE01 magnetic dipole resonance of the rods results in a negative magnetic permeability in the Mie gap spectral region19. The physics of the PhC–MM transition is displayed by the gap map (Fig. 3) that relates the Bragg and Mie gaps positions to the rod permittivity e for given r/a ¼ 0.25. These illustrate essential features such as strong decreasing of Mie resonance frequencies with e increase followed by intersection of the Mie and Bragg bands. The Mie resonances of individual rods form both non-dispersive ﬂat bands in the photonic structure and Mie photonic gaps in the energy spectrum (Figs 2 and 3). For low-contrast PhCs, all Mie resonances are located is a frequency range higher than the lower Bragg gap. With increasing rod The photonic band structures for the 2D square lattice of circular rods were computed numerically using the plane wave expansion method28. The square 2D lattice has a square Brillouin zone, with the three special points G, X and M corresponding to k ¼ 0, k ¼ p a ^x, and k ¼ p a ^x þ p a ^y, respectively. We restrict here our discussion to the case for which the wave vector k of the eigenmodes is oriented in the G-X direction of the ﬁrst Brillouin zone displaying the lowest Bragg frequencies in comparison with the G-M direction (Fig. 2). The photonic band structures were obtained with 16,384 (128 128) plane waves. Results A i The far-ﬁeld scattering can be expanded into orthogonal electromagnetic dipolar and multipolar modes, described by circular Lorenz–Mie resonant coefﬁcients an (transverse-electric NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 (TE) polarization) and bn (transverse-magnetic (TM) polari- zation) corresponding to electric and magnetic moments, respectively15. Figure 2 presents the numerically calculated spectra of the Mie scattering efﬁciency Qsca;n ¼ 2 x an j j2 (ref. 26) for the dipole TE0 and multipole TEn n 1 ð Þ modes in the range of dimensionless frequencies a/l from 0 to 0.8. Each mode consists of an inﬁnite set of quasi-equidistant modes TEnk (ref. 27). The Mie modes of the rod are denoted as TEnk, where n is integer related to the symmetry of the mode, and k 1 identiﬁes resonance number for the modes with ﬁxed symmetry n. The calculations reveal a strong decrease of the Mie eigenfrequencies and narrowing of the resonant Mie bands with e increasing, as can be clearly seen for the lowest TE01 magnetic Mie resonance in Fig. 2. (TE) polarization) and bn (transverse-magnetic (TM) polari- zation) corresponding to electric and magnetic moments, respectively15. Figure 2 presents the numerically calculated spectra of the Mie scattering efﬁciency Qsca;n ¼ 2 x an j j2 (ref. 26) for the dipole TE0 and multipole TEn n 1 ð Þ modes in the range of dimensionless frequencies a/l from 0 to 0.8. Each mode consists of an inﬁnite set of quasi-equidistant modes TEnk (ref. 27). The Mie modes of the rod are denoted as TEnk, where n is integer related to the symmetry of the mode, and k 1 identiﬁes resonance number for the modes with ﬁxed symmetry n. The calculations reveal a strong decrease of the Mie eigenfrequencies and narrowing of the resonant Mie bands with e increasing, as can be clearly seen for the lowest TE01 magnetic Mie resonance in Fig. 2. The transmission spectra were calculated using the CST Microwave Studio software for the wave vector of the incident beam parallel to the G-X direction. We considered the 2D square lattice with different thicknesses along x axis that leads to creation of the photonic bandgaps, including thicknesses of 10 lattice layers as shown in Fig. 2. Results A i TE01 TE11 =9 Bragg d e f TE01 TE01 TE11 TE01 TE02 TE11 TE21 TE01 TE11 Frequency (a /λ) 0.0 0.2 0.4 0.6 0.8 0.0 0.2 0.4 0.6 0.8 0.0 0.2 0.4 0.6 0.8 =12.8 =19.5 =40 =25 =4 =9 TE01 TE02 TE11 TE12 TE21 TE31 1 2 4 3 0 Field intensity 1 0 Wavevector Transmission X Γ Γ Γ Γ 1 2 4 3 0 Field intensity 1 0 Wavevector Transmission X TE01 TE02 TE11 TE21 Mie Mie Bragg Bragg Mie + Bragg Mie + Bragg a b c d e f g h i j k l m n o p q r Figure 2 \| Numerical calculations. (a), (d), (g), (j), (m), (p) Calculated Mie scattering efﬁciency Qsca,n for an isolated dielectric circular rod for TEn (nZ0) modes. (b), (e), (h), (k), (n), (q) The band structure for 2D square lattice of rods with r ¼ 0.25a in air (eair ¼ 1) for the TE polarization. The band structure is shown between the G point (wave vector k ¼ 0) and the X point (\|k\| ¼ p/a along the x direction). (c), (f), (i), (l), (o), (r) The transmittance calculated for 10 lattice layers of the 2D square structure of rods in air for the TE polarization. The frequency and wave vector are plotted in dimensionless units a/l, where l is vacuum wavelength and a denotes the lattice constant. (a)–(c) e ¼ 4, (d)–(f) e ¼ 9, (g)–(i) e ¼ 12.8, (j)–(l) e ¼ 19.5, (m)–(o) e¼ 25, (p)–(r) e¼ 40. NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications 3 TE01 0.0 0.2 0.4 0.6 0.8 =4 Bragg a b c d b c a i TE01 TE02 TE11 TE21 =19.5 Mie + Bragg j k l j l h k 8 g r =40 TE01 TE02 TE11 TE12 TE21 TE31 Γ Γ 1 2 4 3 0 Field intensity 1 0 Wavevector Transmission X Mie p q r p Transmission Figure 2 \| Numerical calculations. (a), (d), (g), (j), (m), (p) Calculated Mie scattering efﬁciency Qsca,n for an isolated dielectric circular rod for TEn (nZ0) modes. (b), (e), (h), (k), (n), (q) The band structure for 2D square lattice of rods with r ¼ 0.25a in air (eair ¼ 1) for the TE polarization. Results A i The band structure is shown between the G point (wave vector k ¼ 0) and the X point (\|k\| ¼ p/a along the x direction). (c), (f), (i), (l), (o), (r) The transmittance calculated for 10 lattice layers of the 2D square structure of rods in air for the TE polarization. The frequency and wave vector are plotted in dimensionless units a/l, where l is vacuum wavelength and a denotes the lattice constant. (a)–(c) e ¼ 4, (d)–(f) e ¼ 9, (g)–(i) e ¼ 12.8, (j)–(l) e ¼ 19.5, (m)–(o) e¼ 25, (p)–(r) e¼ 40. NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 20 30 40 50 60 10 0.0 0.2 0.4 0.6 TE01 TE11 TE12 TE02 TE21 TE0k TE1k TE2k TE3k b c TE31 Wavevector X Γ X TE TM Metamaterial Photonic crystal Bragg Mie a/λ a Γ Figure 3 \| Bandgap diagram for the 2D square lattice of circular rods. (a) The bandgap diagram obtained from the photonic band structures calculated for 1 e 60 with steps of De ¼ 1. The rods are embedded in air, r/a ¼ 0.25, and TE polarization is considered. The Bragg and Mie gaps obtained from the band structure calculations are marked by dark green. (b,c) The photonic band structure for the 2D square lattice of rods with e ¼ 60 for TE and TM polarization, respectively, for the G-X scan of the wave vector k. x y z E H TE y z Figure 4 \| Experimental set-up. An anechoic ch composed of 10 tubes in length (x axis) and 5 t 50 tubes in total. Each tube has a length of 1 m radius of 2 cm and an inner radius of 1.7 cm. Al closed series circuit together with a water heate stabilizer. Insert: schematic illustration of the tra 20 30 40 50 60 10 0.0 0.2 0.4 0.6 TE01 TE11 TE12 TE02 TE21 TE0k TE1k TE2k TE3k b c TE31 Wavevector X Γ X TE TM Metamaterial Photonic crystal Bragg Mie a/λ a Γ Figure 3 \| Bandgap diagram for the 2D square lattice of circular rods. (a) The bandgap diagram obtained from the photonic band structures calculated for 1 e 60 with steps of De ¼ 1. The rods are embedded in air, r/a ¼ 0.25, and TE polarization is considered. Results A i The Bragg and Mie gaps obtained from the band structure calculations are marked by dark green. (b,c) The photonic band structure for the 2D square lattice of rods with e ¼ 60 for TE and TM polarization, respectively, for the G-X scan of the wave vector k. 20 30 40 50 60 10 0.0 0.2 0.4 0.6 TE01 TE11 TE12 TE02 TE21 TE0k TE1k TE2k TE3k b c TE31 Wavevector X Γ X TE TM Metamaterial Photonic crystal Bragg Mie a/λ a Γ x y z E H TE x y z 2r a a Figure 4 \| Experimental set-up. An anechoic chamber with a metacrystal composed of 10 tubes in length (x axis) and 5 tubes in width (y axis), 50 tubes in total. Each tube has a length of 1 m (along z axis), an outer radius of 2 cm and an inner radius of 1.7 cm. All tubes are connected in a closed series circuit together with a water heater and a temperature stabilizer. Insert: schematic illustration of the transformation of the 2D square lattice composed of dielectric rods as the lattice constant a increase. The scheme shows the square lattice from above. The rods with dielectric permittivity e and ﬁxed radius r are embedded in air (eair ¼ 1). The material is homogeneous along the z direction and periodic along x and y. x y z E H TE x y z 2r a a b E Wavevector X Γ Figure 3 \| Bandgap diagram for the 2D square lattice of circular rods. (a) The bandgap diagram obtained from the photonic band structures calculated for 1 e 60 with steps of De ¼ 1. The rods are embedded in air, r/a ¼ 0.25, and TE polarization is considered. The Bragg and Mie gaps obtained from the band structure calculations are marked by dark green. (b,c) The photonic band structure for the 2D square lattice of rods with e ¼ 60 for TE and TM polarization, respectively, for the G-X scan of the wave vector k. Figure 4 \| Experimental set-up. An anechoic chamber with a metacrystal composed of 10 tubes in length (x axis) and 5 tubes in width (y axis), 50 tubes in total. Each tube has a length of 1 m (along z axis), an outer radius of 2 cm and an inner radius of 1.7 cm. Results A i Armed with the results of our calculations, we verify our concept experimentally by engineering a metacrystal composed of plastic circular tubes ﬁlled with water and forming a 2D square lattice with variable lattice constant a (Fig. 4). The structure of the metacrystal enables us to perform two types of experiments according to the r/a- and e-scenarios. The r/a-scenario was realized using a special construction of the metacrystal that makes it possible to change the distance between tubes synchronously in x and y directions keeping the 2D square symmetry of the lattice (Fig. 4). For the e-scenario, we employ the advantages of strong temperature dependence of the dielectric permittivity of water that is characterized by e ¼ 80 at 20 C and e ¼ 50 at 90 C in the microwave frequencies range from 1 to 6 GHz (refs 29,30). On the basis of the calculated results and the parameters of the metacrystal, we chose intervals of most interest for each scenario, namely 60 e 80 at 0:7 r=a 0:8 for the e-scenario and 0:65 r=a 1:9 at e ¼ 60 for the r/a-scenario. Results A i Two crossover scenarios for a metacrystal. Armed with the results of our calculations, we verify our concept experimentally by engineering a metacrystal composed of plastic circular tubes ﬁlled with water and forming a 2D square lattice with variable lattice constant a (Fig. 4). The structure of the metacrystal enables us to perform two types of experiments according to the r/a- and e-scenarios. The r/a-scenario was realized using a special construction of the metacrystal that makes it possible to change the distance between tubes synchronously in x and y directions keeping the 2D square symmetry of the lattice (Fig. 4). For the e-scenario, we employ the advantages of strong temperature dependence of the dielectric permittivity of water that is characterized by e ¼ 80 at 20 C and e ¼ 50 at 90 C in the microwave frequencies range from 1 to 6 GHz (refs 29,30). On the basis of the calculated results and the parameters of the metacrystal, we chose intervals of most interest for each scenario, namely 60 e 80 at 0:7 r=a 0:8 for the e-scenario and 0:65 r=a 1:9 at e ¼ 60 for the r/a-scenario. The transmission spectra of the metacrystal installed in an anechoic chamber were measured in the 1–3-GHz range. A rectangular horn antenna (TRIM 0.75–18 GHz; DR) connected to a transmitting port of the vector network analyser Agilent E8362C was used to approximate plane wave excitation. A similar horn antenna was employed as a receiver. For the electromagnetic wave incident perpendicular to the tube axis z, the two transverse polarizations appear decoupled and the vector wave problem can be reduced to two independent scalar equations. As a result, the modes of 2D PhCs can be classiﬁed as either TE modes (Ex, Ey and Hz) with the electric ﬁeld conﬁned to the x y plane and the magnetic ﬁeld polarized along the axes of the rods (Fig. 4) or TM modes (Hx, Hy and Ez) for which the magnetic ﬁeld is oriented perpendicular to the rod axis z. For the TM polarization, such structures can not show any magnetic activity31, so we only consider TE polarization. Fi 5 d t t ll t t b t th Two crossover scenarios for a metacrystal. Discussion h Figure 6a shows our main result: the PhC–MM phase diagram for the 2D square lattice of circular rods. The axes represent the dielectric permittivity e and ﬁlling ratio r/a. The phase diagram was obtained based on 50 gap maps (such as in Fig. 3a) calculated in the permittivity region 1 e 100 with the step r/a ¼ 0.01 for TE polarization. The diagram is divided into two areas, which represent the PhC and MM phases. The transition from PhC phase to MM phase can be identiﬁed by critical points, which are deﬁned by a special combination of permittivity e and ﬁlling ratio r/a. The curvature of the second dispersion curve around the G point decreases and ﬁnally becomes perfectly ﬂat along the GX path (at e ¼ 19.5, r/a ¼ 0.25, Fig. 2k). With future increase in e or change in r/a, the TE01 Mie gap completely splits from complicated coupled Mie–Bragg band becoming the lowest gap in the spectrum and the MM phase appears. Within this Mie gap, the magnetic permeability is negative and all waves in the medium are evanescent. Since all the Bragg gaps are located at higher frequencies, any diffraction losses in the vicinity of the Mie resonance are absent. The transition from the PhC phase to the MM phase is completed and the medium is classiﬁed as the MM with mo0. A phase transition is usually characterized by an order parameter that basically arises from symmetry breaking but can also be deﬁned for non-symmetry-breaking transitions32. Here The transmission spectra of the metacrystal installed in an anechoic chamber were measured in the 1–3-GHz range. A rectangular horn antenna (TRIM 0.75–18 GHz; DR) connected to a transmitting port of the vector network analyser Agilent E8362C was used to approximate plane wave excitation. A similar horn antenna was employed as a receiver. For the electromagnetic wave incident perpendicular to the tube axis z, the two transverse polarizations appear decoupled and the vector wave problem can be reduced to two independent scalar equations. As a result, the modes of 2D PhCs can be classiﬁed as either TE modes (Ex, Ey and Hz) with the electric ﬁeld conﬁned to the x y plane and the magnetic ﬁeld polarized along the axes of the rods (Fig. 4) or TM modes (Hx, Hy and Ez) for which the magnetic ﬁeld is oriented perpendicular to the rod axis z. Results A i All tubes are connected in a closed series circuit together with a water heater and a temperature stabilizer. Insert: schematic illustration of the transformation of the 2D square lattice composed of dielectric rods as the lattice constant a increase. The scheme shows the square lattice from above. The rods with dielectric permittivity e and ﬁxed radius r are embedded in air (eair ¼ 1). The material is homogeneous along the z direction and periodic along x and y. Figure 4 \| Experimental set-up. An anechoic chamber with a metacrystal composed of 10 tubes in length (x axis) and 5 tubes in width (y axis), 50 tubes in total. Each tube has a length of 1 m (along z axis), an outer radius of 2 cm and an inner radius of 1.7 cm. All tubes are connected in a closed series circuit together with a water heater and a temperature stabilizer. Insert: schematic illustration of the transformation of the 2D square lattice composed of dielectric rods as the lattice constant a increase. The scheme shows the square lattice from above. The rods with dielectric permittivity e and ﬁxed radius r are embedded in air (eair ¼ 1). The material is homogeneous along the z direction and periodic along x and y. permittivity e, the Mie resonances of single rods and corres- pondingly Mie bands of the structure demonstrate a shift from the higher to low frequencies, crossing the strongly dispersive Bragg bands. The creation of the lowest Mie gap driving the artiﬁcial magnetism is clearly seen both in the band structure and in the transmission spectra (Figs 2 and 3). e dependencies. For the r/a-scenario, we started our experiment from the PhC phase (r/a ¼ 0.08) and compressed the structure by decreasing the lattice constant a (Fig. 5a). The lowest Bragg gap demonstrates remarkable shift towards high-frequency region, then nearly almost disappears at r/aE0.09 when it approaches the unchanging TE01 Mie gap from the low-frequency side, and ﬁnally appears again at r/aE0.095 on the high-frequency side of the Mie gap. As a result, the lowest Bragg band and the TE01 Mie band change their positions in the energy scale inaugurating the transition into MM phase. In contrast, for the e-scenario, the Bragg gap position changes very slowly as opposed to the strong shift of the TE01 Mie band that ﬁnally leads to the same general result (Fig. 5d). NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 The calculated (b) and experimentally measured (c) transmission spectra of the metacrystal as a function of the ﬁlling ratio r/a. The calculated spectra are shown in the range 0:05 r=a 0:21, the experimental spectra are shown in the range 0:08 r=a 0:19. (d–f) e-scenario, r/a ¼ 0.08. The calculated (e) and experimentally measured (f) transmission spectra of the metacrystal as a function of the dielectric permittivity. The calculated spectra are shown in the range 30 e 94, the experimental spectra are shown in the range 62 e 94. The spectra are shifted vertically by the constant value. Photonic crystal Metamaterial 0.0 40 60 80 100 20 0.1 0.2 0.3 0.4 0.5 0.6 Ge Te Ge2Sb2Te5 H2O at 0 °C H2O at 90 °C Si Inverted structure 1 50 100 0 0.1 0.2 0.3 0.4 0.5 0 0.05 0.10 0.15 0.20 0.25 Order parameter b a r/a r/a Figure 6 \| PhC–MM phase diagram. (a) PhC–MM phase diagram for a square lattice of dielectric circular rods. TE polarization. Blue circles indicate the points where the TE01 Mie gap splits from Bragg band becoming the lowest gap in the spectrum (Fig. 2). The experimentally investigated regions are marked by red. The dielectric permittivities e of different materials are marked by horizontal lines. (b) The dependence of the order parameter Z on the dielectric permittivity e and the ﬁlling factor r/a. The order parameter is deﬁned as a difference in the energy between the TE01 Mie and Bragg gaps in the MM phase. 1 50 100 0 0.1 0.2 0.3 0.4 0.5 0 0.05 0.10 0.15 0.20 0.25 Order parameter b r/a Photonic crystal Metamaterial 0.0 40 60 80 100 20 0.1 0.2 0.3 0.4 0.5 0.6 Ge Te Ge2Sb2Te5 H2O at 0 °C H2O at 90 °C Si Inverted structure a r/a b a r/a Figure 6 \| PhC–MM phase diagram. (a) PhC–MM phase diagram for a square lattice of dielectric circular rods. TE polarization. Blue circles indicate the points where the TE01 Mie gap splits from Bragg band becoming the lowest gap in the spectrum (Fig. 2). The experimentally investigated regions are marked by red. The dielectric permittivities e of different materials are marked by horizontal lines. (b) The dependence of the order parameter Z on the dielectric permittivity e and the ﬁlling factor r/a. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 r/a-scenario (=62) -scenario (r/a =0.08) Transmission (arb.units) Frequency (GHz) Frequency (GHz) Bragg Bragg Bragg TE01 TE01 TE01 TE01 TE01 1.5 2.0 2.5 1.5 1.0 2.0 1.5 2.0 2.5 1.5 1.0 2.0 Bragg Bragg Bragg Theory Experiment Theory Experiment b a c e d f Model Model Bottom: r /a =0.08 Bottom: r/a =0.05 Top: r/a =0.21 Top: r/a =0.19 Theory Experiment TE01 Theory Top: =94 Bottom: =30 Experiment Bottom: =62 Top: =72 TE01 TE01 TE01 Figure 5 \| Transmission spectra for a square lattice of circular rods. TE polarization. (a–c) r/a-scenario, e ¼ 62. The calculated (b) and experimentally measured (c) transmission spectra of the metacrystal as a function of the ﬁlling ratio r/a. The calculated spectra are shown in the range 0:05 r=a 0:21, the experimental spectra are shown in the range 0:08 r=a 0:19. (d–f) e-scenario, r/a ¼ 0.08. The calculated (e) and experimentally measured (f) transmission spectra of the metacrystal as a function of the dielectric permittivity. The calculated spectra are shown in the range 30 e 94, the experimental spectra are shown in the range 62 e 94. The spectra are shifted vertically by the constant value. Frequency (GHz) Figure 5 \| Transmission spectra for a square lattice of circular rods. TE polarization. (a Figure 5 \| Transmission spectra for a square lattice of circular rods. TE polarization. (a–c) r/a-scenario, e ¼ 62. The Figure 5 \| Transmission spectra for a square lattice of circular rods. TE polarization. (a–c) r/a-scenario, e ¼ 62. The calculated (b) and experimentally measured (c) transmission spectra of the metacrystal as a function of the ﬁlling ratio r/a. The calculated spectra are shown in the range 0:05 r=a 0:21, the experimental spectra are shown in the range 0:08 r=a 0:19. (d–f) e-scenario, r/a ¼ 0.08. The calculated (e) and experimentally measured (f) transmission spectra of the metacrystal as a function of the dielectric permittivity. The calculated spectra are shown in the range 30 e 94, the experimental spectra are shown in the range 62 e 94. The spectra are shifted vertically by the constant value. Figure 5 \| Transmission spectra for a square lattice of circular rods. TE polarization. (a–c) r/a-scenario, e ¼ 62. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 The order parameter is deﬁned as a difference in the energy between the TE01 Mie and Bragg gaps in the MM phase. Identiﬁcation of candidates for dielectric low-loss materials is critical for designing a practical MM for applications in different ranges of the electromagnetic spectrum. As an example, in Fig. 6a, we mark the regions of the MM phase for water in the microwave range (region 55 e 87)31, the chalcogenide glass Ge2Sb2Te5 (eE30)33 and tellurium (eE25)34 in infrared. Thus, the well-studied 2D square lattice of germanium (eE16) and silicon (eE13)34 rods has only PhC phase. for the unusual phase transition PhC-MM, we deﬁne the order parameter Z as a measure of splitting in the energy scale between the TE01 Mie and Bragg gaps. It is zero in the PhC phase and nonzero in the MM phase. Figure 6b presents the order parameter depending on both external variables e and r/a. p p g Now, we review some features of the PhC–MM phase diagram. At low ﬁlling ratio r/ao0.05, the concentration of the completely isolated rods in the lattice is too small for achieving negative average permeability at reasonable values of e. In contrast, when distance between rods become very small (r/a40.45), the inter-rod interaction become strong and the hopping of the Mie photons from one rod to another dominates. This means that Mie resonances are not localized anymore and they ﬁnally exhibit the character of propagating Bloch waves. All requirements for the appearance of the MM phase are broken and the PhC phase extends its region of existence to very high e. We notice that for the metacrystals with a small lattice constant a, the frequency of the Mie resonance is increasing. Therefore, there is no transition to the MM phase in this limit. Discussion h For the TM polarization, such structures can not show any magnetic activity31, so we only consider TE polarization. Figure 5 demonstrates excellent agreement between the measured and calculated transmission spectra both for r/a and NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications References 1. Joannopoulos, J. D., Johnson, S. G., Winn, J. N. & Meade, R. D. Photonic Crystals: Molding the Flow of Light 2nd edn (Princeton Univ. Press, 2008). 1. Joannopoulos, J. D., Johnson, S. G., Winn, J. N. & Meade, R. D. Photonic Crystals: Molding the Flow of Light 2nd edn (Princeton Univ Press 2008) 27. Rybin, M. V. et al. Mie scattering as a cascade of Fano resonances. Opt. Express 21, 30107–30113 (2013). 2. Limonov, M. F. & De La Rue, R. M. (eds) Optical Properties of Photonic Structures: Interplay of Order and Disorder (CRC Press, Taylor & Francis Group, 2012). 28. Johnson, S. G. & Joannopoulos, J. D. 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Mie resonance-based dielectric metamaterials. Mater. Today 12, 60–69 (2009). 18. Andryieuski, A. et al. Homogenization of resonant chiral metamaterials. Phys. Rev. B 82, 235107 (2010). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms10102 MM with a wide range of applications at infrared and visible frequencies. 25. Notomi, M. Theory of light propagation in strongly modulated photonic crystals: Refractionlike behaviour in the vicinity of the photonic band gap. Phys. Rev. B 62, 10696 (2000). 26. Rybin, M. V., Filonov, D. S., Belov, P. A., Kivshar, Y. S. & Limonov, M. F. Switching from visibility to invisibility via Fano resonances: theory and experiment. Sci. Rep. 5, 8774 (2015). Conclusion We have studied a metacrystal composed of dielectric circular rods and introduced the concept of a phase diagram in the permittivity-ﬁlling ratio plane. The boundaries of the MM phase have been obtained theoretically and conﬁrmed experimentally. On the basis of the proposed approach, one can obtain different PhC–MM phase diagrams by altering the dimension, symmetry, composition, size and geometry of the structural elements within a unit cell. The underlying mechanism of the PhC–MM transition provides unique criteria for goal-oriented search of novel low-loss URE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications 5 NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. 19. O’Brien, S. & Pendry, J. B. Photonic band-gap effects and magnetic activity in dielectric composites. J. Phys. Condens. Matter 14, 4035 (2002). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 20. Yannopapas, V. & Moroz, A. Negative refractive index metamaterials from inherently non-magnetic materials for deep infrared to terahertz frequency ranges. J. Phys. Condens. Matter 17, 3717 (2005). How to cite this article: Rybin, M. V. et al. Phase diagram for the transition from photonic crystals to dielectric metamaterials. Nat. Commun. 6:10102 doi: 10.1038/ncomms10102 (2015). How to cite this article: Rybin, M. V. et al. Phase diagram for the transition from photonic crystals to dielectric metamaterials. Nat. Commun. 6:10102 doi: 10.1038/ncomms10102 (2015). g y 21. Wheeler, M. S., Aitchison, J. S. & Mojahedi, M. Three-dimensional array of dielectric spheres with an isotropic negative permeability at infrared frequencies. Phys. Rev. B 72, 193103 (2005). 22. Schuller, J. A., Zia, R., Taubner, T. & Brongersma, M. L. Dielectric metamaterials based on electric and magnetic resonances of silicon carbide particles. Phys. Rev. Lett. 99, 107401 (2007). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 23. Vynck, K. et al. All-dielectric rod-type metamaterials at optical frequencies. Phys. Rev. Lett. 102, 133901 (2009). 24. Foteinopoulou, S. Photonic crystals as metamaterials. Physica B 407, 4056 (2012). 24. Foteinopoulou, S. Photonic crystals as metamaterials. Physica B 407, 4056 (2012). NATURE COMMUNICATIONS \| 6:10102 \| DOI: 10.1038/ncomms10102 \| www.nature.com/naturecommunications 6
https://openalex.org/W2809193200	https://europepmc.org/articles/pmc6023459?pdf=render	English	null	Adverse Effects of Direct Acting Antivirals in HIV/HCV Coinfected Patients: A 4-Year Experience in Miami, Florida	Diseases	2,018	cc-by	4,617	Received: 24 May 2018; Accepted: 18 June 2018; Published: 19 June 2018 Abstract: Introduction: The new direct acting antivirals (DAA) have demonstrated low rates of adverse effects in controlled studies. However, real world-studies have disclosed emerging toxicities and drug-drug interactions in special populations. Methods: We conducted a retrospective review of HIV/HCV coinfected patients who were treated with DAA at Jackson Memorial Hospital from 2014 to 2017. Our aim was to determine the adverse effects (AE) and factors that are associated with AE in HIV/HCV individuals who are treated with DAA. Results: There were 78 coinfected patients treated with DAA. AE that were secondary to DAA were reported by 21 (26.9%) patients. The most common AE were fatigue (47.6%), gastrointestinal symptoms (38.1%), anemia (14.3%), and headache (14.3%). In comparison with the rest of the study cohort, the patients who developed AE were more often Caucasian (33.3% vs. 10.5%, p = 0.017) and were more frequently treated with PrOD/Ribavirin (9.5% vs. 0%, p = 0.018). In terms of antiretroviral therapy (ART), there was a trend towards a more frequent use of TDF/FTC + NNRTI (33.3% vs. 14%, p = 0.055). Conclusions: These ﬁndings demonstrated good tolerability of DAAs in HIV/HCV coinfected patients. More real-world studies are needed to explore the variables that are associated with AE. Keywords: adverse effects; Hepatitis C; HIV; direct acting antivirals 1. Introduction The new direct acting antivirals (DAA) have revolutionized the treatment of hepatitis C, providing cure rates of 95–99% and showing a much better safety proﬁle in comparison to old interferon-based regimens [1]. Low rates of adverse effects that are secondary to DAA have been reported in controlled studies; however, real world-studies have disclosed emerging toxicities and drug-drug interactions in special populations [2,3]. There is also very limited information about the factors that are associated with the adverse effects (AE) of DAA in HIV/HCV cohorts. Here, we contribute with data about the tolerability of DAA in 4 years of experience treating HIV/HCV coinfected patients in our clinical practice. diseases diseases diseases Received: 24 May 2018; Accepted: 18 June 2018; Published: 19 June 2018 Communication Adverse Effects of Direct Acting Antivirals in HIV/HCV Coinfected Patients: A 4-Year Experience in Miami, Florida Jose Armando Gonzales Zamora ID Jose Armando Gonzales Zamora Division of Infectious Diseases, Department of Medicine, University of Miami. Miller School of Medicine, Miami, FL 33136, USA; jxg1416@med.miami.edu or jgonzales2010@hotmail.com; Tel.: +1-706-284-3510; Fax: +1-305-243-4037 Diseases 2018, 6, 51; doi:10.3390/diseases6020051 3. Results There were 78 HIV/HCV coinfected patients treated with DAA, of which 25 (32.1%) were females and 53 (67.9%) were males, with a mean age of 55.6 (SD ± 7.88) years. The majority of patients were African American (57.7%). Most patients (98.6%) had undetectable HIV viral load, and the mean CD4 count was 637.68 cells/uL (SD ± 334.35). Antiretroviral therapy was received by 96.2% of patients. Tenofovir disoproxil fumarate (TDF)/Emtricitabine (FTC) plus protease inhibitor (26.92%), TDF/FTC plus integrase inhibitor (19.23%), and TDF/FTC plus non-nucleoside reverse transcriptase inhibitor (19.23%) were the most common antiretroviral regimens. HCV Genotype 1a was the most prevalent (61%). Advanced liver disease and cirrhosis were found in 28 (35.89%) and 12 (15.4%) patients, respectively. Most individuals were treated with Ledipasvir/Sofosbuvir (71.8%) and Simeprevir/Sofosbuvir (15.4%). Other regimens that were used were Elbasvir/Grazoprevir, Paritaprevir/ritonavir/ombitasvir/dasabuvir (PROD) plus ribavirin, and Ledipasvir/Sofosbuvir plus ribavirin, with two patients in each case. PROD and Sofosbuvir/Velpatasvir were received by 1 patient in both cases. The overall rate of sustained virologic response at 12 weeks post-treatment (SVR12) was 82.1% (Table 1). Adverse effects that were secondary to DAA were reported by 21 (26.9%) patients. The most common AE were fatigue (47.6%), gastrointestinal symptoms (38.1%), anemia (14.3%), and headache (14.3%). Dyspnea was observed in only 2 patients. No serious AE were reported. No patients discontinued HCV treatment due to AE. Of the 56 patients who were treated with Ledipasvir/Sofosbuvir, 8 (14.3%) patients developed fatigue, 3 (5.4%) patients developed headache, 2 (3.6%) patients had gastrointestinal side effects, and 1 (1.8%) patient presented dyspnea. Simeprevir/sofosbuvir was received by 12 patients, of whom 4 (33.3%) developed gastrointestinal side effects, 2 (16.7%) developed fatigue, and 1 (8.3%) presented dyspnea. Only 2 patients were treated with Elbasvir/Grazoprevir and one of them developed gastrointestinal side effects. Of the 2 patients who were treated with PROD/Ribavirin, both developed anemia and one patient developed gastrointestinal side effects (Figure 1). In comparison with the rest of the study cohort, the patients who developed AE were more often Caucasian (33.3% vs. 10.5%, p = 0.017) and were treated more frequently with PrOD/Ribavirin (9.5% vs. 0%, p = 0.018). In terms of antiretroviral therapy (ART), there was a trend towards a more frequent use of TDF/FTC + NNRTI (33.3% vs. 14%, p = 0.055). 2. Material and Methods We conducted a retrospective review of HIV/HCV coinfected patients that received treatment with DAA at the Ryan White Clinic of Jackson Memorial Hospital in Miami, Florida, USA. Our aim was to evaluate the adverse effects that are secondary to DAA in HIV/HCV coinfected patients from January 2014 to December 2017, and to identify the factors associated with the DAA adverse effects. We adopted the deﬁnitions of adverse effects of the U.S. Food and Drug Administration. An adverse effect was deﬁned as any untoward medical occurrence associated with the use of a We conducted a retrospective review of HIV/HCV coinfected patients that received treatment with DAA at the Ryan White Clinic of Jackson Memorial Hospital in Miami, Florida, USA. Our aim was to evaluate the adverse effects that are secondary to DAA in HIV/HCV coinfected patients from January 2014 to December 2017, and to identify the factors associated with the DAA adverse effects. We adopted the deﬁnitions of adverse effects of the U.S. Food and Drug Administration. An adverse effect was deﬁned as any untoward medical occurrence associated with the use of a Diseases 2018, 6, 51; doi:10.3390/diseases6020051 www.mdpi.com/journal/diseases 2 of 8 Diseases 2018, 6, 51 Diseases 2018, 6, 51 drug in humans, whether or not it was considered drug-related. Severe adverse effects were deﬁned as death, any life-threatening event, hospital admission, prolonged hospitalization, persistent or signiﬁcant incapacity or substantial disruption of the ability to conduct normal life functions, congenital anomaly/birth defect, or any event considered serious based upon appropriate medical judgement [4]. For this study, we included all of the population of HIV/HCV coinfected patients treated with DAA in our clinic (78 patients). Clinical records were reviewed in order to collect demographic, clinical, laboratory, and treatment data. Patients with AE were compared to the rest of the study cohort using Chi-square for categorical variables and t-Student for continuous variables. All tests were 2-tailed, and a p value < 0.05 was considered statistically signiﬁcant. The odds ratio (OR) was calculated with a 95% conﬁdence interval (CI). SPSS version 22 statistical software (IBM Corp, Armonk, NY, USA) was used for analysis. 3. Results The higher frequency of AE did not correlate with hemoglobin, transaminases, total bilirubin, albumin, platelet count, CD4 count, cure rates, or degree of liver ﬁbrosis (Table 1). 3 of 8 Diseases 2018, 6, 51 Table 1. Clinical, laboratory, and treatment characteristics of HIV/HCV coinfected patients and a comparison by the development of DAA adverse effects. Variable HIV/HCV Patients No Adverse Effects Adverse Effects p OR (95% CI) (n = 78) (n = 57) (n = 21) Age 55.64 ± 7.88 55.19 ± 8.26 56.86 ± 6.77 0.41 - Sex (male) 53 (67.9%) 41 (71.9%) 12 (57.1%) 0.22 0.63 (0.31–1.29) Race (a) Black 45 (57.7%) 35 (61.4%) 10 (47.6%) 0.27 0.67 (0.32–1.38) (b) White 13 (16.7%) 6 (10.5%) 7 (33.3%) 0.017 2.5 (1.26–4.96) (c) Hispanic 20 (25.6%) 16 (28.1%) 4 (19.0%) 0.42 0.68 (0.26–1.79) HAART 75 (96.2%) 56 (98.2%) 19 (90.5%) 0.11 0.38 (0.16–0.93) Hemoglobin 13.72 ± 1.63 13.8 ± 1.56 13.54 ± 1.82 0.53 - AST 74.44 ± 67.471 78.12 ± 76.35 64.43 ± 32.55) 0.43 - ALT 77.46 ± 82.57 83.39 ± 94.77 61.38 ± 26.96 0.3 - Albumin 4.113 ±0.63 4.18 ± 0.63 3.94 ± 0.60 0.14 - Total bilirubin 0.96 ± 0.92 0.99 ± 0.94 0.90 ± 0.89 0.72 - Platelet count 188.92 ± 70.18 188.72 ± 69.60 189.48 ± 73.45 0.97 - CD4 count 637.68 ± 334.35 610.54 ± 312.63 711.33 ± 386.73 0.24 - CD4/CD8 0.92 ± 0.63 0.92 ± 0.625 0.94 ± 0.65 0.87 - CD4% 30.14 ± 11.26 29.65 ± 11.54 31.48 ± 10.59 0.53 - CD4 count < 500 29 (37.2%) 22 (38.6%) 7 (33.3%) 0.94 0.85 (0.39–1.85) ART regimen (a) TDF/FTC + NNRTI 15 (19.2%) 8 (14.0%) 7 (33.3%) 0.055 2.10 (1.03–4.28) (b) TDF/FTC + PI 21 (26.9%) 17 (29.8%) 4 (19.0%) 0.34 0.64 (0.24–1.68) (c) TDF/FTC + InSTI 15 (19.2%) 12 (21.1%) 3 (14.3%) 0.5 0.70 (0.24–2.07) (d) TAF + InSTI 4 (5.1%) 3 (5.3%) 1 (4.8%) 0.93 0.93 (0.16–5.26) (e) ABC/3TC + InSTI 7 (9.0%) 6 (10.5%) 1 (4.8%) 0.43 0.51 (0.08–3.23) (f) ABC/3TC + PI 4 (5.1%) 3 (5.3%) 1 (4.8%) 0.93 0.93 (0.16–5.26) (g) Other regimens 9 (11.5%) 7 (12.3%) 2 (9.5%) 0.74 0.81 (0.22–2.91) Prior Tx with IFN 22 (28.2%) 15 (26.3%) 7 (33.3%) 0.54 1.27 (0.59–2.73) Liver biopsy 33 (42.3%) 22 (38.6%) 11 (52.4%) 0.27 1.50 (0.72–3.11) Elastography 15 (19.2%) 13(22.8%) 2 (9.5%) 0.19 0.44 (0.12–1.70) 4 of 8 Diseases 2018, 6, 51 Table 1. 4. Discussion The o era 4. Discussion The overall rate of adverse effects in our study was 26.9%, which is much lower than those reported in other real-world studies. The study conducted by Hawkins et al. on HIV/HCV coinfected patients revealed that 48% of patients experienced at least one adverse event, which is similar to the findings reported by Bruno et al., who found a rate of 59.7% [5,6]. Regarding severe adverse effects, the frequency ranges from 0% to 3.2% [6]. We did not observe any severe adverse effects in our population. Additionally, treatment discontinuation that was secondary to adverse effects was not seen, which suggests good tolerability of DAA in HIV/HCV coinfected patients. This finding was in concordance to other studies, in which the development of side effects did not lead to treatment interruptions or discontinuations [5,7]. We observed a predominant use of Sofosbuvir/Ledipasvir in our center. In this group of patients, The overall rate of adverse effects in our study was 26.9%, which is much lower than those reported in other real-world studies. The study conducted by Hawkins et al. on HIV/HCV coinfected patients revealed that 48% of patients experienced at least one adverse event, which is similar to the ﬁndings reported by Bruno et al., who found a rate of 59.7% [5,6]. Regarding severe adverse effects, the frequency ranges from 0% to 3.2% [6]. We did not observe any severe adverse effects in our population. Additionally, treatment discontinuation that was secondary to adverse effects was not seen, which suggests good tolerability of DAA in HIV/HCV coinfected patients. This ﬁnding was in concordance to other studies, in which the development of side effects did not lead to treatment interruptions or discontinuations [5,7]. We observed a predominant use of Sofosbuvir/Ledipasvir in our center. In this group of patients, the most common adverse effects were fatigue, headache, and gastrointestinal symptoms. These findings were similar to the ones reported in controlled studies [8]. In our cohort, fatigue was observed in 14.3% of patients on Sofosbuvir/Ledipasvir, which stands within the range that is reported in clinical trials [9]. Regarding headache, we have found a rate of 5.4%, which is lower than the rate described in the literature (11–17%). Another important difference was the development of gastrointestinal symptoms. Our study showed a rate of 3.6%, which was lower than the frequency that was noted in controlled studies (10–16%) [8]. 3. Results Cont. Variable HIV/HCV Patients No Adverse Effects Adverse Effects p OR (95% CI) (n = 78) (n = 57) (n = 21) Genotype (a) 1a 47 (61.0%) 34 (59.6%) 13 (61.9%) 0.86 1.07 (0.50–2.23) (b) 1b 25 (32.5%) 18 (31.6%) 7 (33.3%) 0.88 1.06 (0.49–2.30) (c) Others 6 (7.7%) 5(8.8%) 1(4.8%) 0.56 0.60 (0.09–3.73) HCV10log 6.18 ± 0.76 6.137 ± 0.82 6.30 ± 0.56 0.4 - Creatinine 1.05 ± 0.38 1.09 ± 0.41 0.95 ± 0.24 0.14 - Advanced liver disease (F3, F4) 28 (35.9%) 20 (35.1%) 8 (38.1%) 0.81 1.10 (0.52–2.33) Cirrhosis 12 (15.4%) 7 (12.3%) 5 (23.8%) 0.21 1.72 (0.78–3.80) HCV treatment (a) Ledipasvir/Sofosbuvir 56 (71.8%) 44 (77.2%) 12 (57.1%) 0.08 0.52 (0.26–1.07) (b) Simeprevir/Sofosbuvir 12 (15.4%) 7 (12.3%) 5 (23.8%) 0.21 1.72 (0.78–3.80) (c) PROD/RBV 2 (2.6%) 0 (0%) 2 (9.5%) 0.018 4.00 (2.71–5.90) (d) Elbasvir/Grazoprevir 2 (2.6%) 1 (1.8%) 1 (4.8%) 0.46 1.90 (0.45–7.99) (e) Ledipasvir/Sofosbuvir + RBV 2 (2.6%) 1 (1.8%) 1 (4.8%) 0.46 1.90 (0.45–7.99) (f) Sofosbuvir + RBV 2 (2.6%) 2 (3.5%) 0 (0%) 0.39 - (g) PROD 1 (1.3%) 1 (1.8%) 0 (0%) 0.54 - (h) Sofosbuvir/Velpatasvir 1 (1.3%) 1 (1.8%) 0 (0%) 0.54 - Tx duration (12 weeks) 71 (91.0%) 53 (98.1%) 18 (90.0%) 0.11 0.38 (0.16–0.93) SVR12 (ITT) 64 (82.1%) 44 (77.2%) 20 (95.2%) 0.07 4.38 (0.64–29.94) Completed HCV Tx 73 (93.6%) 54 (94.7%) 19 (90.5%) 0.5 0.65 (0.21–2.03) Lost to follow-up 4 (5.1%) 3 (5.3%) 1 (4.8%) 0.93 0.93 (0.16–5.26) ART = antiretroviral therapy, AST = aspartate aminotransferase, ALT = alanine aminotransferase, TDF = tenofovir disoproxil fumarate, FTC = emtricitabine, NNRTI = non-nucleoside reverse transcriptase inhibitor, PI = protease inhibitor, InSTI = Integrase inhibitor, ABC = abacavir, 3TC = lamivudine, Tx = treatment, IFN = interferon, PROD = Paritaprevir/ritonavir/ombitasvir/dasabuvir, RBV = ribavirin, SVR12 = sustained virologic response at 12 weeks post-treatment, ITT = intention to treat. The p values that achieved statistical signiﬁcance (p < 0.05) are in bold. 5 of 8 5 of 8 Diseases 2018, 6, 51 Diseases 2018, 6, x Figure 1. Adverse effects of DAA by the regimen of HCV treatment. PROD = Paritaprevir/ritonavir/ombitasvir/dasabuvir, RBV = Ribavirin, GI = Gastrointestinal. 0 1 2 3 4 5 6 7 8 9 Ledipasvir/Sofosbuvir Simeprevir/Sofosbuvir PROD/RBV Elbasvir/Grazoprevir Ledipasvir/Sofosbuvir/RBV Adverse Effects by HCV Regimen Dyspnea Headache Anemia GI symptoms Fatigue Figure 1. Adverse effects of DAA by the regimen of HCV treatment. 3. Results PROD = Paritaprevir/ritonavir/ ombitasvir/dasabuvir, RBV = Ribavirin, GI = Gastrointestinal. Adverse Effects by HCV Regimen Figure 1. Adverse effects of DAA by the regimen of HCV treatment. PROD = Paritaprevir/ritonavir/ombitasvir/dasabuvir, RBV = Ribavirin, GI = Gastrointestinal. Figure 1. Adverse effects of DAA by the regimen of HCV treatment. PROD = Paritaprevir/ritonavir/ ombitasvir/dasabuvir, RBV = Ribavirin, GI = Gastrointestinal. 4. Discussion The o era 4. Discussion It is worth mentioning that newly diagnosed or worsening pulmonary arterial hypertension have been reported in case-series; however, it is difficult to establish true causality [2]. Cases of lactic acidosis have also been described, and the risk seems to increase in patients with severe liver disease [3]. No cases of pulmonary hypertension or lactic acidosis were reported in our study. Simeprevir/sofosbuvir was the second most frequent DAA regimen used in our cohort In the We observed a predominant use of Sofosbuvir/Ledipasvir in our center. In this group of patients, the most common adverse effects were fatigue, headache, and gastrointestinal symptoms. These ﬁndings were similar to the ones reported in controlled studies [8]. In our cohort, fatigue was observed in 14.3% of patients on Sofosbuvir/Ledipasvir, which stands within the range that is reported in clinical trials [9]. Regarding headache, we have found a rate of 5.4%, which is lower than the rate described in the literature (11–17%). Another important difference was the development of gastrointestinal symptoms. Our study showed a rate of 3.6%, which was lower than the frequency that was noted in controlled studies (10–16%) [8]. It is worth mentioning that newly diagnosed or worsening pulmonary arterial hypertension have been reported in case-series; however, it is difﬁcult to establish true causality [2]. Cases of lactic acidosis have also been described, and the risk seems to increase in patients with severe liver disease [3]. No cases of pulmonary hypertension or lactic acidosis were reported in our study. Simeprevir/sofosbuvir was the second most frequent DAA regimen used in our cohort. In the patients who were treated with this regimen, gastrointestinal symptoms were the leading adverse effects, reported in 33.3% of cases. Fatigue and dyspnea developed in 16.7% and 8.3% of patients, respectively. These findings are similar to those disclosed in clinical trials, in which fatigue and nausea were two of the most common side effects [9]. Dyspnea is reported infrequently in controlled Simeprevir/sofosbuvir was the second most frequent DAA regimen used in our cohort. In the patients who were treated with this regimen, gastrointestinal symptoms were the leading adverse effects, reported in 33.3% of cases. Fatigue and dyspnea developed in 16.7% and 8.3% of patients, respectively. These ﬁndings are similar to those disclosed in clinical trials, in which fatigue and nausea 6 of 8 Diseases 2018, 6, 51 were two of the most common side effects [9]. 4. Discussion The o era 4. Discussion Dyspnea is reported infrequently in controlled and real-world studies, with a rate that can be as high as 4%, which is lower than the rate found in our patients (8.3%) [10]. A relatively common adverse effect seen in controlled studies is headache, which occurs in approximately 20% of patients on Simeprevir/sofosbuvir; however, none of our patients developed headaches during their treatment with this regimen [9]. Other clinically signiﬁcant adverse effects that are reported in the literature are pruritus (14%), rash (16%), and photosensitivity (5%) [9,11]. None of these side effects was reported in our study cohort. In terms of treatment adherence, while some real-world studies have identiﬁed low adherence to antiretroviral therapy in HIV-infected patients, poor compliance to DAA has not been described as a limiting factor in the treatment of HCV in HIV/HCV coinfected patients [12]. We have found good compliance with HCV treatment in our clinical practice, with a treatment completion rate of 93.6%. Only four patients were lost to follow-up (5.1%), and one patient did not ﬁnish his treatment due to other reasons (a motor vehicle accident). We had 6 patients who completed their treatment but did not have laboratory studies at 12 weeks post treatment to assess for cure. This limitation inﬂuenced the overall HCV cure rate or SVR12 reported in our center, which was 82.1%—a percentage lower than those reported in controlled and recent real world-studies [5]. Studies comparing DAA adverse effects between HCV monoinfected and HIV/HCV coinfected patients are very limited and have shown contradictory results. The study conducted by Bruno et al. disclosed a higher rate of adverse effects in HIV/HCV patients when compared with HCV monoinfected patients (59.7% vs. 57.2%, p = 0.03) [6]. Fatigue was observed in 17% of coinfected patients and 10% of monoinfected patients—a difference that achieved statistical signiﬁcance (p = 0.003). Jaundice was also identiﬁed as an adverse effect that was more frequently developed by HIV/HCV coinfected individuals (9.7% vs. 1.3%, p < 0.0001) [6]. On the other hand, a study aimed to evaluate the safety and effectiveness of sofosbuvir/simeprevir reported a more frequent development of adverse effects in HCV monoinfected patients when compared with HIV/HCV coinfected individuals (54.2% vs. 51.7%, p = 0.04) [13]. Our center is exclusively oriented to the treatment of HIV-infected individuals; therefore, comparison with HCV monoinfected patients was not feasible. 4. Discussion The o era 4. Discussion Regarding factors associated with adverse effects, we have found a higher frequency of Caucasian ethnicity in patients that developed these events. There are no reports of this association in the literature. We evaluated the DAA regimen of these patients, looking for an association, and we did not ﬁnd any predominant regimen that could explain this ﬁnding. Although our population was small, we believe ethnicity is a factor that should be explored in bigger studies. In terms of the DAA regimen, we found a higher frequency of PROD/ribavirin use in patients that developed adverse effects. Only two patients received this regimen in our study, making it very difﬁcult to establish a real association; however, we observed that both patients developed anemia. In our opinion, it was clear that ribavirin was the culprit, because anemia is a frequent side effect of this medication that can occur in up to 35% of patients [14,15]. The overall frequency of anemia in our study was low (3.8%) and was found exclusively in patients that received ribavirin as part of their treatment. It is worth mentioning that the only absolute indication to add ribavirin to a DAA regimen is decompensated cirrhosis, as stated by current guidelines [16]. In our study, two patients who received ribavirin-containing regimens were non-cirrhotic and one patient had compensated cirrhosis. Of note, these patients were treated in 2015, when the availability of second generation DAAs was still limited in our center and before the release of the new HCV treatment guidelines [16]. Our study also revealed a trend towards a more frequent use of TDF/FTC plus non-nucleoside reverse transcriptase inhibitor (NNRTI) in patients with adverse effects (p = 0.055). Of the patients treated with TDF/FTC plus NNRTI, three were treated with ledipasvir/sofosbuvir, three were treated with simeprevir/sofosbuvir, and one was treated with Elbasvir/Grazoprevir. When treating patients with HIV/HCV coinfection, one of the main parameters to assess is drug-drug interaction. Several pharmacokinetic studies have detected higher levels of TDF in patients treated with ledipasvir, which could potentially increase the risk of nephrotoxicity [17]. In our study, we did not observe any cases 7 of 8 Diseases 2018, 6, 51 Diseases 2018, 6, 51 of acute renal failure, and the higher rate of adverse effects was only seen when TDF was given with NNRTI. We did not identify any signiﬁcant difference when TDF was used with protease or integrase inhibitors. References 1. Assoumou, S.A.; Huang, W.; Young, K.; Horsburgh, C.R.; Linas, B.P. Real-world Outcomes of Hepatitis C Treatment during the Interferon-free Era at an Urban Safety-net Hospital. J. Health Care Poor Underserved 2017, 28, 1333–1344. [CrossRef] [PubMed] 2. Renard, S.; Borentain, P.; Salaun, E.; Benhaourech, S.; Maille, B.; Darque, A.; Bregigeon, S.; Colson, P.; Laugier, D.; Gaubert, M.R.; et al. Severe Pulmonary Arterial Hypertension in Patients Treated for Hepatitis C with Sofosbuvir. Chest 2016, 149, e69–e73. [CrossRef] [PubMed] 3. Welker, M.W.; Luhne, S.; Lange, C.M.; Vermehren, J.; Farnik, H.; Herrmann, E.; Welzel, T.; Zeuzem, S.; Sarrazin, C. Lactic acidosis in patients with hepatitis C virus cirrhosis and combined ribavirin/sofosbuvir treatment. J. Hepatol. 2016, 64, 790–799. [CrossRef] [PubMed] 3. Welker, M.W.; Luhne, S.; Lange, C.M.; Vermehren, J.; Farnik, H.; Herrmann, E.; Welzel, T.; Zeuzem, S.; Sarrazin, C. Lactic acidosis in patients with hepatitis C virus cirrhosis and combined ribavirin/sofosbuvir treatment. J. Hepatol. 2016, 64, 790–799. [CrossRef] [PubMed] p 4. Code of Federal Regulations Title 21. Available online: https://www.accessdata.fda.gov/scripts/cdrh/ cfdocs/cfcfr/CFRSearch.cfm (accessed on 23 May 2018). p 4. Code of Federal Regulations Title 21. Available online: https://www.accessdata.fda.gov/scripts/cdrh/ cfdocs/cfcfr/CFRSearch.cfm (accessed on 23 May 2018). 5. Hawkins, C.; Grant, J.; Ammerman, L.R.; Palella, F.; Mclaughlin, M.; Green, R.; Mcgregor, D.; Stosor, V. High rates of hepatitis C virus (HCV) cure using direct-acting antivirals in HIV/HCV-coinfected patients: A real-world perspective. J. Antimicrob. Chemother. 2016, 71, 2642–2645. [CrossRef] [PubMed] 5. Hawkins, C.; Grant, J.; Ammerman, L.R.; Palella, F.; Mclaughlin, M.; Green, R.; Mcgregor, D.; Stosor, V. High rates of hepatitis C virus (HCV) cure using direct-acting antivirals in HIV/HCV-coinfected patients: A real-world perspective. J. Antimicrob. Chemother. 2016, 71, 2642–2645. [CrossRef] [PubMed] 6. Bruno, G.; Saracino, A.; Scudeller, L.; Fabrizio, C.; Dell’Acqua, R.; Milano, E.; Milella, M.; Ladisa, N.; Monno, L.; Angarano, G. HCV mono-infected and HIV/HCV co-infected individuals treated with direct-acting antivirals: To what extent do they differ? Int. J. Infect. Dis. 2017, 62, 64–71. [CrossRef] [PubMed] 7. Milazzo, L.; Lai, A.; Calvi, E.; Ronzi, P.; Micheli, V.; Binda, F.; Ridolfo, A.L.; Gervasoni, C.; Galli, M.; Antinori, S.; et al. Direct-acting antivirals in hepatitis C virus (HCV)-infected and HCV/HIV-coinfected patients: Real-life safety and efﬁcacy. HIV Med. 2017, 18, 284–291. [CrossRef] [PubMed] 8. Harvoni (Ledipasvir/Sofosbuvir) [Product Monograph]. Gilead Sciences Inc.: Mississauga, ON, Canada, March 2015. Available online: https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/ 205834s001lbl.pdf (accessed on 9 June 2018). 9. 4. Discussion The o era 4. Discussion In terms of interactions between simeprevir and NNRTI, this DAA is contraindicated in patients receiving efavirenz, nevirapine, or etravirine [18]. However, no drug-drug interactions are expected with rilpivirine, which is the drug used in our study. We did not observe any concomitant use of contraindicated NNRTIs with simeprevir in our patients. Other factors besides drug-drug interaction could have potentially played a role in the higher frequency of adverse effects with the concomitant use of TDF/FTC and NNRTI. 5. Conclusions These ﬁndings demonstrated good tolerability of DAAs in HIV/HCV coinfected patients who were treated in our practice. White race, treatment with PrOD/Ribavirin, and possibly the concomitant use of TDF/FTC plus NNRTI were variables that were associated with DAA adverse effects. More real-world studies are needed to explore these possible associations. Funding: This research had no external funding. Funding: This research had no external funding. Acknowledgments: APC was sponsored by MDPI. Conﬂicts of Interest: The corresponding author states that there is no conﬂict of intere Conﬂicts of Interest: The corresponding author states that there is no conﬂict of interest. References Lawitz, E.; Matusow, G.; DeJesus, E.; Yoshida, E.M.; Felizarta, F.; Ghalib, R.; Godofsky, E.; Herring, R.W.; Poleynard, G.; Sheikh, A.; et al. Simeprevir plus sofosbuvir in patients with chronic hepatitis C virus genotype 1 infection and cirrhosis: A phase 3 study (OPTIMIST-2). 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Simeprevir plus sofosbuvir, with or without ribavirin, to treat chronic infection with hepatitis C virus genotype 1 in non-responders to pegylated interferon and ribavirin and treatment-naive patients: The COSMOS randomised study. Lancet 2014, 384, 1756–1765. [CrossRef] 16. Infectious Disease Society of America (IDSA); American Association for the Study of Liver Diseases (AASLD). HCV Guidance: Recommendations for Testing, Managing, and Treating Hepatitis C. 2017. Available online: https://www.hcvguidelines.org/ (accessed on 23 May 2018). 17. MacBrayne, C.; Fierer, D.S.; Marks, K.M. Ledipasvir/sofosbuvir raises tenofovir diphosphate concentrations in red cells. References In Proceedings of the Conference of Retrovirus and Opportunistic Infections, Seattle, WA, USA, 13–16 February 2017. 18. Schlabe, S.; Rockstroh, J.K. Advances in the treatment of HIV/HCV coinfection in adults. Expert Opin. Pharmacother. 2018, 19, 49–64. [CrossRef] [PubMed] © 2018 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 (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2219206660	https://actaneurocomms.biomedcentral.com/track/pdf/10.1186/s40478-015-0268-1	English	null	The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation	Acta neuropathologica communications	2,015	cc-by	7,293	* Correspondence: lesnik@lumc.nl †Equal contributors 4Department of Clinical Genetics, K5-R, Leiden University Medical Center, PO Box 9600 2300 RC, Leiden, The Netherlands Full list of author information is available at the end of the article The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation Julie W. Rutten1, Roselin R. Klever1, Ingrid M. Hegeman2, Dana S. Poole3, Hans G. Dauwerse1,4, Ludo A. M. Broos1, Cor Breukel1, Annemieke M. Aartsma-Rus1, J. Sjef Verbeek1, Louise van der Weerd1,3, Sjoerd G. van Duinen2, Arn M. J. M. van den Maagdenberg1,5† and Saskia A. J. Lesnik Oberstein4† © 2015 Rutten et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH Open Access The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation Julie W. Rutten1, Roselin R. Klever1, Ingrid M. Hegeman2, Dana S. Poole3, Hans G. Dauwerse1,4, Ludo A. M. Broos1, Cor Breukel1, Annemieke M. Aartsma-Rus1, J. Sjef Verbeek1, Louise van der Weerd1,3, Sjoerd G. van Duinen2, Arn M. J. M. van den Maagdenberg1,5† and Saskia A. J. Lesnik Oberstein4† Rutten et al. Acta Neuropathologica Communications (2015) 3:89 DOI 10.1186/s40478-015-0268-1 RESEARCH Open Access The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation Julie W. Rutten1, Roselin R. Klever1, Ingrid M. Hegeman2, Dana S. Poole3, Hans G. Dauwerse1,4, Ludo A. M. Broos1, Cor Breukel1, Annemieke M. Aartsma-Rus1, J. Sjef Verbeek1, Louise van der Weerd1,3, Sjoerd G. van Duinen2, Arn M. J. M. van den Maagdenberg1,5† and Saskia A. J. Lesnik Oberstein4† Rutten et al. Acta Neuropathologica Communications (2015) 3:89 DOI 10.1186/s40478-015-0268-1 Rutten et al. Acta Neuropathologica Communications (2015) 3:89 DOI 10.1186/s40478-015-0268-1 RESEARCH Open Access Correspondence: lesnik@lumc.nl †Equal contributors 4Department of Clinical Genetics, K5-R, Leiden University Medical Center, PO Box 9600 2300 RC, Leiden, The Netherlands Full list of author information is available at the end of the article © 2015 Rutten et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Introduction but are not a reliable predictor of disease progression [16]. Changes in magnetic resonance diffusion histo- grams are a better predictor of disease progression, but have only been studied in symptomatic patients [16, 17]. Ideally, CADASIL therapies would be initi- ated in the pre-symptomatic disease phase, i.e. in young adults with a proven familial NOTCH3 muta- tion. Vascular NOTCH3 protein accumulation could be an interesting therapeutic biomarker for CADASIL, as increased vascular NOTCH3 staining and GOM are consistently found in skin arterioles of pre- symptomatic patients, decades before onset of stroke and cognitive decline [18, 19]. but are not a reliable predictor of disease progression [16]. Changes in magnetic resonance diffusion histo- grams are a better predictor of disease progression, but have only been studied in symptomatic patients [16, 17]. Ideally, CADASIL therapies would be initi- ated in the pre-symptomatic disease phase, i.e. in young adults with a proven familial NOTCH3 muta- tion. Vascular NOTCH3 protein accumulation could be an interesting therapeutic biomarker for CADASIL, as increased vascular NOTCH3 staining and GOM are consistently found in skin arterioles of pre- symptomatic patients, decades before onset of stroke and cognitive decline [18, 19]. Cerebral Autosomal Dominant Arteriopathy with Sub- cortical Infarcts and Leukoencephalopathy (CADASIL) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to mid-adult onset stroke and dementia [1]. CADASIL is characterized by accu- mulation of the extracellular domain of the NOTCH3 protein (NOTCH3ECD) in the media of small- to medium- sized arterioles [2]. In addition, electron dense deposits (granular osmiophilic material, GOM) are seen in close vicinity to the vascular smooth muscle cells (VSMCs) [3]. The arteriopathy is systemic but most pronounced in the brain where it leads to degeneration of VSMCs [3] and a disturbed cerebral blood flow regulation [4]. This causes recurrent ischemic strokes and cognitive decline, starting at a mean age of 45–50 years [5]. To date, there is no ther- apy to prevent or delay symptoms in CADASIL. In this study, we set out to generate a novel, trans- lational CADASIL mouse model and to develop a relevant biomarker in this model. We generated a series of human NOTCH3 transgenic mouse strains, with various expres- sion levels of mutant NOTCH3. Abstract Acta Neuropathologica Communications (2015) 3:89 Abstract Introduction: CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to toxic NOTCH3 protein accumulation in the small- to medium sized arterioles. The accumulation is systemic but most pronounced in the brain vasculature where it leads to clinical symptoms of recurrent stroke and dementia. There is no therapy for CADASIL, and therapeutic development is hampered by a lack of feasible clinical outcome measures and biomarkers, both in mouse models and in CADASIL patients. To facilitate pre-clinical therapeutic interventions for CADASIL, we aimed to develop a novel, translational CADASIL mouse model. Results: We generated transgenic mice in which we overexpressed the full length human NOTCH3 gene from a genomic construct with the archetypal c.544C > T, p.Arg182Cys mutation. The four mutant strains we generated have respective human NOTCH3 RNA expression levels of 100, 150, 200 and 350 % relative to endogenous mouse Notch3 RNA expression. Immunohistochemistry on brain sections shows characteristic vascular human NOTCH3 accumulation in all four mutant strains, with human NOTCH3 RNA expression levels correlating with age at onset and progression of NOTCH3 accumulation. This finding was the basis for developing the ‘NOTCH3 score’, a quantitative measure for the NOTCH3 accumulation load. This score proved to be a robust and sensitive method to assess the progression of NOTCH3 accumulation, and a feasible biomarker for pre-clinical therapeutic testing. Conclusions: This novel, translational CADASIL mouse model is a suitable model for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation, using the NOTCH3 score as a biomarker. Keywords: CADASIL, NOTCH3, Transgenic mouse model, Biomarker * Correspondence: lesnik@lumc.nl †Equal contributors 4Department of Clinical Genetics, K5-R, Leiden University Medical Center, PO Box 9600 2300 RC, Leiden, The Netherlands Full list of author information is available at the end of the article © 2015 Rutten et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 10 Page 2 of 10 Page 2 of 10 Rutten et al. Introduction These mice develop cere- brovascular NOTCH3 accumulation characteristic of CADASIL at an early age, and the NOTCH3 expression level correlates with both the age at onset and progression of vascular NOTCH3 accumulation. We developed a quantitative measure for the vascular NOTCH3 accumu- lation load, which we show to be a sensitive and robust biomarker for CADASIL in these mice. py p y y p NOTCH3 targeting therapies are in the pre-clinical phase of development (Rutten et al., unpublished, pa- tent no. WO 2010085151 A2). The hitherto available CADASIL mouse models have important limitations with respect to their feasibility for testing such thera- peutic strategies. Available models include transgenic models overexpressing human NOTCH3 from a cDNA construct [6–8] or rat Notch3 from a genomic construct [9], and models in which a mutation was introduced into the endogenous Notch3 gene [10, 11]. The first and often only sign of CADASIL in these models is the presence of NOTCH3 accumulation in the vasculature [12], and in all human NOTCH3 transgenic models, the NOTCH3 accu- mulation only becomes apparent at a high age [6–8]. Only the mouse model that expresses mutant rat Notch3 pro- tein from a genomic DNA construct shows early onset vascular Notch3 accumulation with subsequent develop- ment of brain parenchymal lesions [9]. However, this model is less suitable as a translational CADASIL model due to the species difference, which creates an additional hurdle in bringing therapeutic compounds to clinical tri- als. For example, this would be the case for antisense therapeutic strategies targeting mutated pre-mRNA, a therapeutic approach which is being developed for in- creasing numbers of CNS disorders [13]. Materials and methods Generation of NOTCH3 transgenic mice Generation of NOTCH3 transgenic mice For transgenesis, a 142,63 kb BAC clone was used (RP11-456 N16 BAC, Bacpac resources, Oakland, USA) (Ensemble release 59). The BAC contains the full- length human genomic NOTCH3 gene and 44 kb of up- stream and 67 kb of downstream sequence, including flanking genes SYDE1, ILVBL, EPHX3 and a part of the BRD4 gene (Fig. 1a). The c.544C > T (p.Arg182Cys) mutation was introduced using two-step Red-mediated recombination as previously described [20]. BAC con- structs were injected into fertilized C57BL/6 J Ico oocytes. Positive transgenic founder mice were identified by PCR on DNA isolated from mouse ears using human specific primers (for primer sequences see Additional file 1: Table S1). The presence of the mutation was con- firmed by direct Sanger sequencing analysis of PCR products (Fig. 1b). Five transgenic mouse strains were generated: one carrying the wild-type NOTCH3 trans- gene (tgN3WT) and four carrying the mutant NOTCH3 transgene (tgN3MUT). In each strain, integration of the BAC was confirmed by PCR analysis of NOTCH3 and the flanking genes SYDE1, ILVBL and EPHX3 (for pri- mer sequences see Additional file 1: Table S1). All transgenic mouse strains bred normally. All experi- ments described in this study were approved by the local ethical committee for animal experimentation. For therapeutic development, feasible clinical outcome measures and biomarkers are imperative, both in mouse disease models and in patients. In CADASIL patients, the variability in age at onset and progression of clinical symptoms, including the major symptoms of stroke and cognitive decline, limits their use as an outcome meas- ure in clinical trials, because of the large number of patients that would have to be included to detect a treat- ment effect within a typical trial-timeframe of 2 years [14]. White matter lesions, detected on T2 weighted brain MRI images, are present prior to the onset of clin- ical symptoms and correlate with disease severity [15], Rutten et al. Acta Neuropathologica Communications (2015) 3:89 Page 3 of 10 Fig. 1 Generation of transgenic human NOTCH3 mice. a Schematic representation of the BAC construct containing the human NOTCH3 gene and flanking regions, used for generation of tgN3WT and tgN3MUT (c.544C > T, p.Arg182Cys) mice. b Sequencing analysis of PCR products of the human NOTCH3 gene in transgenic mice confirmed the presence of the c.544C > T mutation in tgN3MUT mice. c qPCR analysis of human and mouse NOTCH3 expression in brain. Materials and methods Generation of NOTCH3 transgenic mice In strain tgN3WT, human NOTCH3 expression was comparable to endogenous mouse Notch3 expression. The four mutant strains showed human NOTCH3 expression levels of 350, 200, 150 and 100 %, as compared to endogenous mouse Notch3 expression. Endogenous mouse Notch3 expression was comparable between the transgenic mouse strains Fig. 1 Generation of transgenic human NOTCH3 mice. a Schematic representation of the BAC construct containing the human NOTCH3 gene and flanking regions, used for generation of tgN3WT and tgN3MUT (c.544C > T, p.Arg182Cys) mice. b Sequencing analysis of PCR products of the human NOTCH3 gene in transgenic mice confirmed the presence of the c.544C > T mutation in tgN3MUT mice. c qPCR analysis of human and mouse NOTCH3 expression in brain. In strain tgN3WT, human NOTCH3 expression was comparable to endogenous mouse Notch3 expression. The four mutant strains showed human NOTCH3 expression levels of 350, 200, 150 and 100 %, as compared to endogenous mouse Notch3 expression. Endogenous mouse Notch3 expression was comparable between the transgenic mouse strains NOTCH3 expression analysis in NOTCH3 transgenic mice Total RNA was extracted from a brain hemisphere using RNA-Bee (Tel-test Inc., Friendswood, USA). For RT-PCR analysis, first-strand cDNA was synthe- sized using oligo (dT) primers. RT-PCR analysis was performed with primers across the human NOTCH3 transcript (exons 2–4, exons 14–16, exons 30–32, exons 32–33, and the 3′UTR). For qPCR analysis, cDNA synthesis was performed with random hexamer primers, using the Revert Aid H Minus first strand kit (Thermo Scientific, Waltham, USA). Quantitative PCR was performed in four 10-week-old male and fe- male transgenic mice and non-transgenic littermates, using both human- (exons 7–9, 216 bps) and mouse- specific (exons 6–8, 220 bps) primers. Mouse Gapdh was used as a reference gene, and human NOTCH3 expression levels were calculated relative to endogen- ous mouse Notch3 expression levels. A possible effect of differences in primer efficiencies of human- and mouse-specific NOTCH3/Notch3 primersets was ex- cluded by LinregPCR [21]. Brain MRI of NOTCH3 transgenic mice Brain MRI was performed in 15 mice at 20 months of age; six TgN3MUT350 mice, five tgN3WT mice, and four non-transgenic littermates. Mice were anesthetised by inhalation of 2 % isoflurane in a 1:1 mixture of oxygen and air. Respiration rate was monitored with a respira- tory pad and kept between 50 and 80 respirations per minute by adjusting the isoflurane concentration. T2 weighted imaging was performed on a 7 Tesla Bruker Pharmascan using a 23 mm quadrature coil with the following parameters: TE = 12 ms, RARE factor = 8, ef- fective TE = 48 ms, TR = 4 s, 8 averages. Field-of-view = 19x19 mm, matrix = 196x196, resulting in an in-plane resolution of 97 μm. Slice thickness = 0.5 mm, with 32 slices. NOTCH3 immunohistochemistry and quantification in human material We used paraffin embedded frontal lobe brain sections from three deceased CADASIL patients (I: female age 59, p.Arg153Cys; II: female age 57, p.Arg153Cys; III: male age 70, p.Cys446Phe) and three deceased controls with no known cerebrovascular disorders (I: male age 67, II: male age 58, III: male age 53). Sections were de- waxed, rinsed with ethanol and blocked with methanol/ H2O2. After heat-induced antigen retrieval in 0.01 M cit- rate buffer pH 6, slices were washed three times with PBS, and incubated overnight at room temperature with a 1:1 cocktail of anti-NOTCH3ECD (dilution 1:500) and anti-CD31 (Dako, Glostrup, Denmark; dilution 1:50). The following day, sections were washed and incubated for 1 hour at room temperature with a 1:1 cocktail of anti-rabbit Envision/HRP (Dako) and goat anti-mouse alkaline phosphatase (Vector Laboratories, Burlingame, CA, USA; dilution 1:25). Finally, sections were sequen- tially developed with 3,3′-diaminobenzidine solution and Vector Blue (Vector laboratories). Per individual, four images were taken at a 400× magnification on a Leica IM 500 microscope and analysed using ImageJ software. The vessel area was selected manually based on a posi- tive CD31 staining. Within the vessel area, the NOTCH3 score was calculated using an intensity threshold of 100. NOTCH3 immunohistochemistry and quantification in NOTCH3 transgenic mice Vascular NOTCH3 accumulation in brain was analysed prospectively in groups of three mice, at age 4, 6, 12, 24, 52 and 82 weeks. In addition, vascular NOTCH3 ac- cumulation was assessed in heart, aorta, liver, kidney, skin and tail at age 20 months. NOTCH3 immunohis- tochemistry was performed on cryosections that were fixated in acetone and incubated overnight with an antibody directed against the human NOTCH3ECD (Novus Biologicals, Littleton, USA; dilution 1:2000). The following day, sections were incubated with peroxidase labelled polymer conjugated to anti-rabbit immunoglobu- lins (Envision kit, Dako, Glostrup, Denmark), and devel- oped using 3,3′-diaminobenzidine. Quantification of the vascular NOTCH3 accumulation load was performed on brain cryosections. Per time point (6, 24, 52 and 82 weeks), three tgN3MUT350 mice and three tgN3MUT150 mice were analysed. For each mouse, four frontal lobe brain sections were NOTCH3 immunostained simultaneously. Sec- tions were scanned using the Ultra Fast Scanner (Philips, Rutten et al. Acta Neuropathologica Communications (2015) 3:89 Rutten et al. Acta Neuropathologica Communications (2015) 3:89 Page 4 of 10 JEM-1011 electron microscope (Advanced Microscopy Techniques, Woburn, USA). JEM-1011 electron microscope (Advanced Microscopy Techniques, Woburn, USA). JEM-1011 electron microscope (Advanced Microscopy Techniques, Woburn, USA). Eindhoven, The Netherlands), from which 10 images representative for the NOTCH3 accumulation observed in that mouse were obtained. To exclude a possible bias in image selection, a second, blinded observer was asked to obtain images independently (Additional file 1: Figure S1). ImageJ analysis was performed as follows: the image was converted into an 8-bit image, and filtered using the unsharp mask filter (radius 1, mask 0.60). Next, a threshold was set to a signal intensity of 150 to deter- mine the NOTCH3-positive area. Within the NOTCH3- positive area, individual NOTCH3 particles were identified based on size and circularity (size = 0-30, circularity = 0.50-1.00). Finally, the NOTCH3 score was determined by quantifying the total area of the NOTCH3 positive parti- cles within the image. The average of the three mice per time point was plotted and used for further statistical analysis. Statistical analysis Statistical analyses were performed using Graphpad Prism. Differences in NOTCH3 score between the two mouse strains at a given time point were analysed using the unpaired student’s t-test. Differences in NOTCH3 score between time points were analysed using One- Way ANOVA and Fishers least significant difference post-hoc analysis. Differences in slope (i.e. the rate of in- crease of the NOTCH3 score) over time were analysed using linear regression. Analysis of brain parenchyma in NOTCH3 transgenic mice mice After MRI, anesthetised mice were sacrificed using car- dial perfusion with ice-cold phosphate buffered saline. One brain hemisphere was formalin fixated and paraffin embedded. Sections were stained with hematoxylin and eosin (H&E) to analyse the presence of infarctions, with Kluver Barrera Luxol fast blue to visualise myelin and with Perl’s iron to assess the presence of microbleeds. Astrogliosis was analysed using an anti-glial fibrillary acidic protein (GFAP) antibody (rabbit anti-GFAP, Dako; dilution 1:1000), which was incubated overnight at room temperature. As a secondary antibody, biotin labelled swine-anti Rabbit (Dako; dilution 1:600) was used, this was incubated 1 hour at room temperature. Finally, sections were incubated with avidin-biotin complex (Vectastain ABC-Elite Kit, Vector Lab, Burlin- game, USA) for 30 minutes at room temperature and developed in 3,3′-diaminobenzidine solution. The de- tection of macrophages and myelin was performed using the Animal Research Kit peroxidase (Dako). Bio- tinylated primary antibodies against CD68 (anti-CD68 clone KP-1, Dako; dilution 1:1000) and myelin proteoli- pid protein (anti-PLP, clone plpc-1, Serotec, Kidlington, Four mutant human NOTCH3 transgenic mouse strains with distinct NOTCH3 expression levels Four mutant human NOTCH3 transgenic mouse strains with distinct NOTCH3 expression levels Using qPCR analysis on RNA isolated from brain of 10- week old mice, we found that the four mutant NOTCH3 p.Arg182Cys transgenic mouse strains (tgN3MUT) had human NOTCH3 expression levels of 100, 150, 200 and 350 %, respectively, compared to endogenous mouse Notch3 expression (Fig. 1c). Human NOTCH3 expres- sion in the lowest expressing tgN3MUT strain, was com- parable to that in the wild-type strain (tgN3WT), i.e. ~100 %. RT-PCR analysis with multiple NOTCH3 primer sets spanning the complete transcript, showed that the complete human NOTCH3 cDNA was present in the transgenic transcript (Additional file 1: Figure S2). There was no difference in endogenous mouse Notch3 ex- pression between transgenic and non-transgenic mice (Additional file 1: Figure S3). Development of the NOTCH3 score, a quantitative biomarker for CADASIL As we observed such an early and clear age- and NOTCH3 expression level- dependent vascular NOTCH3 accumulation load, we set out to objectify this by develop- ing a quantitative measure for NOTCH3 staining. This was accomplished by capturing and measuring the surface area of CADASIL specific granular NOTCH3 deposits within brain sections using ImageJ software (Fig. 3a), which we called the ‘NOTCH3 score’. This quantification was first performed in tgN3MUT350 mice, which clearly showed that the NOTCH3 score increased with age, confirming our qualitative observations (Fig. 3b). Next, we validated the NOTCH3 score in a second mouse strain, tgN3MUT150, in which the same age-dependent increase in the NOTCH3 score was seen. At each time-point, the NOTCH3 score was lower for the tgN3MUT150 mice compared to the tgN3MUT350 mice, reflecting the correlation between NOTCH3 expression level and NOTCH3 accumula- tion load (score at age 20 months: 659 ± 51 vs. 1150 ± 107, p = 0.002) (Fig. 3c). Furthermore, progression of NOTCH3 accumulation was slower in tgN3MUT150 mice compared to tgN3MUT350 mice, as shown by a significant difference in the slope of the NOTCH3 scores between the two mouse strains (11.1 ± 0.5 vs. 6.2 ± 0.3, p = 0.002). Finally, we tested the approach in brain sections of three unrelated CADASIL patients. Measurement of the NOTCH3 accumulation load using the NOTCH3 score was technically feasible in human tissue (Fig. 3d) and showed a significantly higher NOTCH3 score in patients than in controls (score 3.81 ± 1.85 vs. 0.24 ± 0.17, p = 0.02) (Fig. 3e). Electron microscopy in NOTCH3 transgenic mice Electron microscopy in NOTCH3 transgenic mice Brain tissue was fixed in 1.5 % glutaraldehyde and 1 % paraformaldehyde in 0.1 M cacodylate buffer, post-fixed in a solution of 2 % osmium tetroxide and 2 % potassium ferrocyanide, dehydrated and embed- ded in epon 812 (LX112). After selection of areas of interest on 1 μm toluidine stained sections, ultrathin sections were cut, contrasted with 3 % uranylacetate and Reynolds lead citrate and examined with a JEOL Page 5 of 10 Page 5 of 10 Rutten et al. Acta Neuropathologica Communications (2015) 3:89 in the aorta (Fig. 2c, data not shown). Overall, the NOTCH3 accumulation observed in the extra-cerebral arterioles was less pronounced than in the brain. Electron microscopy of brain arterioles revealed char- acteristic electron dense deposits within the basement membrane (Fig. 2d) reminiscent of GOM deposits seen in CADASIL patients (Fig. 2e). Neither GOM nor increased cerebrovascular NOTCH3 staining was found in tgN3WT mice at 20 months of age (Fig. 2a). Taken together, these analyses show that transgenic human NOTCH3 p.Arg182Cys mice develop an early and progressive systemic arteriopathy which closely resembles the vascular pathology seen in CADASIL patients, with age-at-onset correlating with the re- spective levels of mutant human NOTCH3 expression. UK; dilution 1:500) were incubated overnight after heat-induced antigen retrieval in 0.01 M EDTA pH 8.0. The following day, sections were incubated with HRP- conjugated streptavidin for 30 minutes at room temperature and developed in 3,3′-diaminobenzidine solution. UK; dilution 1:500) were incubated overnight after heat-induced antigen retrieval in 0.01 M EDTA pH 8.0. The following day, sections were incubated with HRP- conjugated streptavidin for 30 minutes at room temperature and developed in 3,3′-diaminobenzidine solution. Age at onset of vascular NOTCH3 protein accumulation correlates with NOTCH3 expression levels To analyse the presence and onset of a CADASIL vascu- lar phenotype, NOTCH3 immunohistochemistry was performed on brain slices from mice between the ages of 4 weeks and 20 months. This showed that all tgN3MUT strains developed cerebrovascular NOTCH3 accumula- tion, as seen by a positive, granular NOTCH3 staining of the vessel wall (Fig. 2a), similar to that which is seen in CADASIL patients (Fig. 2b). There was a considerable difference in age at onset of positive NOTCH3 staining per mouse strain, ranging from 6 weeks in tgN3MUT350 mice to 12 months in tgN3MUT100 mice. The age at on- set directly correlated with the level of human NOTCH3 RNA expression for all four tgN3MUT strains i.e. the higher the NOTCH3 RNA expression level, the earlier the onset of NOTCH3 accumulation (Table 1). Further- more, in each mutant strain, the positive NOTCH3 im- munostaining became progressively more intense and granular with age (Fig. 2a). The individual granular NOTCH3 deposits increased not only in number, but also in size. This was most prominent in mice with the highest NOTCH3 RNA expression level (tgN3MUT350), in which the NOTCH3 protein accumulation progres- sively evolved to a vessel wall packed with intense and big granular NOTCH3 deposits at age 20 months. Char- acteristic granular NOTCH3 staining was also present in arterioles of the heart, liver, kidney, skin and tail, but not No clear brain parenchyma phenotype in tgN3MUT mice Finally, to determine whether we could correlate the NOTCH3 score to a brain phenotype in tgN3MUT mice, we performed brain MRI and histopathology in mice Rutten et al. Acta Neuropathologica Communications (2015) 3:89 Page 6 of 10 See legend on next page.) Fig. 2 (See legend on next page.) Fig. 2 (See legend on next page.) Page 7 of 10 Rutten et al. Acta Neuropathologica Communications (2015) 3:89 (See figure on previous page.) Fig. 2 Vascular NOTCH3 protein accumulation and GOM deposits in transgenic human NOTCH3 p.Arg182Cys mice. a NOTCH3 immunostaining on brain sections of human NOTCH3 transgenic mice. All four tgN3MUT mouse strains developed a characteristic granular NOTCH3 staining pattern in the brain vasculature. TgN3WT mice showed only a weak, diffuse NOTCH3 staining pattern, which did not increase with age (comparable to non-transgenic litter-mates, data not shown). Age at onset of vascular NOTCH3 protein accumulation correlates with NOTCH3 expression levels The NOTCH3 accumulation load in the tgN3MUT strains correlates well with the NOTCH3 expression level and increases with age; in tgN3MUT350 mice, first granular staining is already visible at 6 weeks of age; at 20 months of age nearly the whole vessel wall is packed with big granular NOTCH3 deposits. In the strains with a lower NOTCH3 expression level, the NOTCH3 accumulation starts at a later age and the granular deposits remain smaller. b Positive NOTCH3 staining in a brain vessel of a CADASIL patient. c NOTCH3 immunostaining of extra-cerebral arteries of 20-month-old tgN3MUT350 mice showing clear granular NOTCH3 staining in vessels of the heart, liver and skin. The aortic wall shows a diffuse and faint NOTCH3 staining pattern comparable to that seen in non-transgenic littermates, whereas the smaller vessels around the aorta do show characteristic granular NOTCH3 staining. d Electron microscopy on brain vessels from 12-month-old tgN3MUT350 mice shows characteristic electron dense deposits reminiscent of granular osmiophilic material (GOM). GOM deposits were first seen at 5–6 months of age. e Electron microscopy on brain tissue from a deceased CADASIL patient shows pathognomonic GOM deposits, adjacent to the basement membrane surrounding the VSMCs. * = granular osmiophilic material (GOM), BM = basement membrane, VSMC = vascular smooth muscle cell aged 20 months (Additional file 1, Figure S4, S5). In two of the six tgN3MUT350 mice, hyperintensities were seen on T2 weighted images, cranial to the cor- pus callosum and around the ventricles in the frontal lobe. However, similar hyperintensities were found in one non-transgenic littermate. Histopathological examination did not show any signs of astrogliosis or white matter le- sions in the mutant mice. The absence of consistent and specific brain abnormalities in tgN3MUT mice prohibited the testing of a potential correlation between NOTCH3 score and brain phenotype. be used for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation. Cerebrovascular NOTCH3 accumulation was se- lected as a potential biomarker because it was con- sistently and specifically found in tgN3MUT mice, and showed an early age at onset and clear progression. Also, NOTCH3 accumulation is a plausible surrogate marker for CADASIL because it is universally present in the cerebrovasculature of CADASIL patients and is be- lieved to play an important role in disease pathophysiology [22]. Because of the lack of consistent brain abnormalities in our mice, we were unable to correlate the NOTCH3 score to a brain phenotype. Age at onset of vascular NOTCH3 protein accumulation correlates with NOTCH3 expression levels However, an early age at onset of NOTCH3 accumulation has previously been found to be associated with the development of brain parenchymal damage in mice [9]. Moreover, age is one of the most im- portant predictors of CADASIL disease severity and pro- gression [14, 23], implicating that the age-dependent increase in NOTCH3 score is a relevant surrogate marker for disease progression. Sample size calculations we per- formed show that the NOTCH3 score is a feasible bio- marker for pre-clinical therapeutic studies, as an effect on NOTCH3 accumulation can be assessed in relatively small groups of mice. For example, treatment of 7 mice allows for the detection of a 50 % effect on the progression of the NOTCH3 score, when treating from 6 to 24 weeks of age. Discussion To facilitate the testing of pre-clinical therapeutic in- terventions for CADASIL, we generated a transla- tional, human genomic NOTCH3 transgenic mouse model with an early vascular phenotype, and devel- oped a biomarker in this model. The mutant mice re- capitulate the CADASIL vascular phenotype with early onset and progressive cerebrovascular NOTCH3 accumu- lation and GOM deposits in arterioles. The respective NOTCH3 RNA expression levels in the four mutant mouse strains correlate strongly and consistently with the age at onset and progression of NOTCH3 protein accu- mulation, with the highest expressing mouse strain devel- oping vascular NOTCH3 accumulation as early as 6 weeks of age. The quantitative biomarker we developed, the NOTCH3 score, allows for a sensitive and objective measure of NOTCH3 accumulation, which can therefore g g We found that the NOTCH3 score can also be mea- sured in the cerebrovasculature of deceased CADASIL patients. Evidently, a NOTCH3 score in brain sections is Table 1 The NOTCH3 RNA expression level correlates with the age at onset of cerebrovascular NOTCH3 protein accumulation Mouse strain NOTCH3 expression levela Age at onset NOTCH3 accumulationb tgN3MUT 350 350 % 6 weeks tgN3MUT 200 200 % 3 months tgN3MUT 150 150 % 5 months tgN3MUT 100 100 % 12 months a mRNA NOTCH3 expression levels relative to endogenous mouse Notch3 expression levels b first sign of positive, granular NOTCH3 staining in brain vessels, as determined by an experienced neuropathologist (S.v.D) Rutten et al. Acta Neuropathologica Communications (2015) 3:89 Page 8 of 10 Fig. 3 Quantitative analysis of vascular NOTCH3 protein accumulation in transgenic human NOTCH3 p.Arg182Cys mice and in brain tissue of CADASIL patients. a ImageJ processing of NOTCH3-immunostained brain sections of tgN3MUT350 mice. The images were filtered to reduce background signal and a standardised threshold was applied to determine the NOTCH3- positive area composed of individual granular NOTCH3 deposits, resulting in the NOTCH3 score. b Quantitative analysis of NOTCH3 accumulation in tgN3MUT350 mice. The NOTCH3 score shows an age-dependent increase and allows for a sensitive discrimination between age groups (One-Way ANOVA, Fishers least significant difference). c Validation of the NOTCH3 score in tgN3MUT150 mice, also showing an age-dependent increase. At each time point, the score is lower in tgN3MUT150 than in tgN3MUT350 mice (unpaired t-test), reflecting the correlation between NOTCH3 RNA expression and NOTCH3 protein accumulation. Data represent the average +/−SD of the three mice analysed per time point. Discussion d ImageJ analysis of human brain sections double stained with NOTCH3 and CD31. The vessel area was selected based on the staining with the endothelial cell marker CD31, and within this area, the NOTCH3 score was determined. e CADASIL patients show a significantly higher NOTCH3 score than age-matched controls. (unpaired t-test) Data represent the average +/−SD of three CADASIL patients and three control individuals Fig. 3 Quantitative analysis of vascular NOTCH3 protein accumulation in transgenic human NOTCH3 p.Arg182Cys mice and in brain tissue of CADASIL patients. a ImageJ processing of NOTCH3-immunostained brain sections of tgN3MUT350 mice. The images were filtered to reduce background signal and a standardised threshold was applied to determine the NOTCH3- positive area composed of individual granular NOTCH3 deposits, resulting in the NOTCH3 score. b Quantitative analysis of NOTCH3 accumulation in tgN3MUT350 mice. The NOTCH3 score shows an age-dependent increase and allows for a sensitive discrimination between age groups (One-Way ANOVA, Fishers least significant difference) c Validation of the NOTCH3 score in tgN3MUT150 mice, also showing an age-dependent increase. At each time point, the score is lower in tgN3MUT150 than in tgN3MUT350 mice (unpaired t-test), reflecting the correlation between NOTCH3 RNA expression and NOTCH3 protein accumulation. Data represent the average +/−SD of the three mice analysed per time point. d ImageJ analysis of human brain sections double stained with NOTCH3 and CD31. The vessel area was selected based on the staining with the endothelial cell marker CD31, and within this area, the NOTCH3 score was determined. e CADASIL patients show a significantly higher NOTCH3 score than age-matched controls. (unpaired t-test) Data represent the average +/−SD of three CADASIL patients and three control individuals not a feasible biomarker in clinical trials. However, vas- cular NOTCH3 accumulation has been extensively dem- onstrated in skin biopsies of CADASIL patients and is detectable decades before the onset of clinical symptoms [18, 19]. In a single family study, an age-dependent in- crease in GOM deposits in skin biopsies was found up to 50 years of age [24]. Although previous studies did not find a correlation between skin biopsy NOTCH3 im- munostaining and disease severity, these studies were limited by a qualitative assessment of the NOTCH3 staining intensity [18, 19]. Whether such a correlation can be established using our quantitative NOTCH3 score, will have to be assessed in future prospective studies. Conclusions 5. Chabriat H, Joutel A, Dichgans M, Tournier-Lasserve E, Bousser MG. Cadasil. Lancet Neurol. 2009;8:643–53. 5. Chabriat H, Joutel A, Dichgans M, Tournier-Lasserve E, Bousser MG. Cadasil. Lancet Neurol. 2009;8:643–53. In conclusion, we developed a novel, unique human NOTCH3 transgenic mouse model and a NOTCH3 score which is a robust and sensitive biomarker for CADASIL. This translational model is ideally suited for pre-clinical testing of therapeutic strategies aimed at delaying or re- versing NOTCH3 protein accumulation. 6. Arboleda-Velasquez JF, Manent J, Lee JH, Tikka S, Ospina C, Vanderburg CR, et al. PNAS Plus: Hypomorphic Notch 3 alleles link Notch signaling to ischemic cerebral small-vessel disease. Proc Natl Acad Sci U S A. 2011;108:E128–35. 7. Monet-Lepretre M, Bardot B, Lemaire B, Domenga V, Godin O, Dichgans M, et al. Distinct phenotypic and functional features of CADASIL mutations in the Notch3 ligand binding domain. Brain. 2009;132:1601–12. 7. Monet-Lepretre M, Bardot B, Lemaire B, Domenga V, Godin O, Dichgans M, et al. Distinct phenotypic and functional features of CADASIL mutations in the Notch3 ligand binding domain. Brain. 2009;132:1601–12. 8. Ruchoux MM, Domenga V, Brulin P, Maciazek J, Limol S, Tournier- Lasserve E, et al. Transgenic mice expressing mutant Notch3 develop vascular alterations characteristic of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Am J Pathol. 2003;162:329–42. Authors’ contributions JWR, AMJMvdM and SAJLO designed the study. JWR, RRK, IMH, DSP, HGD, LAMB and CB performed experiments. Generation of transgenic mice: HGD, CB, AAR, JSV, AMJMvdM and SAJLO. Molecular characterization: JWR, RRK, HD, LAMB and AMJMvdM. Pathological analysis and quantification: JWR, RRK, IMH, SGvD and SAJLO. MRI analysis: JWR, DP, LvdW and SAJLO. JWR and SAJLO wrote the first draft of the manuscript. JWR, AMJMvdM and SAJLO revised the final draft of the manuscript. All authors read and approved the final manuscript. 13. Evers MM, Toonen LJ, van Roon-Mom WM. Antisense oligonucleotides in therapy for neurodegenerative disorders. Adv Drug Deliv Rev. 2015; 87:90–103. 14. Peters N, Herzog J, Opherk C, Dichgans M. A two-year clinical follow-up study in 80 CADASIL subjects: progression patterns and implications for clinical trials. Stroke. 2004;35:1603–8. SAJLO wrote the first draft of the manuscript. JWR, AMJMvdM and SAJLO revised the final draft of the manuscript. All authors read and approved the final manuscript. 15. Dichgans M, Filippi M, Bruning R, Iannucci G, Berchtenbreiter C, Minicucci L, et al. Quantitative MRI in CADASIL: correlation with disability and cognitive performance. Neurology. 1999;52:1361–7. Competing interests NOTCH3 antisense therapies have been patented by the Leiden University Medical Center. As co-inventors on this patent HD, AAR and SAJLO are entitled to a share of potential royalties. 12. Joutel A. Pathogenesis of CADASIL: transgenic and knock-out mice to probe function and dysfunction of the mutated gene, Notch3, in the cerebrovasculature. Bioessays. 2011;33:73–80. Received: 5 November 2015 Accepted: 14 December 2015 Received: 5 November 2015 Accepted: 14 December 2015 21. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37:e45. Discussion This novel CADASIL mouse model is especially suit- able for testing therapeutic strategies for a number of reasons. The presence of the human NOTCH3 gene in our mouse model allows for testing compounds specific- ally directed at human NOTCH3, thereby avoiding an additional hurdle in the translation from pre-clinical to clinical trials. The fact that we used a genomic NOTCH3 construct allows for testing therapeutic interventions that target mutant NOTCH3 at the genomic or (pre-) Page 9 of 10 Rutten et al. Acta Neuropathologica Communications (2015) 3:89 Page 9 of 10 mRNA level. Such interventions, for example using antisense oligonucleotides to reduce or modify mutant NOTCH3 protein, are being developed in our lab (Rutten et al., unpublished, patent no. WO 2010085151 A2). Another practical advantage is that in this early onset model, treatment can be initiated at an early age. 2. Joutel A, Andreux F, Gaulis S, Domenga V, Cecillon M, Battail N, et al. The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients. J Clin Invest. 2000;105:597–605. 3. Ruchoux MM, Guerouaou D, Vandenhaute B, Pruvo JP, Vermersch P, Leys D. Systemic vascular smooth muscle cell impairment in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Acta Neuropathol. 1995;89:500–12. 4. Pfefferkorn T, von Stuckrad-Barre S, Herzog J, Gasser T, Hamann GF, Dichgans M. Reduced cerebrovascular CO(2) reactivity in CADASIL: A transcranial Doppler sonography study. Stroke. 2001;32:17–21. Additional file 1: Supplemental materials. (DOCX 2768 kb) 9. Joutel A, Monet-Lepretre M, Gosele C, Baron-Menguy C, Hammes A, Schmidt S, et al. Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease. J Clin Invest. 2010;120:433–45. Abbreviations CADASIL: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy; NOTCH3ECD: Extracellular domain of the NOTCH3 protein; GOM: Granular osmiophillic material; VSMCs: Vascular smooth muscle cells; MRI: Magnetic resonance imaging. 10. Lundkvist J, Zhu S, Hansson EM, Schweinhardt P, Miao Q, Beatus P, et al. Mice carrying a R142C Notch 3 knock-in mutation do not develop a CADASIL-like phenotype. Genesis. 2005;41:13–22. 11. Wallays G, Nuyens D, Silasi-Mansat R, Souffreau J, Callaerts-Vegh Z, Van NA, et al. Notch3 Arg170Cys knock-in mice display pathologic and clinical features of the neurovascular disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Arterioscler Thromb Vasc Biol. 2011;31:2881–8. Acknowledgements g This work was financially supported by the Netherlands Organisation for Health Research and Development (ZonMw) (JWR, AAR and SAJLO) and the Netherlands Genomics Initiative (NGI)/NOW (AMJMvdM). The authors would like to thank Jos van der Kaa for oocyte injections, Nathalie Rieff and Stephany Koelewijn for technical assistance, Sandra van Heiningen for mouse breeding, employees of the LUMC animal facility for mouse upkeep, and Peter Neeskens for electron microscopy sample preparation. This work was financially supported by the Netherlands Organisation for Health Research and Development (ZonMw) (JWR, AAR and SAJLO) and the Netherlands Genomics Initiative (NGI)/NOW (AMJMvdM). The authors would like to thank Jos van der Kaa for oocyte injections, Nathalie Rieff and Stephany Koelewijn for technical assistance, Sandra van Heiningen for mouse breeding, employees of the LUMC animal facility for mouse upkeep, and Peter Neeskens for electron microscopy sample preparation. This work was financially supported by the Netherlands Organisation for Health Research and Development (ZonMw) (JWR, AAR and SAJLO) and the Netherlands Genomics Initiative (NGI)/NOW (AMJMvdM). The authors would like to thank Jos van der Kaa for oocyte injections, Nathalie Rieff and 16. Holtmannspotter M, Peters N, Opherk C, Martin D, Herzog J, Bruckmann H, et al. Diffusion magnetic resonance histograms as a surrogate marker and predictor of disease progression in CADASIL: a two-year follow-up study. Stroke. 2005;36:2559–65. Stephany Koelewijn for technical assistance, Sandra van Heiningen for mouse breeding, employees of the LUMC animal facility for mouse upkeep, and Peter Neeskens for electron microscopy sample preparation. 17. Molko N, Pappata S, Mangin JF, Poupon F, LeBihan D, Bousser MG, et al. Monitoring disease progression in CADASIL with diffusion magnetic resonance imaging: a study with whole brain histogram analysis. Stroke. 2002;33:2902–8. 1. Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, et al. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature. 1996;383:707–10. Author details 1 18. Joutel A, Favrole P, Labauge P, Chabriat H, Lescoat C, Andreux F, et al. Skin biopsy immunostaining with a Notch3 monoclonal antibody for CADASIL diagnosis. Lancet. 2001;358:2049–51. 1Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. 2Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands. 3Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. 4Department of Clinical Genetics, K5-R, Leiden University Medical Center, PO Box 9600 2300 RC, Leiden, The Netherlands. 5Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands. 19. Lesnik Oberstein SA, van Duinen SG, van den Boom R, Maat-Schieman ML, van Buchem MA, van Houwelingen HC, et al. Evaluation of diagnostic NOTCH3 immunostaining in CADASIL. Acta Neuropathol. 2003;106:107–11. 20. Tischer BK, von Einem J, Kaufer B, Osterrieder N. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques. 2006;40:191–7. 23. Dichgans M, Mayer M, Uttner I, Bruning R, Muller-Hocker J, Rungger G, et al. The phenotypic spectrum of CADASIL: clinical findings in 102 cases. Ann Neurol. 1998;44:731–9. Rutten et al. Acta Neuropathologica Communications (2015) 3:89 24. Brulin P, Godfraind C, Leteurtre E, Ruchoux MM. Morphometric analysis of ultrastructural vascular changes in CADASIL: analysis of 50 skin biopsy specimens and pathogenic implications. Acta Neuropathol. 2002;104:241–8. proteins by excess Notch3ECD: a new pathomechanism in CADASIL. Brain. 2013;136:1830–45. proteins by excess Notch3ECD: a new pathomechanism in CADASIL. Brain. 2013;136:1830–45. 23. Dichgans M, Mayer M, Uttner I, Bruning R, Muller-Hocker J, Rungger G, et al. The phenotypic spectrum of CADASIL: clinical findings in 102 cases. Ann Neurol. 1998;44:731–9. 24. Brulin P, Godfraind C, Leteurtre E, Ruchoux MM. Morphometric analysis of ultrastructural vascular changes in CADASIL: analysis of 50 skin biopsy specimens and pathogenic implications. Acta Neuropathol. 2002;104:241–8. proteins by excess Notch3ECD: a new pathomechanism in CADASIL. Brain. 2013;136:1830–45. References 1. Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, et al. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature. 1996;383:707–10. 22. Monet-Lepretre M, Haddad I, Baron-Menguy C, Fouillot-Panchal M, Riani M, Domenga-Denier V, et al. Abnormal recruitment of extracellular matrix Page 10 of 10 Rutten et al. Acta Neuropathologica Communications (2015) 3:89 • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W4381686252	https://link.springer.com/content/pdf/10.1140/epjp/s13360-023-04158-z.pdf	English	null	Evaluation of chia and flax mucilages as consolidants of paint films and as hydrogels used in the cleaning of canvases reverses: first results	The European physical journal plus	2,023	cc-by	10,142	a e-mail: marinpal@ucm.es (corresponding author) Evaluation of chia and ﬂax mucilages as consolidants of paint ﬁlms and as hydrogels used in the cleaning of canvases reverses: ﬁrst results a Prietoa , Sonia Santos Gómez , Marta Pérez-Estébanez , José Manuel De la Roja Received: 28 July 2022 / Accepted: 4 June 2023 © The Author(s) 2023 Abstract In the ﬁeld of conservation of cultural heritage, animal glues, synthetic resins, and some polysaccharides are used to consolidate pictorial layers. Meanwhile, in order to clean the obverse and the reverse of paintings, organic solvents and aqueous systems are sometimes employed in the form of gels. In this work, vegetable mucilages have been tested for both applications. This research has been carried out in several phases. Firstly, mucilages were extracted from ﬂax and chia seeds. The efﬁcacy of the obtained products was tested on two kinds of mock-ups. As consolidantes, the mucilages were applied on a board with a ﬁlm of powdery tempera painting. In the case of their use as cleaning gels, two linen cloths were prepared: one with animal glue and the other with gacha, an adhesive used in the Mediterranean Basin. In the last step, the efﬁcacy and safety of the treatments were determined through microscopic observations and FTIR-ATR measurements. Colour and gloss characterisation were also performed in the consolidated pictorial layers. According to the ﬁrst results shown in this work, ﬂax mucilage can consolidate disintegrated layers of rabbit-skin glue tempera producing minimum colour and gloss changes, whereas chia mucilage has less efﬁcacy to consolidate the tempera. Regarding the efﬁcacy of mucilages as hydrogels, both have shown promising results. With these ﬁrst results, future work will focus on the study of the long-term behaviour of vegetable mucilages through accelerated artiﬁcial ageing and on laying the foundations for their use in other conservation treatments, such as the cleaning of pictorial layers. Eur. Phys. J. Plus (2023) 138:555 https://doi.org/10.1140/epjp/s13360-023-04158-z Regular Article 1 Introduction In any case, the simplest way to differentiate them is though their biological function: gums are substances exuded by plants in response to an 12 3 555 Page 2 of 13 555 Page 2 of 13 Eur. Phys. J. Plus (2023) 138:555 external physical threat or other adverse conditions, so their function is to seal the injured area [9], whilst mucilages are part of the metabolism of the plant, the seed or the organism in general. external physical threat or other adverse conditions, so their function is to seal the injured area [9], whilst mucilages are part of the metabolism of the plant, the seed or the organism in general. Several functions are attributed to mucilages, such as retaining water to prevent desiccation of plant tissues [8]. They also constitute a reservoir of nutrients for the plant, due to its polysaccharide nature. Mucilages also favour germination because they increase the contact surface between the seed and the earth substrate, increasing water diffusion [10, 11]. For their use as thickeners in the production of hydrogels, two properties are interesting: their ability to retain water [10], and their capacity to form highly viscous solutions at low concentrations, due to the presence of branched structures which contain hydroxyl groups that can form hydrogen bonds with water [11–13]. On the other hand, mucilages can be used in the elaboration of water-based glues due to their adhesive properties [11, 12], which make them interesting as consolidants of disintegrated paint ﬁlms. In this work, the authors propose the application of some mucilages obtained from vegetal organisms, rather than synthetic resins, in the ﬁeld of conservation and restoration of cultural heritage, in accordance with the current trend to use biodegradable materials whose processing does not imply a signiﬁcant carbon footprint. Therefore, the aim of this research is to explore the possibilities of mucilages extracted from not expensive seeds, easily acquired in the market, like ﬂax and chia, (as opposed to baba de nopal or tzauhtli, whose raw materials are more difﬁcult to ﬁnd in Spain), to obtain products that could be suitable to consolidate tempera pictorial layers and to make hydrogels to remove natural adhesives in the reverse of paintings. The study of these possibilities can determine if they could constitute new and good alternatives to the products currently used for these purposes. 1 Introduction The use of mucilages in the artistic ﬁeld is not new. In the past, some pre-Columbian cultures already used certain mucilages in the creation of their art works before and during the Viceroyalty. One example of this kind of materials is tzauhtli, a mucilage obtained from the pseudobulbs of certain endemic orchids in Mexico. This product was used as an adhesive by Mexican artisans in the manufacture of feather mosaics, as a binder for certain pigments in mural paintings and in the paste to create some light sculptures, such as those popularly known as Cristos de maíz [1]. Over the years, they fell into disuse, at the beginning, due to the animal glues brought from Europe and, later on, to the appearance of synthetic adhesives [2]. In the ﬁeld of conservation–restoration of cultural heritage, synthetic polymers and animal glues have been commonly used for consolidation or cleaning purposes. However, products such as agar–agar or funori, polysaccharides obtained from algae, which are not considered part of the plant kingdom, are ﬁnding gradually more use nowadays [3]. Nevertheless, only a few examples of the use of mucilages from vegetal origin as conservation materials can be found. The adhesive known as tzauhtli has been tested recently in Mexico as consolidant for highly degraded fabrics with satisfactory results [4]. Another example is the treatment applied to Bandera Coronela del Batallón de Infantería Rey Fernando, located in the Army Museum (Toledo, Spain), in which it was used a mixture of tzauhtli, Methocel™(hydroxypropylmethylcellulose) and sorbitol, applied by impregnation with a brush (personal communication). On the other hand, the mucilage extracted from nopal, popularly known as baba de nopal, is used, especially in Mexico, as an additive in mortars in the ﬁeld of building rehabilitation [5]. Mucilages can be found in different organs of the plants (bulbs, roots, stems, leaves, and ﬂowers) and in seeds, especially in the outer tegument. They are heterogeneous polysaccharides constituted by macromolecules which consist of some monosaccharides, such as galactose, mannose, glucose and other glucid derivatives, linked by glycosidic bonds [6–8]. It is worth noting that there is some confusion between mucilages and vegetal gums since they are both polysaccharides with similar composition. Jean Bruneton recommends grouping all these plants products under the term hydrocolloids [6]. 2 Materials and methods The methodology applied in this work can be summarised in four steps: 1- Extraction of the mucilages from the seeds. 1- Extraction of the mucilages from the seeds. 2- Characterisation of the obtained products. 3- Preparation of mock-ups to test the mucilages as consolidants of pigments in tempera paintings and natural adhesives. Three mock-ups, imitating different artistic objects, were prepared. One of them (Mock-up A) was made to simulate a ﬁlm of rabbit glue tempera painting with a deﬁciency in the binder that was applied on panel. The objective for this mock-up was testing the effectiveness of chia and linseed mucilages as consolidantes to restore the cohesion of glue tempera pictorial layers, given the adhesive capacity of mucilages. The other two mock-ups were made imitating canvas paintings reverses, to which two different types of natural adhesives, widely used in the Mediterranean area, were applied: rabbit skin glue (Mock-up BI) and gacha (Mock-up BII). In this case, mucilages were used as gels due to their ability for absorbing water, to test their capacity for eliminating such adhesives. In both cases, the aim was to make a ﬁrst approximation of the necessary methodology to check their of conservation of cultural heritage, making an incipient assessment of the method of application, in the c 4- To study the mucilage´s efﬁcacy for such treatments and their safety by analysing the possible changes in colour and gloss of the surfaces, as well as the presence of rests of gacha, rabbit skin glue and mucilages after the treatments. p g g g To prepare the mock-ups, the materials summarised in Table 1 were used: To prepare the mock-ups, the materials summarised in Table 1 were used: 2.1 Equipment 2.1 Equipment To characterise the extracted mucilages and to analyse the cleaned surfaces, Fourier transform infrared spectroscopy (FTIR) with Attenuated Total Reﬂection (ATR) has been used with a Fourier transform infrared spectrometer (Thermo Scientiﬁc Nicolet 380) with a DTGS (deuterated triglycine sulphate) temperature-stabilized coated detector and equipped with an attenuated total reﬂection Table 1 Materials used in the elaboration of mock-ups and commercial brand in brackets Mock-up Consolidation: Mock-up A Aqueous cleaning gel: Mock-up BI Mock-up BII Support Plywood board (30×24 cm) Two Velázquez type linen canvases placed on 22×27 cm stretchers Ground Ground made with rabbit-skin glue (CTS) and plaster of Paris (M. Barrero) Mock-up BI: rabbit-skin glue (CTS). Mock-up BII: gacha* Pictorial layer Tempera paint, made with rabbit-skin glue (CTS) and ultramarine blue pigment (Sennelier) –– Gacha is the most common adhesive used in the Mediterranean countries to line canvas paintings. It has been prepared from colletta, wheat ﬂour (Gallo), water and Venetian turpentine (Talens). Colletta was made of animal glue (CTS), water and vinegar (Alipende) Table 1 Materials used in the elaboration of mock-ups and commercial brand in brackets Mock-up Consolidation: Mock-up A Aqueous cleaning gel: Mock-up BI Mock-up BII Support Plywood board (30×24 cm) Two Velázquez type linen canvases placed on 22×27 cm stretchers Ground Ground made with rabbit-skin glue (CTS) and plaster of Paris (M. Barrero) Mock-up BI: rabbit-skin glue (CTS). Mock-up BII: gacha Pictorial layer Tempera paint, made with rabbit-skin glue (CTS) and ultramarine blue pigment (Sennelier) –– Gacha is the most common adhesive used in the Mediterranean countries to line canvas paintings. It has been prepared from colletta, wheat ﬂour (Gallo), water and Venetian turpentine (Talens). Colletta was made of animal glue (CTS), water and vinegar (Alipende) Table 1 Materials used in the elaboration of mock-ups and commercial brand in brackets 12 3 Page 3 of 13 555 13 555 555 (2023) 138:555 Phys. J. Plus (2023) 138:555 Eur. Phys. J. Plus Table 2 Mucilage extraction procedure Table 2 Mucilage extraction procedure Extraction procedure Raw material Chia seeds Flax seeds Origin Brand San Blas. Chile Brand San Blas. Poland Proportion of seed: water (w/w) 1:50 1:40 Extraction The mixture of seeds and water was heated at 70 °C during one hour with constant stirring at 1200 rpm Then, it was introduced in an ultrasound bath for 10 min to accelerate the separation of the mucilage. After that, the mixture was ﬁltered. 2.1 Equipment The solutions were heated at 80 °C with constant agitation, at 1200 rpm, until it reduced its volume to half [16, 17] Puriﬁcation The solutions were heated at 55°C, and ethanol was added in a 1:1 volume ratio. The mixtures were shaken vigorously for 10 s and allowed to stand for approximately 20 min, whilst the mucilages began to coagulate. They were then separated by ﬁltration [18] Drying Mucilages were dried in a stove at 42°C for 24 h and then, milled using a glass mortar Final product appearance Brown and shiny powder Golden and shiny powder Flax seeds Brand San Blas. Poland 1:40 Golden and shiny powder diamond crystal accessory. The spectra were obtained in absorbance mode from 60 scans, in the range 4000–400 cm−1 at 4 cm−1 resolution and were analysed using Omnic v 7.3 and processed with Origin v 7.0. diamond crystal accessory. The spectra were obtained in absorbance mode from 60 scans, in the range 4000–400 cm−1 at 4 cm−1 resolution and were analysed using Omnic v 7.3 and processed with Origin v 7.0. y g p g To study the possible colours and gloss changes occurred after the consolidation treatment, the prepared mock-ups were studied, checking various areas before and after the application. For the colour, it was used a CM-2600d spectrophotometer from Konica- Minolta, in a wavelength range of 400–700 nm and at an interval of 10 nm. This equipment works with the standard illuminant D65, standard 10º observer, reﬂection optical geometry (d/8) and measurement area diameter of 3 mm. The data were acquired by using the CM-S100w 1.91.0002 SpectraMagic software and processed in a spreadsheet. Three colour measurements were made in each area, subsequently calculating their average value. To determine the colour variations experienced, it was used the CIE2000 formula [14]. Possible gloss changes were veriﬁed by using a TRIO NK-Glossmeter from Neurtek, making four measures in each area, choosing the 85º angular gloss. To verify the effectiveness of the mucilages in the elimination of natural adhesives from the mock-ups BI and BII, and the possible rests after the treatment, the surfaces were observed, ﬁrstly, using natural direct and raking light and, subsequently, with a digital video microscope MOD.AM4113 T-FVW from Dino- Lite, using both visible and UV lights. 3.1 Extraction of mucilages Some aspects were considered in the selection of the mucilages, such as their commercial availability and the easy reproducibility of the extraction process. To obtain mucilages from ﬂax and chia seeds, it has been used an aqueous process in all cases, employing deionised water, Some aspects were considered in the selection of the mucilages, such as their commercial availability and the easy reproducibility of the extraction process. To obtain mucilages from ﬂax and chia seeds, it has been used an aqueous process in all cases, employing deionised water, following the method indicated in Table 2. 3.2 Characterisation of mucilages 3.2 Characterisation of mucilages To characterise the products obtained, the samples were subjected to FTIR analyses. The spectra obtained are shown in Fig. 1. They show the characteristic bands of a polysaccharide: the bands in the 950–1150 cm−1 range are attributed to glycosidic (C–O–C) bonds [17]. In this range, bands corresponding to arabinose at 1143 cm−1, mannose around 1070 cm−1 and glucose at 1030 cm−1 are also found [19, 20]. The broad band around 3274 cm−1 can be ascribed to O–H stretching vibrations [20]. Symmetrical C-OO stretching causes bands around 1410 cm−1, and the absorption at 1601 cm−1 is the result of asymmetric C-OO stretching. In the chia mucilage, small bands appear at 1730 cm−1 and 1541 cm−1, which can be attributed to the carboxylate group of uronic acid [21, 22]. Bands at 2926 and 2854 cm−1 refer to C-H absorptions; these include CH, CH2 and CH3 stretching and bending vibrations, both symmetric and asymmetric, and occasionally double O–H overlaps [17]. 12 3 555 Page 4 of 13 Eur. Phys. J. Plus (2023) 138:555 Fig. 1 FTIR-ATR spectra of the chia and ﬂax mucilages obtained 3.3 Elaboration of the mock-ups The mock-ups have been prepared taken into account in which purposes this kind of substances could be more useful in the ﬁeld of conservation of cultural heritage. Fig. 1 FTIR-ATR spectra of the chia and ﬂax mucilages obtained 3.3 Elaboration of the mock-ups 3.3 Elaboration of the mock-ups The mock-ups have been prepared taken into account in which purposes this kind of substances could be more useful in the ﬁeld of conservation of cultural heritage. The mock-ups have been prepared taken into account in which purposes this kind of substances could be more useful in the ﬁeld of conservation of cultural heritage. It is well known that one of the most important difﬁculties in conservation of old tempera paintings, especially when the medium is animal glue, either applied on canvas or wood, is to consolidate these sensitive layers. The product chosen should be effective to agglutinate the pigments, but also it must respect the colour and the brightness of the pictorial layers. The different consolidants that are usually used for this task, with better or worse results as some studies show, can be of synthetic origin, such as Paraloid® B72, Isinglass®, Aquazol® 200, or of natural origin, such as funori [23]. 3.2 Characterisation of mucilages In many cases, one of the most important problems caused by the use of some of these products is about the gloss and colour changes that they produce in the paint ﬁlms. This study tries to check whether the chosen vegetable mucilages could be a better alternative to the current products used. To do so, the authors have chosen a wooden support, in order to get a ﬂat and hard surface which eases the colour and gloss measurement, but a canvas could be also used. On the other side, the authors also wanted to know if the mucilages could be at least as useful as other products used as thickeners in the formulation of gels (like agar–agar or Laponite®) still employed to remove water adhesives of the reverses of art works. Sometimes, when a lined painting on canvas is restored, it is necessary to eliminate the canvas used to reinforce the original one and, after that, also the rests of adhesive (gacha) employed to adhere both cloths. On the other hand, a piece of cloth with rabbit-skin glue is sometimes used to patch tears in the textile support. If it is necessary to eliminate the patch for some reasons—because it is not useful anymore or even because it may be deforming the canvas—it is imperative to eliminate also the rests of the adhesive afterwards and to leave the cloth clean before restoring it. In the elimination of adhesives, it can be stated that the use of gels allows to apply controlled humidity, so that the possibility that the canvas and even the ground of the painting could be too wet, would be reduced. They can also avoid the use of a wet cotton swab, which can cause a deformation in the canvas by rubbing it. In this way, the other purpose of this work is to test if the mucilages obtained from ﬂax and chia could be suitable for these tasks. One mock-up was prepared to test the mucilages effectiveness as consolidants for rabbit-skin glue tempera painting (Mock-up A) and two more in the elaboration of hydrogels to eliminate natural adhesives (Mock-up BI and BII), as summarised in Table 1. For the purposes of these ﬁrst tests, mock-ups were prepared trying to reproduce deteriorated artworks. Since this work includes the ﬁrst results obtained, the authors have tried on mock-ups directly, without being aged. 123 3.2 Characterisation of mucilages To ﬁx the canvases to their wooden bars, staples were used. On one of them (Mock-up BI), a solution of rabbit-skin glue, hydrated in a proportion of 1:12 at 50 °C (w/w), was applied by impregnation with a brush, in two coats. For the other one, Mock-up BII, gacha was prepared according to a common recipe used in Spain. To make it, ﬁrst it was necessary to prepare the Italian colletta, for which 3 kg of strong animal glue were hydrated in 2 l of water during 24 h. Afterwards, the excess of water was removed, and this glue was heated until it was dissolved. Afterwards, the rest of the components was added (vinegar 2 l, ox gall 50 g). Once the mixture was homogeneous, it was poured into trays, 3 cm thick, and left to cool. Once it had gelled, it was cut into small prisms and laid out on a ﬂat surface to dry, turning them for several days, so they would dry properly on all sides. Once the colletta was dried, the gacha was prepared. To do so, 300 g of ﬂour were added to 1 l of water (23 °C) placed in a pot. The mixture was cooked in a bain-marie, and it was beaten with a hand blender to remove any lumps. After that, it was continuously stirred with a wooden spoon. Then, 100 g of colletta, weighed dry and then hydrated, were put to melt in a bain-marie, and 37 g of Venice turpentine were added. Then, this mixture is added to the pot with the water and ﬂour, stirring constantly. Once the gacha is prepared in this way, two coats of it were applied at 50 °C by brush impregnation over the canvas. p p y pp y p g The adhesives in both mock-ups were left to dry before testing the mucilages. The adhesives in both mock-ups were left to dry before testing the mucilages. mock-ups were left to dry before testing the mucilages. 3.4 Testing of mucilages on mock-ups: consolidants and cleaning gels The ﬁrst step was to hydrate the mucilages extracted from the seeds. To evaluate their use as consolidants, different concentrations were tested, as shown in Table 3. 3.2 Characterisation of mucilages Nevertheless, we plan to test mock-ups subjected to artiﬁcial ageing in the next steps of this research. The ﬁrst mock-up (Mock-up A) was elaborated with the intention to imitate a disintegrated tempera painting. A plywood board was used as a support, on which it was ﬁrst applied a layer of rabbit-skin glue previously hydrated in an adhesive: water ratio of 1:12 (w/w). This layer forms the link between the plywood substrate and the subsequent layers of the ground. Once the adhesive coat dried, the ground was applied, with the same glue and with gilder gypsum. To prepare the ground, 150 ml of the same solution of glue and water were mixed with enough amount of gilder gypsum using a glass rod. In order to achieve the effect of poorly bound tempera pictorial layer, the rabbit-skin glue was prepared with lower concentration than usual. Therefore, a glue concentration to 1% in water (w/w) was used to agglutinate the ultramarine blue pigment that was applied on the plywood board. 123 Eur. Phys. J. Plus (2023) 138:555 Page 5 of 13 555 555 Fig. 2 a Plywood board prepared with ground and the overlapping masking tapes adhered to prepare the paint layers, b deposit of the tempera on the board and c spreading it with a wide ﬂat spatula Fig. 2 a Plywood board prepared with ground and the overlapping masking tapes adhered to prepare the paint layers, b deposit of the tempera on the board and c spreading it with a wide ﬂat spatula Table 3 Concentration of mucilages, their viscosity and decision about the convenience of being tested as consolidants Mucilage Chia Flax Concentration of mucilages(wt.%) 2% 1% 0.5% 2% 1% 0.5% 0.2% Viscosity to be used as consolidant High Optimal Optimal High High High Optimal Tested on the mock-up NO YES YES NO NO YES YES Paint layers of around 130 µm thickness were produced by spreading the tempera between two strips of masking tape using a ﬂat spatula (Fig. 2). Finally, before the tempera was dried, the masking tape was removed, leaving as a result 24×10 cm areas of blue rabbit-skin glue tempera. On the other hand, to test the efﬁcacy of mucilages in the elimination of natural adhesives, two mock-ups were made with Velázquez type linen canvas placed on two 22×27 cm stretchers, (Mock-up BI and Mock-up BII). Table 3 Concentration of mucilages, their viscosity and decision about the convenience of being tested as consolidants Fig. 3 Swabs: a treated area with 0.5% ﬂax mucilage, b treated area with 0.2% ﬂax mucilage, c untreated area, d treated area with 1% chia mucilage and e treated area with 0.5% chia mucilage 3.2 Characterisation of mucilages J. us ( 0 3) 38:555 on placing a Japanese paper (Productos de conservación, ref. 25,501, 6 g) on the surface of ted and to apply the mucilages with a ﬂat and soft brush. The placement of the Japanese paper ge was intended to avoid the dilution of the paint during the application of the consolidant. plied consecutively. Immediately after applying the third layer, the Japanese paper was gently in the production of cleaning gels, mucilages from chia and ﬂax were ﬁrst hydrated in a 0 (w/w) during 24 h at room temperature (23 °C) to obtain a homogeneous gel. After 24 h, the The application method consisted on placing a Japanese paper (Productos de conservación, ref. 25,501, 6 g) on the surface of the tempera painting to be consolidated and to apply the mucilages with a ﬂat and soft brush. The placement of the Japanese paper between the tempera and the mucilage was intended to avoid the dilution of the paint during the application of the consolidant. Three layers of consolidant were applied consecutively. Immediately after applying the third layer, the Japanese paper was gently removed to prevent it from adhering. The application method consisted on placing a Japanese paper (Productos de conservación, ref. 25,501, 6 g) on the surface of the tempera painting to be consolidated and to apply the mucilages with a ﬂat and soft brush. The placement of the Japanese paper between the tempera and the mucilage was intended to avoid the dilution of the paint during the application of the consolidant. Three layers of consolidant were applied consecutively. Immediately after applying the third layer, the Japanese paper was gently removed to prevent it from adhering. To test the efﬁcacy of mucilages in the production of cleaning gels, mucilages from chia and ﬂax were ﬁrst hydrated in a mucilage/distilled water ratio of 1:10 (w/w) during 24 h at room temperature (23 °C) to obtain a homogeneous gel. After 24 h, the mucilages were tested, following the methodology described below. A small amount of the mucilages were placed on selected areas, and they were left to act for 10 and 20 min. Afterwards, the mucilages were removed using a soft plastic spatula. Finally, the area was left to dry. 3.2 Characterisation of mucilages The viscosity of the consolidant must be low enough to penetrate the paint ﬁlm, but not so low that it penetrates too deeply and does not remain in the thickness of the pictorial layer. Flax mucilages show higher viscosity than the chia ones hydrated to the same concentration. For this reason, ﬂax mucilage was tested also in a lower concentration (0.2%). After hydrating the mucilage for 24 h, the mixture was heated at 50 °C for approximately 10 min with a constant stirring at 600 rpm, to facilitate the dissolution of chia and ﬂax mucilages. This temperature was selected with the aim of applying the minimum heat needed to the mixture to avoid possible alterations associated with high temperature. Finally, the mucilages were allowed to cool at room temperature. In the case of the 0.5% ﬂax mucilage, one part was allowed to cool until 40 °C and the other to 23 °C (room temperature), to test the effect of the increased temperature in the consolidation process and the possible dissolution of the tempera. 12 3 555 Page 6 of 13 Eur. Phys. J. Plus (2023) 138:555 Fig. 3 Swabs: a treated area with 0.5% ﬂax mucilage, b treated area with 0.2% ﬂax mucilage, c untreated area, d treated area with 1% chia mucilage and e treated area with 0.5% chia mucilage The application method consisted on placing a Japanese paper (Productos de conservación, ref. 25,501, 6 g) on the surface of the tempera painting to be consolidated and to apply the mucilages with a ﬂat and soft brush. The placement of the Japanese paper between the tempera and the mucilage was intended to avoid the dilution of the paint during the application of the consolidant. Three layers of consolidant were applied consecutively. Immediately after applying the third layer, the Japanese paper was gently removed to prevent it from adhering. To test the efﬁcacy of mucilages in the production of cleaning gels, mucilages from chia and ﬂax were ﬁrst hydrated in a mucilage/distilled water ratio of 1:10 (w/w) during 24 h at room temperature (23 °C) to obtain a homogeneous gel. After 24 h, the mucilages were tested, following the methodology described below. A small amount of the mucilages were placed on selected areas, and they were left to act for 10 and 20 min. Afterwards, the 555 Page 6 of 13 Eur. Phys. J. Plus (2023) 138:555 u . ys. 123 Fig. 4 Traces of pictorial ﬁlm on the Japanese paper interposed when ﬂax mucilage at 0.5% was applied. The upper paper corresponds to the one applied at 40 °C and the lower to the one applied at 23 °C Fig. 5 Whitish veil formed where chia mucilage was applied at a 0,5% and b 1% 4 Results and discussion To test the efﬁcacy of mucilages as consolidants on Mock-up A, dry cotton swabs were gently rubbed on the surfaces of the paint ﬁlms before and after consolidation (Fig. 3). In the case of the ﬂax mucilage, it was able to consolidate the pictorial ﬁlm, with both concentrations tested, 0.5% and 0.2%, and with both temperatures, 23 and 40 °C, as show the nearly clean cotton swabs. In contrast, the efﬁcacy of chia as consolidant was much lower than ﬂax mucilage, since the swabs rubbed in both areas where the mucilage was applied at 1% and at 0.5% appeared still rather stained (Fig. 3). The effect of the temperature during the consolidation process was tested using ﬂax mucilage at 0.5% concentration applied at 23 and 40 °C. When the temperature is 40 °C, a high amount of pigment is attached to the Japanese paper, as can be seen in Fig. 4, whilst a substantially smaller amount of blue pigment remains in the paper after the application of both mucilages, chia (1% and 0.5%) and ﬂax (0.5% and 0.2%) at room temperature (23 °C). As a result, it can be stated that the application temperature is a factor to be considered when evaluating the suitability of the application methodology to consolidate tempera painting: the higher the temperature, the faster the tempera is dissolved, making the pigment to adhere to the paper during the consolidant application. 123 Page 7 of 13 555 Eur. Phys. J. Plus (2023) 138:555 of 13 555 555 Fig. 4 Traces of pictorial ﬁlm on the Japanese paper interposed when ﬂax mucilage at 0.5% was applied. The upper paper corresponds to the one applied at 40 °C and the lower to the one applied at 23 °C Fig. 5 Whitish veil formed where chia mucilage was applied at a 0,5% and b 1% Observation of paint ﬁlms after consolidation evidenced a whitish veil, visible to the naked eye, formed in all areas where the chia mucilage was applied at both 1 and 0.5% concentration (Fig. 5). This assessment is corroborated with the measurements of colour variation. Thus, an increase in the colour parameters of (E00 2.18±0.54; E00 2.16±0.18) and gloss to 85º (Gloss 6.4±0.5; Gloss 4.2±0.3) is experienced for the 1 and 0.5% concentrations, respectively. On the other hand, the use of ﬂax mucilage did not produce a whitish veil visible to the naked eye, and only a signiﬁcant variation in gloss occurs when applied at 0.5% (Gloss 4.9±0.5), but colour changes are negligible (E00 <1), no matter the concentration used (Fig. 6). To study the effectiveness of the mucilages as cleaning gels, Mock-ups BI and BII were analysed with FTIR-ATR and observed with direct and raking natural lights after cleaning. Reference FTIR-ATR spectra of the linen canvas support, the rabbit-skin glue, the gacha, the linen canvas support with rabbit-skin Fig. 4 Traces of pictorial ﬁlm on the Japanese paper interposed when ﬂax mucilage at 0.5% was applied. The upper paper corresponds to the one applied at 40 °C and the lower to the one applied at 23 °C Observation of paint ﬁlms after consolidation evidenced a whitish veil, visible to the naked eye, formed in all areas where the chia mucilage was applied at both 1 and 0.5% concentration (Fig. 5). This assessment is corroborated with the measurements of colour variation. Thus, an increase in the colour parameters of (E00 2.18±0.54; E00 2.16±0.18) and gloss to 85º (Gloss 6.4±0.5; Gloss 4.2±0.3) is experienced for the 1 and 0.5% concentrations, respectively. On the other hand, the use of ﬂax mucilage did not produce a whitish veil visible to the naked eye, and only a signiﬁcant variation in gloss occurs when applied at 0.5% (Gloss 4.9±0.5), but colour changes are negligible (E00 <1), no matter the concentration used (Fig. 6). Eur. Phys. J. Plus (2023) 138:555 6 a Variations of colour and b gloss with the application of the mucilages tested as consolidants Fig. 7 FTIR-ATR reference spectra a Linen canvas with gacha, b gacha, c linen canvas with rabbit-skin glue, d rabbit-skin glue and e linen canvas canvas bands corresponding to carbohydrates (1100–900 cm−1) could not be considered, since they overlap with the bands of the mucilages, chia and ﬂax (Fig. 1and Fig. 8a). Consequently, it was necessary to consider other signiﬁcant secondary bands: the band at 1335 cm−1, attributed predominantly to the CH2 wagging vibration in the rabbit-skin glue and gacha [24, 25] and the band at 1315 cm−1, assigned to C-O–H and H-C–C bending vibrations to the linen canvas [26]. Figure 8b shows these bands, normalised to the band at 1315 cm−1, of the linen canvas alone and the mock-up BI (with rabbit-skin glue) before and after being cleaned with both mucilages. Spectra of the rabbit-skin glue alone are added as reference. As can be noticed, the cleaning treatments with both ﬂax and chia mucilages produce a drastic decrease in the band at 1335 cm−1 proving their efﬁcacy in the elimination of the rabbit-skin glue. Moreover, it can be established that the chia mucilage was able to eliminate the glue slightly better, independently of the application time. Fig. 7 FTIR-ATR reference spectra a Linen canvas with gacha, b gacha, c linen canvas with rabbit-skin glue, d rabbit-skin glue and e linen canvas Fig. 7 FTIR-ATR reference spectra a Linen canvas with gacha, b gacha, c linen canvas with rabbit-skin glue, d rabbit-skin glue and e linen canvas canvas bands corresponding to carbohydrates (1100–900 cm−1) could not be considered, since they overlap with the bands of the mucilages, chia and ﬂax (Fig. 1and Fig. 8a). Consequently, it was necessary to consider other signiﬁcant secondary bands: the band at 1335 cm−1, attributed predominantly to the CH2 wagging vibration in the rabbit-skin glue and gacha [24, 25] and the band at 1315 cm−1, assigned to C-O–H and H-C–C bending vibrations to the linen canvas [26]. Figure 8b shows these bands, normalised to the band at 1315 cm−1, of the linen canvas alone and the mock-up BI (with rabbit-skin glue) before and after being cleaned with both mucilages. Spectra of the rabbit-skin glue alone are added as reference. Eur. Phys. J. Plus (2023) 138:555 To study the effectiveness of the mucilages as cleaning gels, Mock-ups BI and BII were analysed with FTIR-ATR and observed with direct and raking natural lights after cleaning. Reference FTIR-ATR spectra of the linen canvas support, the rabbit-skin glue, the gacha, the linen canvas support with rabbit-skin glue and the linen canvas support with gacha (Fig. 7), in addition to chia and ﬂax mucilages (Fig. 1), are used to evaluate the surfaces cleaned with the different hydrogels. Reference FTIR-ATR spectra of the linen canvas support, the rabbit-skin glue, the gacha, the linen canvas support with rabbit-skin glue and the linen canvas support with gacha (Fig. 7), in addition to chia and ﬂax mucilages (Fig. 1), are used to evaluate the surfaces cleaned with the different hydrogels. For the evaluation of the cleaning process, it has been assumed that a greater cleaning effectiveness will mean more intense bands corresponding to the linen canvas, whilst less cleaning means more intense bands of the rabbit-skin glue in Mock-up BI or gacha in Mock-up BII. For the evaluation of the cleaning process, it has been assumed that a greater cleaning effectiveness will mean more intense bands corresponding to the linen canvas, whilst less cleaning means more intense bands of the rabbit-skin glue in Mock-up BI or gacha in Mock-up BII. The signiﬁcant bands of both compounds which did not show strong overlap were chosen. Thus, the stron glue/gacha corresponding to the amide I group and amide II group (1627/1638 cm−1 and 1524/1539 cm− The signiﬁcant bands of both compounds which did not show strong overlap were chosen. Thus, the strong bands of the rabbit-skin glue/gacha corresponding to the amide I group and amide II group (1627/1638 cm−1 and 1524/1539 cm−1, respectively) or linen 12 3 555 Page 8 of 13 Eur. Phys. J. Plus (2023) 138:555 555 Page 8 of 13 Fig. 6 a Variations of colour and b gloss with the application of the mucilages tested as consolidants Fig. 7 FTIR-ATR reference spectra a Linen canvas with gacha, b gacha, c linen canvas with rabbit-skin glue, d rabbit-skin glue and e linen canvas canvas bands corresponding to carbohydrates (1100 900 cm−1) could not be considered since they overlap with the bands of the Fig. 6 a Variations of colour and b gloss with the application of the mucilages tested as consolidants Fig. Eur. Phys. J. Plus (2023) 138:555 As can be noticed, the cleaning treatments with both ﬂax and chia mucilages produce a drastic decrease in the band at 1335 cm−1 proving their efﬁcacy in the elimination of the rabbit-skin glue. Moreover, it can be established that the chia mucilage was able to eliminate the glue slightly better, independently of the application time. 123 123 12 Eur. Phys. J. Plus (2023) 138:555 Page 9 of 13 555 555 Fig. 8 a FTIR-ATR spectra of the rabbit-skin glue surfaces cleaned with the different hydrogels and of the references of linen canvas with rabbit glue adhesive and linen canvas, in the range of 1700–1200 cm−1; b spectra in the 1350–1300 cm−1 region ﬁtted over the 1315 cm−1 band. Fig. 8 a FTIR-ATR spectra of the rabbit-skin glue surfaces cleaned with the different hydrogels and of the references of linen canvas with rabbit glue adhesive and linen canvas, in the range of 1700–1200 cm−1; b spectra in the 1350–1300 cm−1 region ﬁtted over the 1315 cm−1 band. Fig. 9 a FTIR-ATR spectra of the gacha surfaces cleaned with the different hydrogels and of the references of linen canvas with gacha adhesive and linen canvas, in the range of 1700–1200 cm−1; b spectra in the 1350–1300 cm−1 region ﬁtted over the 1335 cm−1 band. Fig. 9 a FTIR-ATR spectra of the gacha surfaces cleaned with the different hydrogels and of the references of linen canvas with gacha adhesive and linen canvas, in the range of 1700–1200 cm−1; b spectra in the 1350–1300 cm−1 region ﬁtted over the 1335 cm−1 band. For the evaluation by FTIR-ATR of the gacha surfaces cleaned with the different hydrogels (Mock-up BII), as in the Mock-up BI, the reference spectra were used (Fig. 9a). As in the previous case, the band at 1335 cm−1 from the linen canvas with gacha was compared to the band at 1315 cm−1 of the linen canvas. In this case, the spectra were normalised to the band at 1335 cm−1 related to the presence of gacha, since the band of the support is of lower intensity due to the covering power of the gacha compared to the rabbit-skin glue, so it can be considered that the increase in intensity of the band at 1315 cm−1, related to the presence of linen canvas, implies a greater removal of gacha during the cleaning procedure. Eur. Phys. J. Plus (2023) 138:555 In this case, the application of ﬂax mucilage for 20 min produced the best result since the band of the linen canvas is more intense. Application of chia hydrogel for 20 min was also able to clean enough to see the support, whilst an application time of 10 min was not enough in any case. For the determination of possible mucilage residues, it has been considered the presence of the low intensity broad band at 1410 cm−1, common to chia and ﬂax mucilage and corresponding to symmetrical C-OO stretching. This band does not overlap with other bands of the reference patterns, although it is close to the band 1403 cm−1 of adhesive rabbit-skin glue and gacha (Fig. 10). All the FTIR-ATR spectra of the rabbit-skin glue surfaces cleaned (Mock-up BI) with the different hydrogels show a very small shoulder at 1410 cm−1, which may indicate the existence of a small amount of chia and ﬂax mucilage residues (Fig. 11a). In the For the determination of possible mucilage residues, it has been considered the presence of the low intensity broad band at 1410 cm−1, common to chia and ﬂax mucilage and corresponding to symmetrical C-OO stretching. This band does not overlap with other bands of the reference patterns, although it is close to the band 1403 cm−1 of adhesive rabbit-skin glue and gacha (Fig. 10). p g , p y 1410 cm−1, common to chia and ﬂax mucilage and corresponding to symmetrical C-OO stretching. This band does not overlap with other bands of the reference patterns, although it is close to the band 1403 cm−1 of adhesive rabbit-skin glue and gacha (Fig. 10). All the FTIR-ATR spectra of the rabbit-skin glue surfaces cleaned (Mock-up BI) with the different hydrogels show a very small shoulder at 1410 cm−1, which may indicate the existence of a small amount of chia and ﬂax mucilage residues (Fig. 11a). In the same way, the FTIR-ATR spectra of the gacha surfaces cleaned with the different hydrogels and application times (Mock-up BII) show the presence of a small shoulder at 1410 cm−1 as well, which also indicates the possible presence of chia and ﬂax mucilage residues, being slightly higher when they were applied for 10 min (Fig. 11b). Eur. Phys. J. Plus (2023) 138:555 All the FTIR-ATR spectra of the rabbit-skin glue surfaces cleaned (Mock-up BI) with the different hydrogels show a very small shoulder at 1410 cm−1, which may indicate the existence of a small amount of chia and ﬂax mucilage residues (Fig. 11a). In the same way, the FTIR-ATR spectra of the gacha surfaces cleaned with the different hydrogels and application times (Mock-up BII) show the presence of a small shoulder at 1410 cm−1 as well, which also indicates the possible presence of chia and ﬂax mucilage residues, being slightly higher when they were applied for 10 min (Fig. 11b). A visual examination of the Mock-up BI with direct light (Fig. 12) revealed that the chia applied for 20 min generated a dark halo around the area (Fig. 12 d), which was not observed after 10 min (Fig. 12 b) nor in the areas where ﬂax mucilage was applied. Under raking natural light, it could be appreciated that all the zones where the mucilages had been applied and had matt appearance, 12 3 Eur. Phys. J. Plus (2023) 138:555 555 Page 10 of 13 555 Page 10 of 13 Eur. Phys. J. Plus (2023) 138:555 Fig. 10 FTIR-ATR spectra of the chia and ﬂax extracted mucilages, linen canvas, line canvas with gacha and linen canvas with rabbit-skin glue, in the range of 1480–1380 cm.−1 Fig. 11 FTIR-ATR spectra of the surfaces with the different hydrogels in the range of 1480–1380 cm−1: a rabbit-skin glue surfaces cleaned; b gacha surfaces cleaned Fig. 10 FTIR-ATR spectra of the chia and ﬂax extracted mucilages, linen canvas, line canvas with gacha and linen canvas with rabbit-skin glue, in the range of 1480–1380 cm.−1 Fig. 10 FTIR-ATR spectra of the chia and ﬂax extracted mucilages, linen canvas, line canvas with gacha and linen canvas with rabbit-skin glue, in the range of 1480–1380 cm.−1 Fig. 11 FTIR-ATR spectra of the surfaces with the different hydrogels in the range of 1480–1380 cm−1: a rabbit-skin glue surfaces cleaned; b gacha surfaces cleaned which indicates the elimination of the adhesive, in contrast to the untreated areas, which had shiny appearance due to the presence of rabbit-skin glue (Fig. 12d1). Visual observation of the treated areas in Mock-up BII shows that the application of the hydrogels produced a change in appearance due to the elimination of the adhesive and the canvas underneath could be observed (Fig. 13). Eur. Phys. J. Plus (2023) 138:555 Images obtained with the microscope using UV light (Fig. 13) showed that, after the application of ﬂax and chia mucilages for 10 min, some clusters of gacha still remain, seen as white areas in the images, whilst when the application time increases to 20 min, both mucilages satisfactorily eliminated the gacha. However, in the case of chia, the surface of the canvas was slightly deformed (Fig. 13). Fig. 10 FTIR-ATR spectra of the chia and ﬂax extracted mucilages, linen canvas, line canvas with gacha and linen canvas with rabbit-skin glue, in the range of 1480–1380 cm.−1 Fig. 12 Final appearance of the Mock-up BI with rabbit-skin glue observed with direct light after the application of: a Flax mucilage for 10’, b chia mucilage for 10’, c ﬂax mucilage for 20’, d chia mucilage for 20’ and d1) this one under raking light 5 Conclusions It can be stated that, under the conditions tested and with the procedure used here, ﬂax mucilage does have the ability to consolidate disintegrated layers of rabbit-skin glue tempera producing minimum colour and gloss changes, whereas chia has less efﬁcacy to consolidate the tempera and produces a whitish veil visible to the naked eye. About the efﬁcacy of mucilages used as thickeners in the production of hydrogels, chia and ﬂax mucilages had the ability to gel the aqueous phase. The hydrogel with chia was able to release more of the aqueous phase after 20 min of action, allowing a greater removal of the adhesive, but with the disadvantage of producing a deformation of the canvas and a dark halo. Therefore, it should probably be used for less time than ﬂax at the concentrations tested. In all cases, it has been observed that traces of adhesives remain after cleaning. 123 123 123 Page 11 of 13 555 Eur. Phys. J. Plus (2023) 138:555 Af h f b h hi d ﬂ il f h li i i f l dh i ll f id h b d d According to the approach made with this research, the main limitation of this methodology is that the mock-ups used were not subjected to artiﬁcial ageing, so it is possible that the results may change slightly. Variables such as action times are affected, in the case of hydrogels, since it is possible that in older adhesive layers, these may be longer, as these will be more rigid, and their removal could be more difﬁcult. So, in the future, it is planned the use of aged mock-ups, to simulate old cultural heritage. After the use of both chia and ﬂax mucilages for the elimination of natural adhesives, small amounts of residues have been detected, hi h k it t t l t d tiﬁi l i t t t d t i th l t b h i f th il i According to the approach made with this research, the main limitation of this methodology is that the mock-ups used were not subjected to artiﬁcial ageing, so it is possible that the results may change slightly. Variables such as action times are affected, in the case of hydrogels, since it is possible that in older adhesive layers, these may be longer, as these will be more rigid, and their removal could be more difﬁcult. So, in the future, it is planned the use of aged mock-ups, to simulate old cultural heritage. According to the approach made with this research, the main limitation of this methodology is that the mock-ups used were not subjected to artiﬁcial ageing, so it is possible that the results may change slightly. Variables such as action times are affected, in the case of hydrogels, since it is possible that in older adhesive layers, these may be longer, as these will be more rigid, and their removal could be more difﬁcult. So, in the future, it is planned the use of aged mock-ups, to simulate old cultural heritage. After the use of both chia and ﬂax mucilages for the elimination of natural adhesives, small amounts of residues have been detected, which makes it necessary to carry out accelerated artiﬁcial aging tests to determine the long-term behaviour of the mucilages, in terms of their stability, chromatic variation and binding power. Eur. Phys. J. Plus (2023) 138:555 555 Fig. 12 Final appearance of the Mock-up BI with rabbit-skin glue observed with direct light after the application of: a Flax mucilage for 10’, b chia mucilage for 10’, c ﬂax mucilage for 20’, d chia mucilage for 20’ and d1) this one under raking light According to the approach made with this research, the main limitation of this methodology is that the mock-ups used were not subjected to artiﬁcial ageing, so it is possible that the results may change slightly. Variables such as action times are affected, in the case of hydrogels, since it is possible that in older adhesive layers, these may be longer, as these will be more rigid, and their removal could be more difﬁcult. So, in the future, it is planned the use of aged mock-ups, to simulate old cultural heritage. After the use of both chia and ﬂax mucilages for the elimination of natural adhesives, small amounts of residues have been detected, which makes it necessary to carry out accelerated artiﬁcial aging tests to determine the long-term behaviour of the mucilages, in terms of their stability, chromatic variation and binding power. These tests could show if the mucilage residues will affect some aspect of the pictorial layers or supports. mitation of this methodology is that the mock-ups used were not y change slightly. Variables such as action times are affected, in yers, these may be longer, as these will be more rigid, and their use of aged mock-ups, to simulate old cultural heritage. f natural adhesives, small amounts of residues have been detected, tests to determine the long-term behaviour of the mucilages, in hese tests could show if the mucilage residues will affect some According to the approach made with this research, the main limitation of this methodology is that the mock-ups used were not subjected to artiﬁcial ageing, so it is possible that the results may change slightly. Variables such as action times are affected, in the case of hydrogels, since it is possible that in older adhesive layers, these may be longer, as these will be more rigid, and their removal could be more difﬁcult. So, in the future, it is planned the use of aged mock-ups, to simulate old cultural heritage. Eur. Phys. J. Plus (2023) 138:555 Requests for material should be made to the corresponding authors. Eur. Phys. J. Plus (2023) 138:555 These tests could show if the mucilage residues will affect some aspect of the pictorial layers or supports. After the use of both chia and ﬂax mucilages for the elimination of natural adhesives, small amounts of residues have been detected, which makes it necessary to carry out accelerated artiﬁcial aging tests to determine the long-term behaviour of the mucilages, in terms of their stability, chromatic variation and binding power. These tests could show if the mucilage residues will affect some aspect of the pictorial layers or supports. 123 123 123 3 555 Page 12 of 13 Eur. Phys. J. Plus (2023) 138:555 Fig. 13 General image of the area of gacha Mock-up BII, where ﬂax and chia mucilages were applied and detail images under 35x, using UV light. a ﬂax and b chia, in both cases left to act for 10’ (images taken from the centre of the treated area); c ﬂax and d chia that were left to act for 20’ (images taken in the limit between the treated and the untreated areas) Fig. 13 General image of the area of gacha Mock-up BII, where ﬂax and chia mucilages were applied and detail images under 35x, using UV light. a ﬂax and b chia, in both cases left to act for 10’ (images taken from the centre of the treated area); c ﬂax and d chia that were left to act for 20’ (images taken in the limit between the treated and the untreated areas) Finally, the authors are also interested in the research of other applications of mucilages in the ﬁeld of conservation of heritage. On the one hand, as thickeners in the elaboration of hydrogels used to clean sensitive pictorial layers in different supports. On the other hand, as consolidants, using other methods to apply them and testing them on other kinds of tempera painting, such as egg or casein. Acknowledgements The authors gratefully acknowledge Lab[Mat], laboratory of materials at the Fine Arts Faculty of Complutense University of Madrid, for allowing the experimental phase to be carried out in their installations. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Data Availability Statement All data generated or analysed during this study are included in this published article. Request o the corresponding authors. Data Availability Statement All data generated or analysed during this study are included in this published article. Declarations Conﬂict of interest All authors certify that they have no afﬁliations with or involvement in any organisation or entity with any ﬁnancial interest or non-ﬁnancial interest in the subject matter or materials discussed in this manuscript. 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 123 12 123 Page 13 of 13 Eur. Phys. J. 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https://openalex.org/W3009811807	https://pub.dzne.de/record/151535/files/6904.pdf	English	null	Mirror Movements in Amyotrophic Lateral Sclerosis: A Combined Study Using Diffusion Tensor Imaging and Transcranial Magnetic Stimulation	Frontiers in neurology	2,020	cc-by	6,420	BRIEF RESEARCH REPORT published: 06 March 2020 doi: 10.3389/fneur.2020.00164 Mirror Movements in Amyotrophic Lateral Sclerosis: A Combined Study Using Diffusion Tensor Imaging and Transcranial Magnetic Stimulation Matthias Wittstock 1, Nora Wilde 1, Annette Grossmann 2, Elisabeth Kasper 3 and Stefan Teipel 3 1 Department of Neurology, University Medicine Rostock, Rostock, Germany, 2 Institute of Diagnostic and Interventional Radiology, University Medicine Rostock, Rostock, Germany, 3 DZNE, German Centre for Neurodegenerative Diseases, Rostock, Germany Objective: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder predominantly affecting the motor system. In a number of patients, mirror movements (MMs) suggest involvement of transcallosal ﬁber tracts in conjunction with upper motor neuron involvement. The aim of the study was to elucidate functional and structural alterations of callosal integrity in ALS patients with MMs. Edited by: Carlos Gómez, University of Valladolid, Spain Reviewed by: Salem Hannoun, American University of Beirut, Lebanon Francesca Trojsi, University of Campania Luigi Vanvitelli, Italy Methods: Nineteen patients with ALS displaying MMs and 20 controls underwent clinical assessment, transcranial magnetic stimulation (TMS), and diffusion tensor imaging (DTI). TBSS (tract based spatial statistics) was performed. We investigated ipsilateral silent period (iSP) as a measure of transcallosal inhibition, and diffusion changes in the corpus callosum and corticospinal tract (CST) as measure of structural integrity. Correspondence: Matthias Wittstock matthias.wittstock@ med.uni-rostock.de Correspondence: Matthias Wittstock matthias.wittstock@ med.uni-rostock.de Results: In ALS patients TMS revealed a longer mean iSP latency than controls. Twelve ALS patients (63.2%) showed loss of iSP, but none of the controls. Using region of interest analysis, fractional anisotropy (FA) values of the CST were signiﬁcantly lower in ALS patients compared with controls, but diffusion parameters of the corpus callosum did not differ between patients and controls. The lack of diffusion changes in the corpus callosum was conﬁrmed in whole brain tract based statistics, assessing FA as well as mean, radial, and axial diffusivity. There was a signiﬁcant negative correlation between resting motor threshold and FA values of the CST, but not between iSP and FA of the corpus callosum. Specialty section: This article was submitted to Applied Neuroimaging, a section of the journal Frontiers in Neurology Received: 26 November 2019 Accepted: 21 February 2020 Published: 06 March 2020 Keywords: amyotrophic lateral sclerosis, diffusion tensor imaging, transcranial magnetic stimulation, mirror movements, corpus callosum Citation: Conclusion: In conclusion the study failed to show microstructural changes in the corpus callosum in conjunction with MMs. One possible reason may be that functional disturbance of transcallosal pathways precede microstructural changes in the corpus callosum. Wittstock M, Wilde N, Grossmann A, Kasper E and Teipel S (2020) Mirror Movements in Amyotrophic Lateral Sclerosis: A Combined Study Using Diffusion Tensor Imaging and Transcranial Magnetic Stimulation. Front. Neurol. 11:164. doi: 10.3389/fneur.2020.00164 Keywords: amyotrophic lateral sclerosis, diffusion tensor imaging, transcranial magnetic stimulation, mirror movements, corpus callosum March 2020 \| Volume 11 \| Article 164 Frontiers in Neurology \| www.frontiersin.org 1 Mirror Movements in ALS Wittstock et al. TABLE 1 \| Demographic and clinical data of all study subjects. ALS (N = 19) Controls (N = 20) p Mean (SD) Range Mean (SD) Range Age (years) 62.6 (10.5) 42–74 67.1 (5.4) 60–75 0.143 Gender (N male/ female) 11/8 5/5 0.714 ALSFRS-R 38.5 (4.7) NA UMNB 9.4 (4.2) NA MOCA 24.4 (3.9) 18–29 27.6 (0.7) 27–29 0.006 Disease duration (month) 34.8 (34.8) NA El Escorial (N NA/poss/prob/def) 3/4/8/4 NA Phenotype (N class/UMN/LMN) 11/3/4 NA ALS, amyotrophic lateral sclerosis; SD, standard deviation; ALSFRS-R, amyotrophic lateral sclerosis functional rating scale—revised; UMNB, upper motor neuron burden; MOCA, Montreal Cognitive Assessment; El Escorial, El Escorial criteria; NA, not applicable; poss, possible; prob, probable; def, deﬁnite; N, number, UMN, upper motor neuron; LMN, lower motor neuron; p, signiﬁcance of Student’s t-test. INTRODUCTION TABLE 1 \| Demographic and clinical data of all study subjects. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease mainly characterized by a motor syndrome with variable expression of lower (LMN) and upper (UMN) motor neuron dysfunction. El Escorial criteria (1) require UMN and LMN involvement in one or more body regions for making the diagnosis of deﬁnitive ALS. UMN signs may be spasticity, enhanced or preserved tendon reﬂexes and extensor plantar response. Mirror movements (MMs) have repeatedly been reported in ALS (2–4), but are still not part of the diagnostic criteria. MMs are involuntary movements contralaterally to an intended ﬁnger movement. MMs can be seen in normal children up to 10 years of age, but their prevalence and intensity declines after this age (5–7) most likely reﬂecting maturation of the corpus callosum (8). Persistence or novel manifestation of MMs in adults can arise from a variety of etiologies. Persistent congenital MMs have been described in diﬀerent conditions ranging from the absence of other neurological abnormalities to severe congenital hemiparesis in cerebral palsy (9). MMs also have been reported in a variety of other acquired conditions such as Parkinson’s disease (PD) (10, 11) or stroke (12, 13). Two main hypothesizes for the development of MMs were discussed: abnormal development of the primary motor system, involving the ipsilateral corticospinal tract, and lack of contralateral motor cortex inhibitory mechanisms, mainly through the corpus callosum (9, 14). Pathophysiological basis of MMs in all these acquired clinical conditions is thought to be the result of a predominant alteration of callosal projecting pathways. Nevertheless, an additional or pre-existing cortical or pyramidal malfunctioning appears to be necessary for the development of MMs (2). Moreover, MMs have been found to be associated with reduced transcallosal inhibition (TI) as measured by transcranial magnetic stimulation (TMS) in ALS, PD, and stroke (3, 4, 15–17). Regardless of the exact pathophysiological mechanism of MMs, the most important clinical aspect is that they are reﬂecting CNS involvement by the underlying pathological condition. Diﬀusion MRI techniques, such as diﬀusion tensor imaging (DTI) have been established to study integrity of neuronal tracts in vivo in the human brain. DTI studies found alterations of ﬁber tract integrity in neurodegenerative diseases like Alzheimer’s disease and ALS (18–21), including callosal involvement (22–24). INTRODUCTION An observational study found a consistent reduction in fractional anisotropy in the corpus callosum of ALS patients, extending rostrally, and bilaterally to the region of the primary motor cortices (23). A more recent study using DTI and TMS for characterization of neurodegeneration in ALS and concluded a complementary role as diagnostic biomarkers of UMN dysfunction (25). Ellis and colleagues found a signiﬁcant increase in the mean diﬀusivity and reduction of fractional anisotropy along the corticospinal tract in ALS patients with correlation to disease severity and UMN involvement (26). None of these studies focused on MMs or functional connectivity in ALS. Therefore, we studied associations of functional TMS measures with diﬀusion markers of structural integrity of h ll d b i l i i ALS, amyotrophic lateral sclerosis; SD, standard deviation; ALSFRS-R, amyotrophic lateral sclerosis functional rating scale—revised; UMNB, upper motor neuron burden; MOCA, Montreal Cognitive Assessment; El Escorial, El Escorial criteria; NA, not applicable; poss, possible; prob, probable; def, deﬁnite; N, number, UMN, upper motor neuron; LMN, lower motor neuron; p, signiﬁcance of Student’s t-test. ALS, amyotrophic lateral sclerosis; SD, standard deviation; ALSFRS-R, amyotrophic lateral sclerosis functional rating scale—revised; UMNB, upper motor neuron burden; MOCA, Montreal Cognitive Assessment; El Escorial, El Escorial criteria; NA, not applicable; poss, possible; prob, probable; def, deﬁnite; N, number, UMN, upper motor neuron; LMN, lower motor neuron; p, signiﬁcance of Student’s t-test. pathophysiological concepts of MMs in ALS. We hypothesized that ALS patients with MMs would show impaired functional integrity of the corpus callosum, associated with decline of structural integrity markers. Such ﬁndings would support the role of callosal dysfunction and structural impairment for MMs in ALS. Frontiers in Neurology \| www.frontiersin.org Subjects Nineteen patients with ALS and 20 healthy control subjects underwent clinical assessment, and MRI examinations, including DTI. Detailed demographic date and clinical characteristic of the entire study cohort are shown in Table 1. All subjects were right- handed as assessed by the Edinburgh handedness inventory (27). At the time point of examination 4 patients showed deﬁnitive ALS, 8 probable ALS and 4 possible ALS according to the revised El Escorial criteria (1). Three patients displayed lower motor neuron variant. Mean disease duration was 34.8 ± 34.8 months. Clinical assessment consisted of neurological examination with special respect of handedness; MMs were evaluated by sequential ﬁnger tapping of one hand without optic control and observation of MMs according to the procedure of Woods and Teuber (28), and the evaluation of the revised ALS functional rating scale (-R) (29). Furthermore, to address the upper motor neuron (UMN) involvement an UMN “burden” (UMNB) was calculated by totalling the number of pathological UMN signs on examination (maximum score 16) (30). For cognitive screening we used the “Montreal Cognitive assessment” (MOCA) (31). The MOCA score ranged between 18 and 29 with an average of 24.5 ± 4 for the ALS patients, and between 27 and 29 with an average of 27.6 ± 0.7 for the controls (P = 0.006). Controls did not have cognitive complaints and scored within 1.5 standard deviations of the MOCA age and education adjusted norm value. Therefore, we studied associations of functional TMS measures with diﬀusion markers of structural integrity of the corpus callosum and cerebrospinal tract in a prospective sample of ALS patients displaying MMs to elucidate the March 2020 \| Volume 11 \| Article 164 Frontiers in Neurology \| www.frontiersin.org 2 Mirror Movements in ALS Wittstock et al. Patients and control individuals were only included in the study if written consent was given. The study was approved by the Institutional Review Board of the Medical Faculty, University of Rostock (A-2011-0026, A 2012-0083). over the contralateral motor cortex; TMS was applied at the POE with 1.5 times resting motor threshold (RMT) while subjects performed a maximum tonic activation of the ipsilateral FDI muscle and while they kept the contralateral FDI muscle relaxed as published in detail previously (3). For determination of iSP latency and duration 10 trials for each hemisphere were used. After oﬄine rectiﬁcation and averaging of EMG signals, iSP parameters were analyzed. Transcranial Magnetic Stimulation g We performed TMS in all patients and in 10 of 20 healthy controls. Central motor conduction time (CMCT), motor evoked potentials (MEP) amplitudes, and contralateral silent period (cSP) were determined in all ALS patients and control subjects. MEPs were recorded from the ﬁrst dorsal interosseus muscle (FDI) and from the anterior tibial muscle (TA) using a standard circular coil (outside diameter 9 cm) connected to a Magstim 200 stimulator (Magstim Co., Whitland, Dyfeld, UK). For data acquisition a commercially available MEP system was employed (Brain Quick System Plus, Inomed, Erlangen, Germany). Investigation of the ipsilateral silent period (iSP) was performed with a focal coil (external loop diameter 7 cm). The coil was oriented to induce a posterior-anteriorly directed current ﬂow to the hand area of the motor cortex; the point of optimal excitability (POE) of the FDI muscle was determined March 2020 \| Volume 11 \| Article 164 Frontiers in Neurology \| www.frontiersin.org MRI Acquisition The images had a matrix size of 128 × 128 mm, slice thickness of 2 mm, the resulting voxel size was 2.0 × 2.0 × 2.0 mm2. For ROI analysis, group comparisons of FA of patients and controls were performed according to the ﬁndings of pathological iSP and non-pathological iSP in an univariate variance analysis and subsequent post-hoc tests. Furthermore, correlations between iSP ﬁndings and ALSFRS and FA parameters of all investigated ROIs was calculated. For correlation analysis Spearman-Rank- correlation were used and signiﬁcance was set at P < 0.05.For whole brain analysis, we investigated group diﬀerences in was performed (ﬂuid attenuation inversion recovery FLAIR, matrix size of 384 × 187, 24 slices with slice thickness of 5.0 mm, TE/TI/TR of 94 ms/2,500 ms/9,000 ms, ﬂip angle 150◦). Diﬀusion-weighted imaging was performed with an echo-planar- imaging sequence (TE/TR 81 ms/12,700 ms) Diﬀusion gradients were applied in 30 diﬀerent spatial directions. The b values were 0 and 1,000 s/mm2. The images had a matrix size of 128 × 128 mm, slice thickness of 2 mm, the resulting voxel size was 2.0 × 2.0 × 2.0 mm2. OIs was calculated. For correlation analysis Spearman-Ran rrelation were used and signiﬁcance was set at P < 0.05.F hole brain analysis, we investigated group diﬀerences FIGURE 2 \| Ipsilateral muscle responses (10 rectiﬁed and superimposed EMG-traces) recorded from the ﬁrst dorsal interosseous muscle (FDI) in ndividual ALS patients and controls. (A) The latency of the iSP was normal in ontrol case 3 (37.8 ms), (B) the iSP latency was prolonged in ALS patient ase 11 (51 ms), and (C) ALS patient case 18 displayed a loss of the iSP. MRI Data Processing FIGURE 2 \| Ipsilateral muscle responses (10 rectiﬁed and superimposed EMG-traces) recorded from the ﬁrst dorsal interosseous muscle (FDI) in individual ALS patients and controls. (A) The latency of the iSP was normal in control case 3 (37.8 ms), (B) the iSP latency was prolonged in ALS patient case 11 (51 ms), and (C) ALS patient case 18 displayed a loss of the iSP. MRI Acquisition MRI acquisitions of the brain were conducted using a 3- Tesla MRI scanner with a 32-channel phased-array head coil and parallel imaging capabilities (Magnetom Verio, Siemens, Erlangen, Germany, software syngo MR B17). Subjects were scanned in a single session without changing their position in the scanner. The following sequences were used: We acquired a sagittal high-resolutionT1-weighted magnetization prepared rapidly acquired gradient echo (MP-RAGE) 3D-sequence, matrix size of 256 × 256 × 192, isometric voxel size 1.0 mm3), TE/TI/TR of 4.82 ms/1,100 ms/2,500 ms, ﬂip angle 7◦. To identify white matter lesions a two-dimensional T2-weighted sequence FIGURE 1 \| The ﬁgure shows the overlap of the mean FA skeleton map of our sample (blue) and the normalized MNI-FA template and the deﬁned regions of interests based on JHU White matter atlas: genu (yellow), body (red), and Splenium (orange) of Corpus callosum, CST both sites (green) and crus posterior of the internal capsula on both sides (light blue). FIGURE 1 \| The ﬁgure shows the overlap of the mean FA skeleton map of our sample (blue) and the normalized MNI-FA template and the deﬁned regions of interests based on JHU White matter atlas: genu (yellow), body (red), and Splenium (orange) of Corpus callosum, CST both sites (green) and crus posterior of the internal capsula on both sides (light blue). TABLE 2 \| TMS ﬁndings in ALS patients and controls. ALS (N = 19) Controls (N = 10) p right left right left right/left RMT 50.9 (16.9) 51.6 (1.3) 41.5 (4.6) 41.1 (3.5) 0.029/0.015 CMCT 8.1 (3.8) 7.9 (4.3) 6.9 (0.7) 6.8 (0.8) 0.123/0.203 iSP latency 41.3 (5.4) 40.9 (5.7) 39.0 (4.6) 37.7 (6.3) 0.006/0.012 iSP duration 18.3 (1.2) 17.2 (4.3) 12.3 (2.1) 14.3 (2.8) 0.113/0.481 iSP loss 9 8 0 0 iSP pathological 12 (68.4%) 0 0.000 cSP 156.8 (45.1) 156.6 (41.2) 155.6 (35.3) 149.0 (28.6) 0.402/0.326 TABLE 2 \| TMS ﬁndings in ALS patients and controls. March 2020 \| Volume 11 \| Article 164 3 Wittstock et al. Mirror Movements in ALS was performed (ﬂuid attenuation inversion recovery FLAIR, matrix size of 384 × 187, 24 slices with slice thickness of 5.0 mm, TE/TI/TR of 94 ms/2,500 ms/9,000 ms, ﬂip angle 150◦). Diﬀusion-weighted imaging was performed with an echo-planar- imaging sequence (TE/TR 81 ms/12,700 ms) Diﬀusion gradients were applied in 30 diﬀerent spatial directions. The b values were 0 and 1,000 s/mm2. MRI Data Processing DTI data were pre-processed using the DTI tool box of the FSL software (http://www.fmrib.ox.ac.uk/fsl/, FMRIB, Oxford, UK, Version 4.1) (32). We ﬁrst applied corrections for eddy currents and head motion. The skull was stripped using Brain Extraction Tool for diﬀerentiation of brain tissue and non-brain tissue using a binary mask (threshold 0.15–0.3) and the diﬀusion tensors were ﬁtted to the data with DTI ﬁt (FMRIB Image Analysis Group, Oxford, UK). Fractional anisotropy (FA), and mean diﬀusivity (MD) maps as well as axial diﬀusivity (AD) and radial diﬀusivity (RD) maps were created. TBSS analysis (tract based spatial statistics) were performed using FMRIB software (version 4.1 www.fmrib.ox.ac.uk/fsl) (33). TBSS allows spatial reorientation of FA maps into a standard space without systematic eﬀects of spatial transformation on ﬁber tract directionality and without the need to select a spatial smoothing kernel that may impact upon the desired eﬀects (22). Processing of diﬀusivity maps consisted of the following steps: FA maps were transformed to MNI (Montreal Neurologic Institute) space. Normalized FA maps of all subjects were averaged and skeletonized with a standardized threshold of 0.2. Based on the study speciﬁc with matter skeletons an individual skeleton for each subject was created. j For speciﬁc analysis of changes in diﬀusivity of individual ﬁber tracts we conducted a region of interest (ROI) analysis. The selection of ROIs from JHU was done as described before (34). Based on the JHU white matter atlas (35) we deﬁned seven regions: corpus callosum with subregions genu, truncus, and splenium, corticospinal tract, and crus posterior of the internal capsula on both sides. Data processing was done by Matlab software 2013a (MathWorks Inc., MA, Natrick, USA) (Figure 1). For analysis of correlation between TMS values and DTI measures we performed a region of interest (ROI) analysis using MarsBaR (36) to extract mean values from corpus callosum and left and right corticospinal tract, respectively. For speciﬁc analysis of changes in diﬀusivity of individual ﬁber tracts we conducted a region of interest (ROI) analysis. The selection of ROIs from JHU was done as described before (34). Based on the JHU white matter atlas (35) we deﬁned seven regions: corpus callosum with subregions genu, truncus, and splenium, corticospinal tract, and crus posterior of the internal capsula on both sides. Data processing was done by Matlab software 2013a (MathWorks Inc., MA, Natrick, USA) (Figure 1). TMS Results TMS investigation in ALS patients revealed a mean iSP latency of 41.3 ± 5.4 ms/40.9 ± 5.7 ms (right/left), which was longer than that of the healthy controls 39.0 ± 4.6/37.7 ± 6.3 (right/left) (p = 0.006/0.012). No diﬀerences were found for the iSP duration between the ALS patients and control subjects. Twelve ALS patients (63.2%), but none of the controls showed loss of iSP. cSP was numerically shorter in ALS patients than in controls without reaching signiﬁcance. ALS patients had a signiﬁcantly lower mean RMT compared to controls (TMS results are shown in detail in Table 2 and Figure 2). controls (Table 4). There was no signiﬁcant correlation between FA values and iSP parameters (Table 4). In contrast, there was a signiﬁcant correlation of ALS-FRS-R scores with FA values of the CST on both sides (r CST-right, ALS-FRS-R = 0.58, p = 0.009 and r CST-left, LS-FRS-R = 0.05, p = 0.02), but not with FA values of the corpus callosum. controls (Table 4). There was no signiﬁcant correlation between FA values and iSP parameters (Table 4). In contrast, there was a signiﬁcant correlation of ALS-FRS-R scores with FA values of the CST on both sides (r CST-right, ALS-FRS-R = 0.58, p = 0.009 and r CST-left, LS-FRS-R = 0.05, p = 0.02), but not with FA values of the corpus callosum. DISCUSSION Lower and upper motor neuron dysfunction is a clinical hallmark of ALS. Besides involvement of primary motor areas, an early callosal dysfunction has been suggested in ALS as well (37). MMs may be a clinical sign of dysfunctional transcallosal pathways and could be observed in ∼30% of ALS patients. Further evidence for the interpretation of MMs as markers of callosal dysfunction comes from a range of TMS studies showing callosal Statistical Analysis ues + ate B MD values AD values RD values ALS (10−4) p ALS (x10−3) p ALS (x10−4) p 93 6.075 0.643 1.057 0.038 3.849 0.068 0.164 49 5.959 0.760 1.067 0.114 3.624 70 7.289 0.942 1.507 0.648 3.404 0.714 55 7.700 0.621 1.536 0.445 3.871 0.876 02 7.108 0.449 1.569 0.909 2.863 0.578 88 6.509 0.066 1.285 0.085 3.338 0.165 74 6.519 0.871 2.879 0.833 3.344 0.658 iate B: UMNB, upper motor neuron burden. TABLE 4 \| Correlations between TMS and DTI. Correlation FA values—RMT CST right CST left r −0.56 −0.52 p 0.021 0.012 Correlation FA values—iSP iSP latency Genu CC Body CC Splenium CC Right r 0.25 0.15 0.18 p 0.26 0.51 0.41 Left r 0.18 0.13 0.35 p 0.44 0.56 0.12 Correlation FA values—ALSFRS-R CST right CST left r 0.58 0.50 p 0.009 0.02 controls (Table 4). There was no signiﬁcant correlation between FA values and iSP parameters (Table 4). In contrast, there was a signiﬁcant correlation of ALS-FRS-R scores with FA values of the CST on both sides (r CST-right, ALS-FRS-R = 0.58, p = 0.009 and r CST-left, LS-FRS-R = 0.05, p = 0.02), but not with FA values of the corpus callosum. fractional anisotropy and mean diﬀusivity using the general linear model on a voxel basis. Whole brain analyses were performed for all diﬀusion parameters. FA, MD, RD, and AD maps were compared by univariate variance analysis (factor: group, covariate: ALSFRS-R and UMNB). To avoid overestimation of signiﬁcance, a test of multiple comparisons was applied (family wise error method) and signiﬁcance was set at P < 0.05. Group Differences in Fractional Anisotropy and Mean Diffusivity Concerning the analyses of pre-speciﬁed ROIs FA values of the CST were signiﬁcantly lower in ALS patients compared with controls. FA values of the diﬀerent ROIs of the corpus callosum (genu, splenium, and body) did not diﬀer between both groups (Table 3). AD and RD values showed similar ﬁndings. Whole brain analysis did not show any diﬀerences in FA, MD, RD, and AD between ALS patients and controls at an FWE corrected level of signiﬁcance. Statistical Analysis Statistical analysis was conducted for the clinical scores, TMS measurements and ROI data in SPSS (version IBM SPSS statistics 20). For the whole brain analysis using the tool box “Randomize” under TBSS in FMRIB (version 4.1 www.fmrib.ox.ac.uk/fsl) was used. All clinical scores and TMS parameters showed a normal distribution, which was tested by the use of Kolmogorov Smirnov test (P > 0.1). Diﬀerences in TMS parameters were compared between groups using Student’s t-test. The results of iSP measurements were dichotomized in pathological vs. non- pathological, and analyzed using Chi² test. FIGURE 2 \| Ipsilateral muscle responses (10 rectiﬁed and superimposed EMG-traces) recorded from the ﬁrst dorsal interosseous muscle (FDI) in individual ALS patients and controls. (A) The latency of the iSP was normal in control case 3 (37.8 ms), (B) the iSP latency was prolonged in ALS patient case 11 (51 ms), and (C) ALS patient case 18 displayed a loss of the iSP. March 2020 \| Volume 11 \| Article 164 Frontiers in Neurology \| www.frontiersin.org 4 Mirror Movements in ALS Wittstock et al. TABLE 3 \| DTI ﬁndings in ALS patients and controls [fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD)]. FA values FA values + covariate A* FA values + covariate B* MD values AD values RD values ROI ALS (N = 19) Controls (N = 20) p P p ALS (10−4) p ALS (x10−3) p ALS (x10−4) p CST right 0.589 0.624 0.026 0.011 0.093 6.075 0.643 1.057 0.038 3.849 0.068 0.164 CST left 0.621 0.648 0.126 0.020 0.049 5.959 0.760 1.067 0.114 3.624 Genu 0.737 0.731 0.604 0.458 0.470 7.289 0.942 1.507 0.648 3.404 0.714 Body 0.698 0.701 0.875 0.346 0.355 7.700 0.621 1.536 0.445 3.871 0.876 Splenium 0.789 0.786 0.604 0.149 0.102 7.108 0.449 1.569 0.909 2.863 0.578 Crus posterior of internal capsule right 0.700 0.716 0.484 0.972 0.188 6.509 0.066 1.285 0.085 3.338 0.165 Crus posterior of internal capsule left 0.699 0.705 0.668 0.705 0.074 6.519 0.871 2.879 0.833 3.344 0.658 *Covariate A: ALSFRS-R, amyotrophic lateral sclerosis functional rating scale-revised; covariate B: UMNB, upper motor neuron burden. Correlation of Clinical Parameter, TMS Parameters, and DTI Measures We found signiﬁcant negative correlations of FA values of the CST with RMT values on both sides in ALS patients, but not in March 2020 \| Volume 11 \| Article 164 Frontiers in Neurology \| www.frontiersin.org 5 Mirror Movements in ALS Wittstock et al. dysfunction as indicated by impaired transcallosal inhibition (2– 4, 15) in conjunction with the presence of MMs in ALS and other conditions like Parkinson’s disease (16). Furthermore, several DTI studies have shown microstructural changes not only in the pyramidal tract, but also in the corpus callosum in ALS (22, 23, 38). Callosal involvement has been demonstrated to be a relatively consistent feature of ALS, even without clinical UMN involvement, and may reﬂect interhemispheric spread of pathology and an impaired structural motor connectivity (23, 24). dysfunction as indicated by impaired transcallosal inhibition (2– 4, 15) in conjunction with the presence of MMs in ALS and other conditions like Parkinson’s disease (16). Furthermore, several DTI studies have shown microstructural changes not only in the pyramidal tract, but also in the corpus callosum in ALS (22, 23, 38). Callosal involvement has been demonstrated to be a relatively consistent feature of ALS, even without clinical UMN involvement, and may reﬂect interhemispheric spread of pathology and an impaired structural motor connectivity (23, 24). TMS changes in ALS, without eﬀects in the corpus callosum. Another explanation of the lack of signiﬁcance of DTI ﬁndings in the corpus callosum in our sample might be the relative great heterogeneity of patients, which represents one limitation of our study. Another limitation is the low number of patients. A major reason for this circumstance might be the relative rarity of MMs, which could be observed in just approximately 30% of ALS patients. Because of limitations in available staﬀresource the study period could not be extended. In conclusion, this is the ﬁrst study investigating a cohort of ALS patients which all were displaying MMs. The present study replicates and extends previous ﬁndings on reduced ﬁber tract integrity in patients with ALS with MMs, but failed to show microstructural changes accompanying mirror movements and disturbed transcallosal inhibition. The aﬀection of the pyramidal tract appears to be an important precondition for development of MMs. This study adds further evidence for the understanding of impaired connectivity in ALS patients. Therefore, further studies in bigger and more homogeneous cohorts are needed. ETHICS STATEMENT The studies involving human participant were reviewed and approved by the Institutional Review Board of the Medical Faculty, University of Rostock (A-2011-0026, A267 2012-0083). The patients/participants provided their written consent to participate in this study. AUTHOR CONTRIBUTIONS MW: conception, design, drafting of manuscript, acquisition of data, and ﬁnal approval of manuscript. NW, AG, and EK: acquisition of data, revision of manuscript, and ﬁnal approval of manuscript. ST: concept, design, analysis, and interpretation of data, drafting of manuscript, revision of manuscript, and ﬁnal approval of manuscript. DATA AVAILABILITY STATEMENT Deidentiﬁed participant data will be shared, as well as the study protocol and statistical analyses, upon reasonable requests. Correlation of Clinical Parameter, TMS Parameters, and DTI Measures In the current study we expected a signiﬁcant correlation of DTI measures and TMS ﬁndings reﬂecting transcallosal inhibition in ALS patients with MMs. Consistent with our expectation, we found an impaired transcallosal inhibition in up to 68% of cases which is in line with previously published data from independent cohorts (3, 4). In contrast, however, ALS patients with MMs in the current sample did not show microstructural involvement of the corpus callosum, which is in contrast with some (22, 34), but not all previous studies (39). One interpretation of the lack of corpus callosum changes in our study may be that our patients exhibited an earlier functional disturbance potentially preceding the microstructural ﬁndings in transcallosal pathways. Thus, the two previous studies ﬁnding corpus callous diﬀusion changes (23, 41) included functionally more advanced cases (mean ALSFRS-R score 33.1 and 36.5, respectively, compared with 38.5 in the current study). A recent study demonstrated increased AD in the corpus callosum and reduction of FA in the right CST in ALS without considering MMs (25). The study of Geraldo et al. had no focus on electrophysiological changes in the transcallosally projecting pathways but cortical excitability and CST conduction properties were tested by investigation of the resting motor threshold (RMT) as measure of cortical excitability and the central motor conduction time (CMCT). Keeping in mind that Geraldo and co-workers used a diﬀerent small hand muscle as target muscle (abductor digiti minimi vs. ﬁrst dorsal interosseus muscle in our study) RMT was slightly lower in our study reﬂecting a greater cortical excitability in our cohort displaying MMs. CMCT measurements displayed comparable values to our study. Extending these previous ﬁndings to ALS patients with MMs, we could replicate FA reduction of the CST associated with 5. Reitz M, Müller K. Diﬀerences between ’congenital mirror movements’ and ’associated movements’ in normal children: a neurophysiological case study. Neurosci Lett. (1998) 256:69–72. doi: 10.1016/S0304-3940(98)00748-4 7. Koerte I, Eftimov L, Laubender RP, Esslinger O, Schroeder AS, Ertl-Wagner B, et al. Mirror movements in healthy humans across the lifespan: eﬀects of development and ageing. Dev Med Child Neurol. (2010) 52:1106–12. doi: 10.1111/j.1469-8749.2010.03766.x 6. Mayston MJ, Harrison LM, Stephens JA. 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(2014) 9:e114543. doi: 10.1371/journal.pone.0114543 22. Sach M, Winkler G, Glauche V, Liepert J, Heimbach B, Koch MA, et al. Diﬀusion tensor MRI of early upper motor neuron involvement in amyotrophic lateral sclerosis. Brain. (2004) 127:340–50. doi: 10.1093/brain/awh041 Conﬂict of Interest: ST has done the listed works below (all in Germany): MSD Sharp & Dohme GmbH, Lindenplatz 1, 85540 Haar 11.09.2018—Quality circle for physicians in Kühlungsborn, Talk: “Dementia and Diabetes—current report” Conﬂict of Interest: ST has done the listed works below (all in Germany): MSD Sharp & Dohme GmbH, Lindenplatz 1, 85540 Haar 11.09.2018—Quality circle for physicians in Kühlungsborn, Talk: “Dementia and Diabetes—current report” 23. Filippini N, Douaud G, Mackay CE, Knight S, Talbot K, Turner MR. Corpus callosum involvement is a consistent feature of amyotrophic lateral sclerosis. Neurology. (2010) 75:1645–52. doi: 10.1212/WNL.0b013e3181f b84d1 14.11.2018—MSD Expert-forum: NAB Alzheimer in Munich, participator as consultant 13.08.2019—Event “Diabetes and Dementia” in Rostock, Talk: “Dementia and Diabetes—current report” ROCHE Pharma AG, Emil-Barell-Str. 1, 79639 Grenzach-Wyhlen 24. Verstraete E, Veldink JH, Mandl RCW, van den Berg LH, van den Heuvel MP. Impaired structural motor connectome in amyotrophic lateral sclerosis. PLoS ONE. (2011) 6:e24239. doi: 10.1371/journal.pone.0024239 12.09.2019-−3. National Advisory-Board in Frankfurt am Main, participation as consultant 25. Geraldo AF, Pereira J, Nunes P, Reimão S, Sousa R, Castelo-Branco M, et al. Beyond fractional anisotropy in amyotrophic lateral sclerosis: the value of mean, axial, and radial diﬀusivity and its correlation with electrophysiological conductivity changes. Neuroradiology. (2018) 60:505–15. REFERENCES doi: 10.1007/s00234-018-2012-6 27.09.2019—ROCHE Symposium at the DGN Congress in Stuttgart, Talk: “Amyloid as target for diagnosis and treatment in Alzheimer’s disease.” 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. 26. Ellis CM, Simmons A, Jones DK, Bland J, Dawson JM, Horsﬁeld MA, et al. Diﬀusion tensor MRI assesses corticospinal tract damage in ALS. Neurology. (1999) 53:1051–8. doi: 10.1212/WNL.53.5.1051 27. Oldﬁeld RC. The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia. (1971) 9:97–113. doi: 10.1016/0028-3932(71)90067-4 Copyright © 2020 Wittstock, Wilde, Grossmann, Kasper and Teipel. 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. 28. Woods BT, Teuber HL. Mirror movements after childhood hemiparesis. Neurology. (1978) 28:1152–7. doi: 10.1212/WNL.28.11.1152 29. Cedarbaum JM, Stambler N, Malta E, Fuller C, Hilt D, Thurmond B, et al. The ALSFRS-R: a revised ALS functional rating scale that incorporates assessments March 2020 \| Volume 11 \| Article 164 Frontiers in Neurology \| www.frontiersin.org 7
https://openalex.org/W3047000081	https://vbn.aau.dk/files/348932887/Meszl_nyi_et_al_2020_Passive_transfer_of_sera_from_als_patients_with_identified_mutations_evokes_an_increased_synaptic_vesicle_number_and_elevation_of_calcium_levels_in_motor_axon_terminals.pdf	English	null	Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients	International journal of molecular sciences	2,020	cc-by	14,758	Passive transfer of sera from als patients with identified mutations evokes an increased synaptic vesicle number and elevation of calcium levels in motor axon terminals, similar to sera from sporadic patients Meszlényi, Valéria; Patai, Roland; Polgár, Tamás F.; Nógrádi, Bernát; Körmöczy, Laura; Kristóf, Rebeka; Spisák, Krisztina; Tripolszki, Kornélia; Széll, Márta; Obál, Izabella; Engelhardt, József I.; Siklós, László Published in: Meszlényi, Valéria; Patai, Roland; Polgár, Tamás F.; Nógrádi, Bernát; Körmöczy, Laura; Kristóf, Rebeka; Spisák, Krisztina; Tripolszki, Kornélia; Széll, Márta; Obál, Izabella; Engelhardt, József I.; Siklós, László Published in: Meszlényi, Valéria; Patai, Roland; Polgár, Tamás F.; Nógrádi, Bernát; Körmöczy, Laura; Kristóf, Rebeka; Spisák, Krisztina; Tripolszki, Kornélia; Széll, Márta; Obál, Izabella; Engelhardt, József I.; Siklós, László Published in: International Journal of Molecular Sciences DOI (link to publication from Publisher): 10.3390/ijms21155566 Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Meszlényi, V., Patai, R., Polgár, T. F., Nógrádi, B., Körmöczy, L., Kristóf, R., Spisák, K., Tripolszki, K., Széll, M., Obál, I., Engelhardt, J. I., & Siklós, L. (2020). Passive transfer of sera from als patients with identified mutations evokes an increased synaptic vesicle number and elevation of calcium levels in motor axon terminals, similar to sera from sporadic patients. International Journal of Molecular Sciences , 21(15), 1-18. Article 5566. https://doi.org/10.3390/ijms21155566 Citation for published version (APA): Meszlényi, V., Patai, R., Polgár, T. F., Nógrádi, B., Körmöczy, L., Kristóf, R., Spisák, K., Tripolszki, K., Széll, M., Obál, I., Engelhardt, J. I., & Siklós, L. (2020). Passive transfer of sera from als patients with identified mutations evokes an increased synaptic vesicle number and elevation of calcium levels in motor axon terminals, similar to sera from sporadic patients. International Journal of Molecular Sciences , 21(15), 1-18. Article 5566. https://doi.org/10.3390/ijms21155566 Aalborg Universitet eneral rights opyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners nd it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain Passive Transfer of Sera from ALS Patients with Identiﬁed Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients Valéria Meszlényi 1,2, Roland Patai 1 , Tamás F. Polgár 1, Bernát Nógrádi 1,2 , Laura Körmöczy 1, Rebeka Kristóf 1, Krisztina Spisák 1, Kornélia Tripolszki 3, Márta Széll 3,4, Izabella Obál 5,6, József I. Engelhardt 6 and László Siklós 1,* 1 Biological Research Centre, Institute of Biophysics, 62 Temesvári krt., H-6726 Szeged, Hungary; mesval13@gmail.com (V.M.); patai.roland@brc.hu (R.P.); polgar.tamas@brc.hu (T.F.P.); bernatnogradi@gmail.com (B.N.); laura.kormoci96@gmail.com (L.K.); crebby32@gmail.com (R.K.); spisakkrisztina96@gmail.com (K.S.) 1 Biological Research Centre, Institute of Biophysics, 62 Temesvári krt., H-6726 Szeged, Hungary; mesval13@gmail.com (V.M.); patai.roland@brc.hu (R.P.); polgar.tamas@brc.hu (T.F.P.); bernatnogradi@gmail.com (B.N.); laura.kormoci96@gmail.com (L.K.); crebby32@gmail.com (R.K.); spisakkrisztina96@gmail.com (K.S.) p g 2 Foundation for the Future of Biomedical Sciences in Szeged, Szeged Scientists Academy, 15 Lechner té H-6721 Szeged, Hungary 2 Foundation for the Future of Biomedical Sciences in Szeged, Szeged Scientists Academy, 15 Lechner tér, H-6721 Szeged, Hungary 3 3 Department of Medical Genetics, University of Szeged, 4/B Sz˝okefalvi-Nagy Béla u., H-6720 Szeged, Hungary; tripolszki.kornelia@med.u-szeged.hu (K.T.); szell.marta@med.u-szeged.hu (M.S.) 3 Department of Medical Genetics, University of Szeged, 4/B Sz˝okefalvi-Nagy Béla u., H-6720 Szeged, Hungary; tripolszki.kornelia@med.u-szeged.hu (K.T.); szell.marta@med.u-szeged.hu (M.S.) 4 Dermatological Research Group, University of Szeged, Hungarian Academy of Sciences, 4/B Department of Medical Genetics, University of Szeged, 4/B Szokefalvi-Nagy Béla u., H-6720 Szeged, Hungary; tripolszki.kornelia@med.u-szeged.hu (K.T.); szell.marta@med.u-szeged.hu (M.S.) 4 Dermatological Research Group, University of Szeged, Hungarian Academy of Sciences, 4/B Sz˝okefalvi-Nagy Béla u., H-6720 Szeged, Hungary 4 Dermatological Research Group, University of Szeged, Hungarian Academy of Sciences, 4/B Sz˝okefalvi-Nagy Béla u., H-6720 Szeged, Hungary 5 Department of Neurology, Aalborg University Hospital, 15 Skovvej Sdr., DK-9000 Aalborg, Denmark; obalizabella@yahoo.com 6 Department of Neurology, University of Szeged, 6 Semmelweis u., H-6725 Szeged, Hungary; eji48dec9@yahoo.com j y * Correspondence: siklos.laszlo@brc.hu; Tel.: +36-62-599-611 respondence: siklos.laszlo@brc.hu; Tel.: +36-62-599-61 General rights C i h d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners nd it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. Y f h di ib h i l i f fi ki i i i l i - Users may download and print one copy of any publication from the public portal for the purpose of private - You may not further distribute the material or use it for any profit-making activity or commercial gain You may not further distribute the material or use it for any profit making activity o - You may freely distribute the URL identifying the publication in the public portal - y y p g y - You may freely distribute the URL identifying the publication in the public portal - International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Amyotrophic lateral sclerosis (ALS) is one of the most common motor neuron diseases, which, according to the historical principles of neurology, primarily aﬀects the upper and lower motor neurons [1]. ALS patients are traditionally sorted into two categories. Familial ALS patients represent 5–10% of the patient population, with more than a dozen, mostly autosomal dominant, mutations in diﬀerent genes [2,3]. The sporadic form, with no familial accumulation, represents the overwhelming majority, i.e., 90–95%, of ALS patients [4]. However, the identiﬁcation of an expansion of the intronic hexanucleotide repeat sequence in chromosome nine open reading frame 72 (C9ORF72) in patients with no family history signiﬁcantly contributed to the decline of the traditional distinction between sporadic and familial ALS, which have identical clinical manifestations [5]. Since the hexanucleotide repeat sequence in C9ORF72 is now considered the most frequent genetic alteration, not just in ALS but in frontotemporal dementia [6], ALS is now considered a multisystem disorder, overlapping mostly with frontotemporal dementia [7]. In addition, regarding disease manifestations on a continuum of frontotemporal dementia, ALS blurs the distinction between two speciﬁc neurodegenerative diseases. Another noteworthy observation is that the identiﬁed pathomechanisms of ALS are not restricted to this syndrome [8]. Indeed, mechanisms including, but not restricted to, excitotoxicity [9,10], oxidative stress [11–13], mitochondrial dysfunction [14,15], immune/inﬂammatory reactions [16–18] and perturbed neuronal calcium homeostasis [19–23] are shown to contribute to the pathobiology of multiple sclerosis, Alzheimer’s, Parkinson’s, Huntington’s, and other diseases as well. ALS plays a distinguished role since, unlike other neurodegenerative diseases aﬀecting the central nervous system, parts of the degenerating nerve cells can be sampled without the necessity of ethically questionable brain biopsies. This special anatomical feature [24] provides the opportunity to compare and validate the results obtained from animal models with appropriate human samples. It is now accepted that the disturbed calcium homeostasis of aﬀected neurons plays a general role in neurodegenerative diseases [19], and particularly in ALS, this mechanism may serve as a central factor, providing positive feedback between the individual components of the complex pathomechanism of the disease [25]. In our pioneering study with electron microscopic analyses, we described an elevated calcium level accompanied by an increased number of synaptic vesicles in the motor axon terminals obtained from sporadic ALS patients [26]. Received: 16 July 2020; Accepted: 31 July 2020; Published: 3 August 2020 Abstract: Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with puriﬁed immunoglobulin G. Later, we conﬁrmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identiﬁed mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identiﬁed mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 diﬀerent ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the eﬀect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identiﬁed mutations is based on a common ﬁnal pathway, in which increasing calcium levels play a central role. Keywords: ALS; passive transfer; intracellular calcium; synaptic vesicles; SOD1 mutation; C9ORF72 mutation y p C9ORF72 mutation Int. J. Mol. Sci. 2020, 21, 5566; doi:10.3390/ijms21155566 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5566 2 of 18 1. Introduction While these results documented for the ﬁrst time the perturbed calcium homeostasis in degenerating human nerve cells, the applied sampling was not uniform, since it was restricted to a subpopulation of ALS patients with sporadic classiﬁcations and no identiﬁed mutations. To complete the study and generalize the results, an analysis of the muscle biopsy samples from patients with identiﬁed mutations was required. Based on our human muscle biopsy study, motor axon terminals could be localized in only 10% of the 71 patients who underwent a routine biopsy of the biceps muscles in a period of 9 months [26]. In view of these numbers, and because of the low incidence of patients with familial history or identiﬁed mutations, similar examinations are not feasible, since according to the current epidemiological data [27], the worldwide prevalence of ALS is 4–8 persons/100,000 inhabitants. Thus, such a study would require a group size of 10 million to provide the same number of patients with a familial history or identiﬁed mutations as in the sporadic study. An indirect approach to disclose information about the underlying mechanisms in patients with ALS-related mutations relies on the passive transfer of their blood-derived samples to experimental animals. Indeed, anomalies, i.e., elevated calcium levels and increased numbers of synaptic vesicles, observed in the motor axon terminals of sporadic ALS patients, can be transferred to mice inoculated with whole serum or even with immunoglobulin G (IgG) puriﬁed from the blood of these patients [28]. If similar alterations are induced in the blood samples from patients with known mutations (mostly familial patients), assuming corresponding changes in the donor patients, a coherent view of the role of calcium in the pathomechanism of ALS could be conﬁrmed. With the present demonstration that increased calcium can be transferred with the sera from ALS patients with identiﬁed mutations, Int. J. Mol. Sci. 2020, 21, 5566 3 of 18 data describing increased calcium in the axon terminals of sporadic ALS patients, increased calcium after passive transfer from sporadic patients and increased calcium in the genetic model of familial ALS could be obtained (Figure 1). Int. J. Mol. Sci. 2020, 21, 5566 3 of 18 patients, increased calcium after passive transfer from sporadic patients and increased calcium in the genetic model of familial ALS could be obtained (Figure 1) Figure 1. 1. Introduction Amyotrophic lateral sclerosis (ALS) patients are represented with green circles, and the ALS models are symbolized with blue circles. With the passive transfer of serum or immunoglobulin G (IgG) from sporadic patients to mice (red arrow), a model of sporadic ALS could be created which reproduces the elevated calcium in the motor axon terminals demonstrated in the patients. ALS patients are represented with green circles, and the ALS models are symbolized with blue circles. A transgenic model of familial ALS, based on the mutations identified in patients, could also be created (curved red arrow). By replicating the sporadic ALS model with the passive transfer of sera from familial patients (shaded area), another model of familial ALS was set up in the present study. Since in each model comparable increases of calcium in the motor axon terminals could be demonstrated (green arrows), an elevated calcium level in the motor axon terminals, similar to that seen in sporadic patients, could be hypothesized for the familial ALS patients (dashed green arrow). Figure 1. Amyotrophic lateral sclerosis (ALS) patients are represented with green circles, and the ALS models are symbolized with blue circles. With the passive transfer of serum or immunoglobulin G (IgG) from sporadic patients to mice (red arrow), a model of sporadic ALS could be created which reproduces the elevated calcium in the motor axon terminals demonstrated in the patients. ALS patients are represented with green circles, and the ALS models are symbolized with blue circles. A transgenic model of familial ALS, based on the mutations identiﬁed in patients, could also be created (curved red arrow). By replicating the sporadic ALS model with the passive transfer of sera from familial patients (shaded area), another model of familial ALS was set up in the present study. Since in each model comparable increases of calcium in the motor axon terminals could be demonstrated (green arrows), an elevated calcium level in the motor axon terminals, similar to that seen in sporadic patients, could be hypothesized for the familial ALS patients (dashed green arrow). Figure 1. Amyotrophic lateral sclerosis (ALS) patients are represented with green circles, and the ALS models are symbolized with blue circles. With the passive transfer of serum or immunoglobulin G (IgG) from sporadic patients to mice (red arrow), a model of sporadic ALS could be created which reproduces the elevated calcium in the motor axon terminals demonstrated in the patients. 1. Introduction Since in each mode comparable increases of calcium in the motor axon terminals could be demonstrated (green arrows an elevated calcium level in the motor axon terminals, similar to that seen in sporadic patients, could b hypothesized for the familial ALS patients (dashed green arrow). Twelve ALS patients with identified mutations, with or without family history (two patients had no family history or known mutations) were introduced to the study. Sera prepared from the patients were intraperitoneally injected into mice for 2 days with daily injections, then the motor axon terminals were analyzed electron microscopically to determine the calcium content and the number of synaptic vesicles. The observed increase in calcium levels and the elevated number of synaptic vesicles in the axon terminals, together with the previous observations of elevated calcium in the genetic model of familial ALS and the passive transfer model of sporadic ALS, provide additional data for the concept that elevated calcium levels play a central role in the pathomechanism of ALS. This common denominator may contribute to the identical clinical manifestation of the disease in its final stage. Twelve ALS patients with identiﬁed mutations, with or without family history (two patients had no family history or known mutations) were introduced to the study. Sera prepared from the patients were intraperitoneally injected into mice for 2 days with daily injections, then the motor axon terminals were analyzed electron microscopically to determine the calcium content and the number of synaptic vesicles. The observed increase in calcium levels and the elevated number of synaptic vesicles in the axon terminals, together with the previous observations of elevated calcium in the genetic model of familial ALS and the passive transfer model of sporadic ALS, provide additional data for the concept that elevated calcium levels play a central role in the pathomechanism of ALS. This common denominator may contribute to the identical clinical manifestation of the disease in its ﬁnal stage. 1. Introduction ALS patients are represented with green circles, and the ALS models are symbolized with blue circles. A transgenic model of familial ALS, based on the mutations identified in patients, could also be created (curved red arrow). By replicating the sporadic ALS model with the passive transfer of sera from familial patients (shaded area), another model of familial ALS was set up in the present study. Since in each model comparable increases of calcium in the motor axon terminals could be demonstrated (green arrows), an elevated calcium level in the motor axon terminals, similar to that seen in sporadic patients, could be hypothesized for the familial ALS patients (dashed green arrow). Figure 1. Amyotrophic lateral sclerosis (ALS) patients are represented with green circles, and the ALS models are symbolized with blue circles. With the passive transfer of serum or immunoglobulin G (IgG) from sporadic patients to mice (red arrow), a model of sporadic ALS could be created which reproduces the elevated calcium in the motor axon terminals demonstrated in the patients. ALS patients are represented with green circles, and the ALS models are symbolized with blue circles. A transgenic model of familial ALS, based on the mutations identiﬁed in patients, could also be created (curved red arrow). By replicating the sporadic ALS model with the passive transfer of sera from familial patients (shaded area), another model of familial ALS was set up in the present study. Since in each model comparable increases of calcium in the motor axon terminals could be demonstrated (green arrows), an elevated calcium level in the motor axon terminals, similar to that seen in sporadic patients, could be hypothesized for the familial ALS patients (dashed green arrow). (curved red arrow). By replicating the sporadic ALS model with the passive transfer of sera from familial patients (shaded area), another model of familial ALS was set up in the present study. Since in each model comparable increases of calcium in the motor axon terminals could be demonstrated (green arrows), an elevated calcium level in the motor axon terminals, similar to that seen in sporadic patients, could be hypothesized for the familial ALS patients (dashed green arrow). arrow). By replicating the sporadic ALS model with the passive transfer of sera from familial patient shaded area), another model of familial ALS was set up in the present study. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera The postsynaptic compartments of the neuromuscular synapses of mice inoculated with sera obtained from different ALS patients displayed no structural alterations: intact muscle fibers and specialized postsynaptic membranes were visible (Figure 2). Compared to the controls, the most obvious alterations of the motor axon terminals of mice injected with sera from ALS patients were the increased number of electron-dense deposits (EDDs), representing the distribution of calcium in the tissue, and the increased number of synaptic vesicles (Figures 2 and 3). Qualitatively, these The postsynaptic compartments of the neuromuscular synapses of mice inoculated with sera obtained from diﬀerent ALS patients displayed no structural alterations: intact muscle ﬁbers and specialized postsynaptic membranes were visible (Figure 2). Compared to the controls, the most obvious alterations of the motor axon terminals of mice injected with sera from ALS patients were the increased number of electron-dense deposits (EDDs), representing the distribution of calcium in the tissue, and the increased number of synaptic vesicles (Figures 2 and 3). Qualitatively, these changes were most Int. J. Mol. Sci. 2020, 21, 5566 4 of 18 prominent in the axon terminals of mice injected with sera from the patient with a C9ORF72 mutation (Figure 2D). Occasionally, swollen mitochondria containing clusters of EDDs were encountered in the axon terminals of the mice (Figure 2C). Int. J. Mol. Sci. 2020, 21, 5566 4 of 18 a C9ORF72 mutation (Figure 2D). Occasionally, swollen mitochondria containing clusters of EDDs were encountered in the axon terminals of the mice (Figure 2C). Figure 2. Electron micrographs of the neuromuscular synapses in the interosseous muscles after oxalate-pyroantimonate fixation. Junctional folds (jf) with no structural alterations are visible around all axon terminals. Axon terminals from an untreated mouse (A), and from a mouse injected with serum from a healthy individual (B) show no sign of structural damage, and contain intact mitochondria (mit). Furthermore, calcium-containing electron-dense deposits (EDDs) are only sparsely visible (arrows). Axon terminals from mice injected with sera from ALS patients, exemplified with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72 (D)) mutations, display an increased amount of EDDs (arrows), particularly in (D). Furthermore, a global increase in synaptic vesicles can be noted. Occasionally, swollen mitochondria (mit) with clusters of EDDs are visible (C). sm: skeletal muscle. Scale bar: 500 nm. Figure 2. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera Axon terminals from mice injected with serum from ALS patients, represented with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72) mutations (D), display an increased number of synaptic vesicles in these regions. The increase in the number of synaptic vesicles is exceptionally high in the axon terminal of a mouse injected with the sera from a patient with a C9ORF72 mutation (D). jf: junctional folds. Scale bar: 200 nm. Figure 3. Enlarged view of motor axon terminals in the vicinity of the active zones of the neuromuscular synapses—oxalate-pyroantimonate ﬁxation. In the axon terminal of an untreated mouse (A), and a mouse injected with serum from a healthy individual (B), only a few synaptic vesicles (arrows) are present. Some of them contain dot-like electron-dense deposits (EDDs). Axon terminals from mice injected with serum from ALS patients, represented with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72) mutations (D), display an increased number of synaptic vesicles in these regions. The increase in the number of synaptic vesicles is exceptionally high in the axon terminal of a mouse injected with the sera from a patient with a C9ORF72 mutation (D). jf: junctional folds. Scale bar: 200 nm. 2.2. Quantitative Analysis of the Change in the Number of Synaptic Vesicles and Increase in Intracellular Calcium in Motor Axon Terminals after an Inoculation with ALS Sera 2.2. Quantitative Analysis of the Change in the Number of Synaptic Vesicles and Increase in Intracellular Calcium in Motor Axon Terminals after an Inoculation with ALS Sera 2.2. Quantitative Analysis of the Change in the Number of Synaptic Vesicles and Increase in Intracellular Calcium in Motor Axon Terminals after an Inoculation with ALS Sera 2.2. Quantitative Analysis of the Change in the Number of Synaptic Vesicles and Increase in Intracellular Calcium in Motor Axon Terminals after an Inoculation with ALS Sera f The intracellular calcium level of motor axon terminals was expressed as the volume occupied by the EDDs relative to the volume of the axon terminal. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera Electron micrographs of the neuromuscular synapses in the interosseous muscles after oxalate-pyroantimonate ﬁxation. Junctional folds (jf) with no structural alterations are visible around all axon terminals. Axon terminals from an untreated mouse (A), and from a mouse injected with serum from a healthy individual (B) show no sign of structural damage, and contain intact mitochondria (mit). Furthermore, calcium-containing electron-dense deposits (EDDs) are only sparsely visible (arrows). Axon terminals from mice injected with sera from ALS patients, exempliﬁed with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72 (D)) mutations, display an increased amount of EDDs (arrows), particularly in (D). Furthermore, a global increase in synaptic vesicles can be noted. Occasionally, swollen mitochondria (mit) with clusters of EDDs are visible (C). sm: skeletal muscle. Scale bar: 500 nm. Figure 2. Electron micrographs of the neuromuscular synapses in the interosseous muscles after oxalate-pyroantimonate fixation. Junctional folds (jf) with no structural alterations are visible around all axon terminals. Axon terminals from an untreated mouse (A), and from a mouse injected with serum from a healthy individual (B) show no sign of structural damage, and contain intact mitochondria (mit). Furthermore, calcium-containing electron-dense deposits (EDDs) are only sparsely visible (arrows). Axon terminals from mice injected with sera from ALS patients, exemplified with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72 (D)) mutations, display an increased amount of EDDs (arrows), particularly in (D). Furthermore, a global increase in synaptic vesicles can be noted. Occasionally, swollen mitochondria (mit) with clusters of EDDs are visible (C). sm: skeletal muscle. Scale bar: 500 nm. Figure 2. Electron micrographs of the neuromuscular synapses in the interosseous muscles after oxalate-pyroantimonate ﬁxation. Junctional folds (jf) with no structural alterations are visible around all axon terminals. Axon terminals from an untreated mouse (A), and from a mouse injected with serum from a healthy individual (B) show no sign of structural damage, and contain intact mitochondria (mit). Furthermore, calcium-containing electron-dense deposits (EDDs) are only sparsely visible (arrows). Axon terminals from mice injected with sera from ALS patients, exempliﬁed with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72 (D)) mutations, display an increased amount of EDDs (arrows), particularly in (D). Furthermore, a global increase in synaptic vesicles can be noted. Occasionally, swollen mitochondria (mit) with clusters of EDDs are visible (C). sm: skeletal muscle. Scale bar: 500 nm. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera Int. J. Mol. Sci. 2020, 21, 5566 I t J M l S i 2020 21 5566 5 of 18 Int. J. Mol. Sci. 2020, 21, 5566 5 of 1 Int. J. Mol. Sci. 2020, 21, 5566 5 of 18 Figure 3. Enlarged view of motor axon terminals in the vicinity of the active zones of the neuromuscular synapses—oxalate-pyroantimonate fixation. In the axon terminal of an untreated mouse (A), and a mouse injected with serum from a healthy individual (B), only a few synaptic vesicles (arrows) are present. Some of them contain dot-like electron-dense deposits (EDDs). Axon terminals from mice injected with serum from ALS patients, represented with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72) mutations (D), display an increased number of synaptic vesicles in these regions. The increase in the number of synaptic vesicles is exceptionally high in the axon terminal of a mouse injected with the sera from a patient with a C9ORF72 mutation (D). jf: junctional folds. Scale bar: 200 nm. 2 2 Quantitative Analysis of the Change in the Number of Synaptic Vesicles and Increase in Intracellular Figure 3. Enlarged view of motor axon terminals in the vicinity of the active zones of the neuromuscular synapses—oxalate-pyroantimonate ﬁxation. In the axon terminal of an untreated mouse (A), and a mouse injected with serum from a healthy individual (B), only a few synaptic vesicles (arrows) are present. Some of them contain dot-like electron-dense deposits (EDDs). Axon terminals from mice injected with serum from ALS patients, represented with superoxide dismutase 1 (SOD1) pAsp90Ala (C) and chromosome 9 open reading frame 72 (C9ORF72) mutations (D), display an increased number of synaptic vesicles in these regions. The increase in the number of synaptic vesicles is exceptionally high in the axon terminal of a mouse injected with the sera from a patient with a C9ORF72 mutation (D). jf: junctional folds. Scale bar: 200 nm. 2.2. Quantitative Analysis of the Change in the Number of Synaptic Vesicles and Increase in Intracellular Figure 3. Enlarged view of motor axon terminals in the vicinity of the active zones of the neuromuscular synapses—oxalate-pyroantimonate fixation. In the axon terminal of an untreated mouse (A), and a mouse injected with serum from a healthy individual (B), only a few synaptic vesicles (arrows) are present. Some of them contain dot-like electron-dense deposits (EDDs). 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera Supporting the qualitative observations, an inoculation with all ALS sera induced a significant calcium increase in the axon terminals (superoxide dismutase 1 (SOD1) pLeu144Phe: 17.31% ± 0.96%; SOD1 pVal14Met: 17.67% ± 1.96%; SOD1 pAsp90Ala: 19.37% ± 1.78%; SOD1 pLys91ArgfsTer8: 21.84% ± 1.23%; C9ORF72: 34.29% ± 2.45%; sequestosome 1 (SQSTM1) pPro392Leu: 17.30% ± 3.41%; G2/mitotic-specific cyclin F (CCNF) The intracellular calcium level of motor axon terminals was expressed as the volume occupied by the EDDs relative to the volume of the axon terminal. Supporting the qualitative observations, an inoculation with all ALS sera induced a signiﬁcant calcium increase in the axon terminals (superoxide dismutase 1 (SOD1) pLeu144Phe: 17.31% ± 0.96%; SOD1 pVal14Met: 17.67% ± 1.96%; SOD1 pAsp90Ala: 19.37% ± 1.78%; SOD1 pLys91ArgfsTer8: 21.84% ± 1.23%; C9ORF72: 34.29% ± 2.45%; sequestosome 1 (SQSTM1) pPro392Leu: 17.30% ± 3.41%; G2/mitotic-speciﬁc cyclin F (CCNF) 6 of 18 6 of 18 Int. J. Mol. Sci. 2020, 21, 5566 I t J M l S i 2020 21 5566 pLeu106Val: 21.06% ± 3.68%; NEK1 (NIMA-related kinase 1) and TBK1 (TANK-binding kinase 1): 20.47% ± 3.19%; UBQLN2 (Ubiquilin 2): 17.62% ± 3.42%; sporadic: 23.60% ± 1.37%) compared to the control groups (untreated: 5.42% ± 0.68%; healthy serum treated: 6.86% ± 1.10% (Figure 4)). pLeu106Val: 21.06% ± 3.68%; NEK1 (NIMA-related kinase 1) and TBK1 (TANK-binding kinase 1): 20.47% ± 3.19%; UBQLN2 (Ubiquilin 2): 17.62% ± 3.42%; sporadic: 23.60% ± 1.37%) compared to the control groups (untreated: 5.42% ± 0.68%; healthy serum treated: 6.86% ± 1.10% (Figure 4)). Figure 4. The ratio of the volume of electron-dense deposits (EDDs) and the volume of the axon terminals after inoculation with sera from ALS patients. A significant elevation in EDDs could be noted in each amyotrophic lateral sclerosis (ALS) serum treated group. Furthermore, this elevation was significantly higher (###: p < 0.001) in the motor axon terminals of mice injected with sera from ALS patients with C9ORF72 mutations compared to all other groups. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one- way analysis of the variance (ANOVA) with the least significant difference post-hoc pairwise comparison. *: p < 0.001. Figure 4. The ratio of the volume of electron-dense deposits (EDDs) and the volume of the axon terminals after inoculation with sera from ALS patients. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera A signiﬁcant elevation in EDDs could be noted in each amyotrophic lateral sclerosis (ALS) serum treated group. Furthermore, this elevation was signiﬁcantly higher (###: p < 0.001) in the motor axon terminals of mice injected with sera from ALS patients with C9ORF72 mutations compared to all other groups. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least signiﬁcant diﬀerence post-hoc pairwise comparison. : p < 0.001. Figure 4. The ratio of the volume of electron-dense deposits (EDDs) and the volume of the axon terminals after inoculation with sera from ALS patients. A significant elevation in EDDs could be noted in each amyotrophic lateral sclerosis (ALS) serum treated group. Furthermore, this elevation was significantly higher (###: p < 0.001) in the motor axon terminals of mice injected with sera from ALS patients with C9ORF72 mutations compared to all other groups. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one- way analysis of the variance (ANOVA) with the least significant difference post-hoc pairwise comparison. : p < 0.001. Figure 4. The ratio of the volume of electron-dense deposits (EDDs) and the volume of the axon terminals after inoculation with sera from ALS patients. A signiﬁcant elevation in EDDs could be noted in each amyotrophic lateral sclerosis (ALS) serum treated group. Furthermore, this elevation was signiﬁcantly higher (###: p < 0.001) in the motor axon terminals of mice injected with sera from ALS patients with C9ORF72 mutations compared to all other groups. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least signiﬁcant diﬀerence post-hoc pairwise comparison. : p < 0.001. To reduce the variability of the results due to the largely inhomogeneous distribution of synaptic vesicles within the axon terminals, a quantitative evaluation of the density of synaptic vesicles was limited to the active zones, the physiologically most relevant regions of the synapses. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera All treatments with ALS sera resulted in significant increases in the synaptic vesicle density (SOD1 pLeu144Phe 110.30 ± 8.81 vesicles/µm3; SOD1 pVal14Met: 134.65 ± 1.82 vesicles/µm3; SOD1 pAsp90Ala: 133.87 ± 10.88 vesicles/µm3; SOD1 pLys91ArgfsTer8: 120.28 ± 11.26 vesicles/µm3; C9ORF72: 201.15 ± 14.29 vesicles/µm3; SQSTM1 pPro392Leu: 108.01 ± 12.74 vesicles/µm3; CCNF pLeu106Val: 124.60 ± 9.91 vesicles/µm3; NEK1 and TBK1: 162.43 ± 3.19 vesicles/µm3; UBQLN2: 138.55 ± 15.02 vesicles/µm3 sporadic: 116.10 ± 7.68 vesicles/µm3), while there were no significant changes in the group treated with healthy sera (89.40 ± 6.05 vesicles/µm3) compared to the control (80.38 ± 1.5 vesicles/µm3 (Figure 5)). To reduce the variability of the results due to the largely inhomogeneous distribution of synaptic vesicles within the axon terminals, a quantitative evaluation of the density of synaptic vesicles was limited to the active zones, the physiologically most relevant regions of the synapses. All treatments with ALS sera resulted in signiﬁcant increases in the synaptic vesicle density (SOD1 pLeu144Phe: 110.30 ± 8.81 vesicles/µm3; SOD1 pVal14Met: 134.65 ± 1.82 vesicles/µm3; SOD1 pAsp90Ala: 133.87 ± 10.88 vesicles/µm3; SOD1 pLys91ArgfsTer8: 120.28 ± 11.26 vesicles/µm3; C9ORF72: 201.15 ± 14.29 vesicles/µm3; SQSTM1 pPro392Leu: 108.01 ± 12.74 vesicles/µm3; CCNF pLeu106Val: 124.60 ± 9.91 vesicles/µm3; NEK1 and TBK1: 162.43 ± 3.19 vesicles/µm3; UBQLN2: 138.55 ± 15.02 vesicles/µm3; sporadic: 116.10 ± 7.68 vesicles/µm3), while there were no signiﬁcant changes in the group treated with healthy sera (89.40 ± 6.05 vesicles/µm3) compared to the control (80.38 ± 1.5 vesicles/µm3 (Figure 5)). 5)). The elevations in the density of the active zone synaptic vesicles and the level of intracellular calcium were noted as significant effects of the inoculation with sera from ALS patients. Since these parameters are closely associated with the activity of the synapses, their cross-relation was analyzed as well (Figure 6). In Figure 6, each point represents patients, except the “untreated control” points which display values derived from individual mice, to illustrate the reproducibility of the histochemical method. The data points are split into three main groups: controls, ALS patients and ALS outliers, representing the patients with the C9ORF72 mutation. A K-means statistical cluster analysis confirmed the presence of these distinct clusters: (i) one represented the untreated mice and the healthy volunteers, (ii) another shows all ALS mutations, except the C9ORF72, (iii) and the last cluster represented the patients with a C9ORF72 mutation (Figure 6). 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera The elevations in the density of the active zone synaptic vesicles and the level of intracellular calcium were noted as signiﬁcant eﬀects of the inoculation with sera from ALS patients. Since these parameters are closely associated with the activity of the synapses, their cross-relation was analyzed as well (Figure 6). In Figure 6, each point represents patients, except the “untreated control” points, which display values derived from individual mice, to illustrate the reproducibility of the histochemical method. The data points are split into three main groups: controls, ALS patients and ALS outliers, representing the patients with the C9ORF72 mutation. A K-means statistical cluster analysis conﬁrmed the presence of these distinct clusters: (i) one represented the untreated mice and the healthy volunteers, (ii) another shows all ALS mutations, except the C9ORF72, (iii) and the last cluster represented the patients with a C9ORF72 mutation (Figure 6). 7 of 18 of 18 7 of 18 Int. J. Mol. Sci. 2020, 21, 5566 I t J M l S i 2020 21 5566 Int. J. Mol. Sci. 2020, 21, 5566 Figure 5. Volume density of synaptic vesicles in the active zones of neuromuscular synapses of mice inoculated with sera from amyotrophic lateral sclerosis (ALS) patients. All sera from ALS patients induced a significant increase in the number of active zone synaptic vesicles. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least significant difference post-hoc pairwise comparison. : p < 0.05; : p < 0.01; : p < 0.001. Figure 5. Volume density of synaptic vesicles in the active zones of neuromuscular synapses of mice inoculated with sera from amyotrophic lateral sclerosis (ALS) patients. All sera from ALS patients induced a signiﬁcant increase in the number of active zone synaptic vesicles. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least signiﬁcant diﬀerence post-hoc pairwise comparison. : p < 0.05; : p < 0.01; : p < 0.001. Figure 5. Volume density of synaptic vesicles in the active zones of neuromuscular synapses of mice inoculated with sera from amyotrophic lateral sclerosis (ALS) patients. All sera from ALS patients induced a significant increase in the number of active zone synaptic vesicles. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least significant difference post-hoc pairwise comparison. : p < 0.05; : p < 0.01; : p < 0.001. Figure 5. Volume density of synaptic vesicles in the active zones of neuromuscular synapses of mice inoculated with sera from amyotrophic lateral sclerosis (ALS) patients. All sera from ALS patients induced a significant increase in the number of active zone synaptic vesicles. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least significant difference post-hoc pairwise comparison. : p < 0.05; : p < 0.01; : p < 0.001. Figure 5. Volume density of synaptic vesicles in the active zones of neuromuscular synapses of mice inoculated with sera from amyotrophic lateral sclerosis (ALS) patients. All sera from ALS patients induced a signiﬁcant increase in the number of active zone synaptic vesicles. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least signiﬁcant diﬀerence post-hoc pairwise comparison. : p < 0.05; : p < 0.01; : p < 0.001. Figure 5. Volume density of synaptic vesicles in the active zones of neuromuscular synapses of mice inoculated with sera from amyotrophic lateral sclerosis (ALS) patients. All sera from ALS patients induced a significant increase in the number of active zone synaptic vesicles. Data are represented as the mean value ± standard error of the mean (s.e.m.). Statistical evaluation was determined using a one-way analysis of the variance (ANOVA) with the least significant difference post-hoc pairwise comparison. : p < 0.05; : p < 0.01; *: p < 0.001. Figure 6. The active zone synaptic vesicle density is plotted against the volume density of the calcium- containing electron-dense deposits (EDDs). Since all amyotrophic lateral sclerosis (ALS) sera treatments resulted in a mutual increase in calcium levels and the density of synaptic vesicles, their combined values could form new groups (blue ellipse) separated from controls (yellow ellipse). 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera The points representing the patients with the chromosome 9 open reading frame 72 (C9ORF72) mutation were sorted into a stand-alone cluster (represented by the red ellipse), and this group is beyond the 99.5% confidence regions of the control and the ALS groups represented by the covered area of the yellow and blue confidence ellipses. Figure 6. The active zone synaptic vesicle density is plotted against the volume density of the calcium- containing electron-dense deposits (EDDs). Since all amyotrophic lateral sclerosis (ALS) sera treatments resulted in a mutual increase in calcium levels and the density of synaptic vesicles, their combined values could form new groups (blue ellipse) separated from controls (yellow ellipse). The points representing the patients with the chromosome 9 open reading frame 72 (C9ORF72) mutation were sorted into a stand-alone cluster (represented by the red ellipse), and this group is beyond the 99.5% confidence regions of the control and the ALS groups represented by the covered area of the yellow and blue confidence ellipses. Figure 6. The active zone synaptic vesicle density is plotted against the volume density of the calcium-containing electron-dense deposits (EDDs). Since all amyotrophic lateral sclerosis (ALS) sera treatments resulted in a mutual increase in calcium levels and the density of synaptic vesicles, their combined values could form new groups (blue ellipse) separated from controls (yellow ellipse). The points representing the patients with the chromosome 9 open reading frame 72 (C9ORF72) mutation were sorted into a stand-alone cluster (represented by the red ellipse), and this group is beyond the 99.5% conﬁdence regions of the control and the ALS groups represented by the covered area of the yellow and blue conﬁdence ellipses. Fi 6 Th ti ti i l d it i l tt d i t th l d it f th l i naptic vesicle density is plotted a containing electron-dense deposits (EDDs). Since all amyotrophic lateral sclerosis (ALS) sera treatments resulted in a mutual increase in calcium levels and the density of synaptic vesicles, their combined values could form new groups (blue ellipse) separated from controls (yellow ellipse). The points representing the patients with the chromosome 9 open reading frame 72 (C9ORF72) mutation were sorted into a stand-alone cluster (represented by the red ellipse), and this group is beyond the 99.5% confidence regions of the control and the ALS groups represented by the covered area of the yellow and blue confidence ellipses. Figure 6. 3. Discussion Calcium, a ubiquitous second messenger, plays an integral physiological role as summarized by Otto Loewi in his famous saying “Yes, calcium is everything” (1959). It has also been proposed to play a role in pathological situations [29]. However, starting from the early 1980s, based on the pioneering studies of Dennis W. Choi on neocortical cultures [30,31], an autonomous “calcium hypothesis” has been developed, since, according to his observations, the “toxicity of glutamate on cortical neurons may depend primarily on the presence of extracellular calcium, probably through a mechanism which is distinct from simple excitotoxicity” [32]. Nowadays it is widely accepted that increased calcium levels are a key factor not only in acute injuries [10,33] but also in the pathobiology of neurodegenerative diseases [19,21,34–37]. The role of calcium in the pathomechanism of diﬀerent neurodegenerative diseases has been directly or indirectly evidenced in their models, such as in Alzheimer’s disease [38], Parkinson’s disease [36], or ALS [39,40]. However, a direct demonstration of altered calcium levels in degenerating human nerve cells has been hampered due to the absence of brain biopsy samples for calcium studies, because of evident ethical concerns, and as autopsy samples are not suitable for this purpose since the fast diﬀusion of intracellular elements signiﬁcantly alters their original distribution. ALS, however, is unique compared to other neurodegenerative diseases, since, due to the anatomy of the motor system, parts of the degenerating motor neurons residing in the skeletal muscles can be sampled with routine muscle biopsies [24]. In our original study, based on the results of muscle biopsies obtained from seven sporadic ALS patients, increased calcium levels and an increased synaptic vesicle number could be demonstrated in the motor axon terminals. These parameters separated the patients from the control population [26]. Similar alterations could be transferred to mice via inoculation of the total sera or IgG obtained from these patients [17,28]. In the present experiment, to prove that similar alterations may exist in the rest of the population of ALS patients, sera from ALS patients with identiﬁed mutations were collected, inoculated into mice, and the motor axon terminals in the hindlimb interosseous muscles were analyzed. For these experiments, instead of IgG, serum was used for injections, as in our previous study [17], because it exhibits more complex and stronger biological eﬀects than the isolated and thus partially inactivated IgG. 2 1 Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera 2.1. Ultrastructural Alterations of the Motor Axon Terminals after Inoculation with ALS Sera The active zone synaptic vesicle density is plotted against the volume density of the calcium- containing electron-dense deposits (EDDs). Since all amyotrophic lateral sclerosis (ALS) sera treatments resulted in a mutual increase in calcium levels and the density of synaptic vesicles, their combined values could form new groups (blue ellipse) separated from controls (yellow ellipse). The points representing the patients with the chromosome 9 open reading frame 72 (C9ORF72) mutation were sorted into a stand-alone cluster (represented by the red ellipse), and this group is beyond the 99.5% confidence regions of the control and the ALS groups represented by the covered area of the yellow and blue confidence ellipses. Figure 6. The active zone synaptic vesicle density is plotted against the volume density of the calcium-containing electron-dense deposits (EDDs). Since all amyotrophic lateral sclerosis (ALS) sera treatments resulted in a mutual increase in calcium levels and the density of synaptic vesicles, their combined values could form new groups (blue ellipse) separated from controls (yellow ellipse). The points representing the patients with the chromosome 9 open reading frame 72 (C9ORF72) mutation were sorted into a stand-alone cluster (represented by the red ellipse), and this group is beyond the 99.5% conﬁdence regions of the control and the ALS groups represented by the covered area of the yellow and blue conﬁdence ellipses. Int. J. Mol. Sci. 2020, 21, 5566 8 of 18 3. Discussion In vitro, our ﬁndings are also compatible with the physiological abnormality of increased miniature endplate potentials, which are depolarizations of the postsynaptic terminal caused by the release of a single vesicle into the synaptic cleft, which could be induced by the passive transfer of IgG [56] or sera [57] from ALS patients. The present ﬁndings demonstrate that, according to the mutual eﬀect of the sera from ALS patients with known mutations on the number of synaptic vesicles and the level of intracellular calcium, the patients can be sorted into clusters that diﬀerentiate them from the controls (Figure 6). Although it lies beyond the scope of the present study to analyze the eﬀect of the sera obtained from patients with diﬀerent mutations, it is noteworthy that the eﬀect of the sera obtained from patients with the C9ORF72 mutation exceeds the eﬀect of all other sera from patients with identiﬁed mutations (Figure 6). The extraordinary eﬀect of this mutation might be attributed to both the loss of function of the C9ORF72 protein and toxic gain of function from the C9ORF72 repeat ribonucleic acid (RNA), or from dipeptide repeat proteins produced by a repeat-associated non-ATG translation [58]. Mutations in this gene have a strong immune/inﬂammatory eﬀect due to a generalized systemic pro-inﬂammatory state, which initiates autoimmunity-related degenerative pathways and microglia activation in the C9ORF72 knock-out model of ALS [59], without a regression of the motor function [60]. In summary, it should be noted that starting from the original observation that calcium is indeed elevated in the degenerating nerve cells of humans [26], which can be replicated in diﬀerent models of ALS [28,61], several lines of study have been initiated, investigating this pathobiological process as a possible neuroprotective strategy in ALS. For example, based on the recognition that certain calcium-binding proteins may lend resistance to neurons [62–64], double transgenic parvalbumin × mutant SOD1 mice were bred, overexpressing the parvalbumin protein in spinal motor neurons, which delayed disease onset in the mice, but could not ultimately rescue them [65]. It was a reasonable assumption that increasing the calcium buﬀering capacity of the cells would increase their tolerance, but, per deﬁnition, sooner or later saturation occurs if the stress endures. However, an alternative therapeutic approach was based on reducing the calcium inﬂux through calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, which has a speciﬁc alteration in ALS [66]. 3. Discussion The degenerative eﬀect of ALS serum partially comes from the presence of autoimmune IgG antibodies, which were characterized in another study where a panel of 20 IgG antibodies speciﬁc for ALS were identiﬁed from the sera of ALS patients [41]. Furthermore, puriﬁed IgG can initiate motoneuronal calcium accumulation, as was demonstrated by Pullen and his coworkers [42,43]. ALS IgG also induced selective motor neuron apoptosis in rat mixed primary spinal cord cultures [44]. Immunoglobulins from patients with sporadic ALS can bind [45] and alter the function of L-type and P-type, as well as other neuronal calcium channels [46–49]. These antibodies induce ultrastructural degeneration in motor neurons and an increase in intracellular calcium [50], as well as an increase in the frequency of miniature endplate potentials [48] and glutamate levels in the cerebrospinal ﬂuid [51]. The intraperitoneal administration of IgG from ALS patients and the immunized goat model of ALS increased TNF-α, IL-6, and IL-10 levels in the spinal cord and the serum of inoculated mice [52]. The pathomechanistic role of ALS IgG in the sera of the patients seems to be essential since, if the IgG is removed by pre-incubating it with anti-human IgG or with the putative target of the antibodies, the pathobiological eﬀects completely vanish [48]. Besides the eﬀect on motor neurons, the various speciﬁc eﬀects of ALS IgG were described in other cell types, also playing a role in the pathomechanism of ALS. These antibodies are capable of inducing oxidative stress and an upregulation of the antioxidative system in a BV-2 microglial cell line [53] and also aﬀect cytosolic calcium homeostasis in cultured rat astrocytes [54]. Nevertheless, sera from two sporadic ALS patients were also included in the present study, which proved that identical results could be obtained from these patients as in the previous study. The present results, together with our earlier ﬁndings [26], provide the basis for the assumption that in ALS, regardless of the primary cause of the disease, calcium plays a central role in the pathomechanism. The increased number of synaptic Int. J. Mol. Sci. 2020, 21, 5566 9 of 18 vesicles and the simultaneously elevated levels of intra-terminal calcium are congruent with the needle electromyography ﬁndings in ALS patients with spontaneous and involuntary discharges, which may arise from the motoneuron or its axon [55]. 3. Discussion After some promising results in SOD1 transgenic mice with the drug Talampanel [67], which was also introduced in clinical trials, it turned out, that its protective eﬀect against calcium overload was eﬀective only if the treatment was started before the appearance of clinical symptoms [68]. Our present results conﬁrm the central role of calcium in the pathomechanism of ALS and probably in other neurodegenerative diseases. This phenomenon may unify the diﬀerent pathomechanisms into a self-perpetuating cascade [25] responsible for the identical clinical manifestation of diﬀerent forms of ALS, regardless of whether the patients possess mutations for the disease, which underlines the necessity of early diagnostic markers for eﬀective treatments. 4.2. Patients Patient mALS 9 with an SQSTM1 mutation, patient mALS 10 with a CCNF mutation, patient mALS 11 with a UBQLN2 mutation, and patient mALS 12 with a NEK1 mutation, together with TBK1 mutations, were evaluated. ALS patients screened for 35 major ALS related genes with known mutations were targeted in next-generation sequencing posteriorly [17]. g y The genomic DNAs were isolated from blood samples. The entire coding region of the SOD1 genes and the ﬂanking introns were ampliﬁed and the polymerase chain reaction products underwent a direct sequencing on an ABI 3100 sequencer (Thermo Scientiﬁc; Waltham, MA, USA) and were compared with the wild-type sequences on the Ensemble Genome Browser [69]. The patients who carried the GGGGCC repeat expansion were also screened for the “risk” haplotype, the rs3849942 variant, which was used as a marker for the “risk” haplotype for the patient and control genotypes. Rs3849942 genotyping was based on allelic discrimination assays using TaqMan chemistry (Life Technologies; Budapest, Hungary) [69]. In patient mfALS 4 in Table 1, the mutation screening revealed a mutation located in the signal peptide (M24I) together with the SOD1 pLeu144Phe mutations. Patient mALS 9 with an SQSTM1 mutation, patient mALS 10 with a CCNF mutation, patient mALS 11 with a UBQLN2 mutation, and patient mALS 12 with a NEK1 mutation, together with TBK1 mutations, were evaluated. ALS patients screened for 35 major ALS related genes with known mutations were targeted in next-generation sequencing posteriorly [17]. The diagnoses fulﬁlled the El Escorial revisited [55] and the Awaji [70] criteria for ALS. The patients were followed up several times and their disease progressions were recorded with the revised ALS functional rating scale (ALSFRS-R) [71]. The blood samples taken from the cubital veins were frozen, partly as whole blood and partly as sera after centrifugation, and stored until use at –80 ◦C. Only patient mALS12 had frontotemporal dementia. The scores of the Mini-Mental State Examination (MMSE) were in the normal range, except patient mALS 12 who had progressive frontotemporal dementia (Table 1). Initial symptoms in four patients with SOD1 mutations appeared as lower extremity weaknesses. In the ﬁfth one, the initial symptoms involved all of the extremities. The disease in patient mALS 2 had progressed over 12 years with the mutation of SOD1 pAsp90Ala. 4.1. Ethics Approval and Consent to Participate Ethical approvals for the studies involving animals were given by (1) The Government Oﬃce in Csongrád-Csanád County, Hungary; (2) The Committee for Animal Experiments of the University of Szeged, Szeged, Hungary I. 74-II/2015 (14 January 2015). All experiments were carried out in accordance with the institutional guidelines for the use and care of experimental animals and the governmental law for animal protection (XXVIII. chapter IV. paragraph 31) which conforms to international laws and policies (EEC Council Directive 86/609, OJL 358 1 DEC. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, United States National Research Council, revised 1996). All eﬀorts were made to minimize animal suﬀering throughout the experiments. Int. J. Mol. Sci. 2020, 21, 5566 10 of 18 Ethical approval for obtaining blood from patients and controls, for research purposes, and storing it anonymously with the written informed consent of patients and controls was given by The Human Investigation Review Board, University of Szeged, Hungary, in agreement with the declaration of the Medical World Federation proclaimed in Helsinki 1964. (Project title: Search for Biomarkers in Neurodegenerative Diseases: Amyotrophic Lateral Sclerosis, Parkinson’s Disease and Alzheimer’s Disease #2557/2009 (29 June 2009, revised 23 January 2012). 4.2. Patients Fourteen ALS patients (Table 1) and three healthy controls participated with informed consent in the study. Five ALS patients possessed mutations in the SOD1 gene. Three patients had a GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene. One patient had a mutation in the SQSTM1 gene, another one had a mutation in the CCNF gene, a further one had a mutation in the UBQLN2 gene and ﬁnally, a patient with ALS and frontotemporal dementia had a double mutation in the NEK1 gene together with a mutation in the TBK1 gene (Table 1). Two patients, composing the sporadic ALS group, were genetically screened for all 35 major ALS genes and proved to be negative (Table 1). For controls, sera from 3 age-matched healthy volunteers were obtained. Untreated animals also served as controls. Fourteen ALS patients (Table 1) and three healthy controls participated with informed consent in the study. Five ALS patients possessed mutations in the SOD1 gene. Three patients had a GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene. One patient had a mutation in the SQSTM1 gene, another one had a mutation in the CCNF gene, a further one had a mutation in the UBQLN2 gene and ﬁnally, a patient with ALS and frontotemporal dementia had a double mutation in the NEK1 gene together with a mutation in the TBK1 gene (Table 1). Two patients, composing the sporadic ALS group, were genetically screened for all 35 major ALS genes and proved to be negative (Table 1). For controls, sera from 3 age-matched healthy volunteers were obtained. Untreated animals also served as controls. The genomic DNAs were isolated from blood samples. The entire coding region of the SOD1 genes and the ﬂanking introns were ampliﬁed and the polymerase chain reaction products underwent a direct sequencing on an ABI 3100 sequencer (Thermo Scientiﬁc; Waltham, MA, USA) and were compared with the wild-type sequences on the Ensemble Genome Browser [69]. The patients who carried the GGGGCC repeat expansion were also screened for the “risk” haplotype, the rs3849942 variant, which was used as a marker for the “risk” haplotype for the patient and control genotypes. Rs3849942 genotyping was based on allelic discrimination assays using TaqMan chemistry (Life Technologies; Budapest, Hungary) [69]. In patient mfALS 4 in Table 1, the mutation screening revealed a mutation located in the signal peptide (M24I) together with the SOD1 pLeu144Phe mutations. 4.2. Patients The patient with the fast progression (6 months) had a novel heterozygous mutation (c.275_276delAA, pLys91ArgfsTer8) located in the fourth exon of the SOD1 gene and led to a frameshift with the insertion of 8 novel amino acids and the formation of a premature stop codon at the new amino acid position 99. Patient mALS 1 had a SOD1 mutation together with an angiogenin (ANG) mutation and lived one year after the onset of symptoms. Patient mfALS 3 and mfALS 4 survived for 2 years and 3 years after the appearance of the symptoms, respectively. The patients with a C9ORF72 repeat expansion had a short disease course initiated with bulbar (6, 8 and 12 months). The remaining four patients with genetic alterations also had a short (6 months) disease course. The two sporadic ALS cases without genetic alterations in the major ALS genes survived 9 months and 1 year after the diagnosis was established. 11 of 18 Int. J. Mol. Sci. 2020, 21, 5566 Table 1. Summary of the clinical data of the patients. Table 1. Summary of the clinical data of the patients. 4.3. Passive Transfer with Human Sera and Tissue Preparation 4.3. Passive Transfer with Human Sera and Tissue Preparation 4.3. Passive Transfer with Human Sera and Tissue Preparation Altogether, 48 male Balb/c mice, obtained from Charles River Appoints AnimaLab Hungary Kft. (Vác, Hungary), were injected intraperitoneally with 1 mL/day serum from ALS patients with diﬀerent mutations (n = 3) or healthy serum (n = 3) for two days. All animals received sera from the same patient during the inoculation period. However, animals treated with serum from diﬀerent patients with the same mutation (patients mfALS 3 and 4; patient mALS 6, 7 and 8) were pooled together, since there was no statistical diﬀerence between the eﬀect of the sera from diﬀerent patients with the same mutation. A similar pooling protocol was applied to animals treated with sera from sporadic patients (patient sALS 1,2). One group of animals did not receive an injection and was used as the untreated control (n = 3). During the two-day inoculation period, animals were housed in plastic cages (5 animals/cage, at most) in a thermoneutral environment (22 ± 3 ◦C) and 12 h light/dark cycle with access to drinking water and regular rodent chow ad libitum. Twenty-four hours after the second serum injection, muscle samples from animals were prepared from the animals for the analytical study of calcium by an electron microscope, as described originally by Borgers and coworkers [72,73]. The method, as adapted and regularly tested for the speciﬁcity of calcium in our laboratory, ensures a good preservation of the tissue suitable for electron microscopy and results in the EDDs due to the precipitation of tissue calcium by the ﬁxative [74–78]. First, animals under terminal anesthesia (avertin; 2,2,2-tribromoethanol, Merck, Darmstadt, Germany; 240 mg/kg body weight in a 1.0 mL volume i.p.) were transcardially perfused with 90 mM potassium oxalate (Merck; pH adjusted to 7.4 with KOH) followed by 3% glutaraldehyde (Polysciences Inc., Warrington, PA, USA; pH adjusted to 7.4 with KOH) containing 90 mM of potassium oxalate (pH 7.4). The hindlimb interosseus muscles were removed and ﬁxed in the same ﬁxative overnight (4 ◦C). Specimens were then rinsed in 7.5% sucrose (Molar Chemicals Kft., Budapest, Hungary) containing 90 mM of potassium oxalate (pH 7.4), postﬁxed with 2% potassium pyroantimonate (Merck) +1% osmic acid (Sigma; pH adjusted to 7.4 with acetic acid (Molar Chemicals Kft.)) for 2 h (4 ◦C). 4.2. Patients Patients Age at Onset (Years) Duration of the Disease at the Study Initial Symptoms Clinical Signs ALS FRS-R MMSE Genetic Alteration Family History Therapy Other Disease mALS 1 63 1 year proximal bilateral lower limb weakness LMN, UMN 32/48 28/30 SOD1 pVal14 Met ANG Met24Ile negative Riluzole, Perindopril, Aspirin, Piracetam, Vinpocetine, Nebivolol atherosclerosis, hypertension mALS 2 75 12 years bilateral lower limb weakness LMN, UMN, B 19/48 29/30 SOD1 pAsp90Ala negative Riluzole cervical and lumbar spondylarthrosis, hyperlipidemia mfALS 3 29 2 years gait disturbance LMN, UMN 36/48 30/30 SOD1 pLeu144Phe grandmother (fraternal) Riluzole - mfALS 4 49 3 years distal weakness of lower limbs LMN, UMN, B, PB 25/48 28/30 SOD1 pLeu144Phe grandmother (maternal) Riluzole, Citalopram depression, lumbar discs’ herniation mALS 5 67 6 months four limbs weakness B, PB, UMN, LMN 39/48 30/30 SOD1 pLys91Arg fs Ter8 negative Riluzole, Atorvastatin, Valsartan breast cancer (irradiated 8 years ago), hypercholesterolemia, cervical and lumbar discs’ protrusion mALS 6 68 6 months bilateral peroneal palsy, dysarthria LMN, B, UMN 44/48 30/30 C9ORF72 repeat expansion negative Alprazolam, Perindopril, Duloxetine hyperparathyroidism (cured), generalized lipomatosis, osteoporosis, hypertension, depression mALS 7 55 1 year dysarthria, dysphagia B, PB, LMN, UMN 37/48 27/30 C9ORF72 repeat expansion negative Riluzole, L-thyroxin Hashimoto’s thyroiditis mfALS 8 56 8 months dysarthria, dysphagia B, PB, LMN, UMN 36/48 30/30 C9ORF72 repeat expansion mother with suspected ALS (not documented) Riluzole, L-thyroxin hypothyroidism mALS 9 54 6 months dyspnea B, PB, LMN, UMN 40/48 30/30 SQSTM1 pPro392Leu negative Valsartan-HCT hypertension mALS 10 61 6 months UMN, LMN lesions in the lower limbs LMN, UMN, B 42/48 30/30 CCNF pLeu106Val negative Valsartan, Riluzole hypertension, cervical and lumbar discs’ protrusion mALS 11 65 6 months four limbs weakness LMN, UMN 43/48 29/30 UBQLN2 pMet392Val negative Riluzole hypertension depression mALS 12 37 6 months four limbs weakness, dysarthria, cognitive deﬁcit UMN, LMN, B, PB 39/48 23/30 NEK1 pSer261His TBK1 pLys631 deletion negative Riluzole, Perindopril, Paroxetine hypertension depression, frontotemporal dementia sALS1 71 1 year weakness of the right arm and leg (peroneal) UMN, LMN 41/48 28/30 - negative Piracetam, Diclofenac, Aspirin, Perindopril, Isosorbide-mononitrate, Bisoprolol hypertension, hypercholesterolemia, atherosclerosis, post zoster neuralgia sALS2 74 9 months dysarthria, dysphagia B, UMN, LMN 39/48 26/26 - negative Amlodipine, Perindopril, Metoprolol, Atorvastatin, Riluzole hypertension, hypercholesterolemia mALS: ALS with identiﬁed mutation; mfALS: familial ALS with identiﬁed mutation; sALS: sporadic ALS; LMN: lower motor neuron; UMN: upper motor neuron; B: bulbar; PB: pseudobulbar; ALSFRS-R: ALS functional rating scale revised; MMSE: Mini-Mental State Examination. 4.2. Patients Int. J. Mol. Sci. 2020, 21, 5566 12 of 18 12 of 18 4.3. Passive Transfer with Human Sera and Tissue Preparation Next, specimens were rinsed in distilled water (pH adjusted to 10 with KOH) for 10 min, dehydrated in a graded series of ethanol (Molar Chemicals Kft.), processed through propylene oxide (Merck), and embedded in Durcupan ACM (Merck). Blocks were polymerized for 48 h at 56 ◦C. Semithin (0.3 µm) sections were cut from the blocks on an Ultracut UCT ultramicrotome (Leica, Wetzlar, Germany), etched [79] and stained [80], and then evaluated under an Eclipse 80i light microscope (Nikon, Tokyo, Japan) to identify the zones of innervation in the muscles. After trimming the blocks to the appropriate regions, to avoid personal bias in determining the ﬁelds for analysis [81], systematic random sets of ultrathin sections (50 nm) were prepared. The distance between the sections was set to 15 µm to circumvent repeated sampling of identical neuromuscular synapses during the collection of electron microscopic images. Sections were mounted on single-hole formvar coated copper grids (Electron Microscopy Sciences, Hatﬁeld, PA, USA), contrasted with uranyl acetate (Electron Microscopy Sciences; 2% in 50% ethanol) [82] and lead citrate (Electron Microscopy Sciences; 2% in distilled water) [83]. 4.4. Quantiﬁcation of the Intracellular Calcium Levels in the Motor Axon Terminals Author Contributions: Conceptualization, J.I.E. and L.S.; Data curation, R.P., T.F.P. and B.N.; Formal analysis, R.P., T.F.P. and K.T.; Funding acquisition, L.S.; Investigation, V.M., T.F.P., B.N., L.K., R.K., K.S., K.T. and I.O.; Methodology, R.P., M.S. and L.S.; Project administration, R.P., M.S. and J.I.E.; Resources, R.P.; Supervision, L.S.; Validation, M.S., J.I.E. and L.S.; Visualization, V.M., L.K., R.K. and K.S.; Writing—original draft, V.M. and R.P.; Writing—review and editing, J.I.E. and L.S. All authors have read and agreed to the published version of the manuscript. 4.5. Quantiﬁcation of the Density of Synaptic Vesicles In addition to the calcium content in the axon terminals, changes in the synaptic vesicle density in the active zones were also analyzed and were expressed quantitatively. Electron microscopic images of 15 axon terminals were evaluated for each animal, where all active zones of the neuromuscular junction were quantiﬁed individually. Although the determination of the active zones for these measurements was arbitrary—by the symmetrical adjustment of a 100 nm wide and 200 nm long rectangle for the postsynaptic junction—this method was consistently applied during quantiﬁcation, and adapted from the analysis of the human motor axon terminals [26]. The volume of the examined region was calculated using the area of the selected rectangle and the known section thickness. Subsequently, the number of synaptic vesicles was determined in the created cuboid. Synaptic vesicles were measured if their center was within the examined volume. Results were averaged per animal, then group averages were calculated using the individual values of the animals, which were expressed as volume densities. 4.4. Quantiﬁcation of the Intracellular Calcium Levels in the Motor Axon Terminals Ultrathin sections were examined using a JEM-1400Flash transmission electron microscope (JEOL, Tokyo, Japan). Sections from each animal were systematically screened at low magniﬁcation (500–2000×) for the presence of neuromuscular synapses until 15 axon terminals could be collected for microscopic analysis. Axon terminals were recorded as 16-bit grayscale images at an instrumental magniﬁcation of 12,000× with a 2k × 2k high-sensitivity scientiﬁc complementary metal-oxide-semiconductor camera (Matataki Flash sCMOS, JEOL) and saved in a tagged image ﬁle format. The relative volume of the axon terminals occupied by the EDDs representing the calcium precipitates was determined by point counting methods [84,85], which were modiﬁed for these unique structures and photographic conditions [75]. The recorded pictures were analyzed with the built-in modules of Image-Pro Plus (Media Cybernetics; Rockville, MD, USA) image analysis software. The tessellation of sampling Int. J. Mol. Sci. 2020, 21, 5566 13 of 18 13 of 18 points was superimposed onto each electron microscopic image, then sampling points hitting the axon terminals in each image served as reference areas and were counted. Sampling points hitting the EDDs within the reference area were counted as well. The corresponding counts obtained in the individual ﬁelds were summed up throughout the series of the identiﬁed axon terminals in each animal [86]. The appropriate ratios expressing the relative amount of the EDDs within these structures were calculated for each animal. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.6. Statistical Analysis To determine the average volume occupied by EDDs within the axon terminals, and the synaptic vesicle density, the data derived from individual electron microscopic ﬁelds were pooled according to animals and passive transfer groups. Fifteen ﬁelds of view for each were analyzed in the motor axon terminals from each animal. Diﬀerences among the multiple means of the volume density of the EDDs were assessed by a one-way analysis of variance (ANOVA), followed by the least signiﬁcant diﬀerence post-hoc test. All of the statistical analyses were performed with R (version 3.6.2, R Foundation for Statistical Computing, Vienna, Austria) and with R Studio Integrated Development Environment (version 1.1.453, RStudio Inc., Boston, MA, USA) for Windows. All data are presented as mean values ± the standard error of the means (s.e.m.). A cluster analysis was performed using the K-means clustering algorithm with WEKA data mining software (v3.8.3., Waikato, New Zealand) to investigate the cross-relationship of the volume density of EDDs and synaptic vesicles in patients with diﬀerent ALS genotypes and controls. Since the analysis resulted in distinct clusters, the Jaccard similarity index was not calculated, but the 99.5% conﬁdence regions of the control and ALS groups were evaluated and represented in Figure 6 as conﬁdence ellipses. Author Contributions: Conceptualization, J.I.E. and L.S.; Data curation, R.P., T.F.P. and B.N.; Formal analysis, R.P., T.F.P. and K.T.; Funding acquisition, L.S.; Investigation, V.M., T.F.P., B.N., L.K., R.K., K.S., K.T. and I.O.; Methodology, R.P., M.S. and L.S.; Project administration, R.P., M.S. and J.I.E.; Resources, R.P.; Supervision, L.S.; Validation, M.S., J.I.E. and L.S.; Visualization, V.M., L.K., R.K. and K.S.; Writing—original draft, V.M. and R.P.; Writing—review and editing, J.I.E. and L.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was partially supported by the National Research, Development and Innovation Oﬃce of Hungary through the GINOP-2.3.2-15-2016-00001, GINOP-2.3.2-15-2016-00034 and GINOP-2.3.3.-15-2016-00001 programs. This research work was conducted with the support of the Szeged Scientists Academy under the sponsorship of the Hungarian Ministry of Human Capacities (EMMI: 11136-2/2019/FIRFIN). R.P. was supported by the “National Talent Programme” with the ﬁnancial aid of the Ministry of Human Capacities (NTP-NFTÖ-18-B-208). Acknowledgments: We thank Erika Bánﬁné Rácz for her assistance in ultramicrotomy, and Jennifer Tusz to correcting the English language of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. 14 of 18 Int. J. Mol. Sci. 4.6. Statistical Analysis 2020, 21, 5566 Abbreviations ALS Amyotrophic lateral sclerosis ALSFRS-R Revised amyotrophic lateral sclerosis functional rating scale AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ANG Angiogenin ANOVA Analysis of variance B Bulbar C9ORF72 Chromosome 9 open reading frame 72 CCNF G2/mitotic-speciﬁc cyclin F EDDs Electron-dense deposits IgG Immunoglobulin G LMN Lower motor neuron mALS Amyotrophic lateral sclerosis with an identiﬁed mutation mfALS Familial amyotrophic lateral sclerosis with an identiﬁed mutation MMSE Mini-Mental State Examination NEK1 NIMA-related kinase 1 PB Pseudobulbar RNA Ribonucleic acid sALS Sporadic amyotrophic lateral sclerosis s.e.m. Standard error of the mean SOD1 Superoxide dismutase 1 SQSTM1 Sequestosome 1 TBK1 TANK-binding kinase 1 UBQLN2 Ubiquilin 2 UMN Upper motor neuron References 1. Rowland, L.P.; Shneider, N.A. Amyotrophic lateral sclerosis. N. Eng. J. Med. 2001, 344, 1688–1700. [CrossRef] [PubMed] 1. 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https://openalex.org/W2771134477	https://mediatum.ub.tum.de/doc/1439080/document.pdf	English	null	Corrigendum to “Fatal Systemic Vasoconstriction in a Case of Metastatic Small-Intestinal NET”	Case reports in gastrointestinal medicine	2,017	cc-by	727	Jochen Stenzel,1 Sebastian Noe,2 Konstantin Holzapfel,3 Franziska Erlmeier,4 and Florian Eyer1 1Department of Clinical Toxicology, Klinikum rechts der Isar, Technical University of Munich, Ismaningerstrasse 2 81675 Munich, Germany Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technical University of Munich, smaningerstrasse 22, 81675 Munich, Germany 4Institute of Pathology, Klinikum rechts der Isar, Technical University of Munich, Ismaningerstrasse 22, 81675 Munich, Germany Correspondence should be addressed to Jochen Stenzel; j.stenzel@tum.de eceived 16 November 2017; Accepted 21 November 2017; Published 3 December 2017 Received 16 November 2017; Accepted 21 November 2017; Published 3 December 2017 Copyright © 2017 Jochen Stenzel 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. Copyright © 2017 Jochen Stenzel 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. In the article titled “Fatal Systemic Vasoconstriction in a Case of Metastatic Small-Intestinal NET” [1], the first and last names of all the authors were reversed. The revised authors’ list is shown above and updated in place. Hindawi Case Reports in Gastrointestinal Medicine Volume 2017, Article ID 8694296, 1 page https://doi.org/10.1155/2017/8694296 References [1] J. Stenzel, S. Noe, K. Holzapfel, F. Erlmeier, and F. Eyer, “Fatal systemic vasoconstriction in a case of metastatic small- intestinal NET,” Case Reports in Gastrointestinal Medicine, vol. 2017, Article ID 9810194, 6 pages, 2017. 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https://openalex.org/W3033696799	https://link.springer.com/content/pdf/10.1007/s00261-020-02599-z.pdf	English	null	Portosystemic shunt surgery in the era of TIPS: imaging-based planning of the surgical approach	Abdominal radiology	2,020	cc-by	7,223	Abdominal Radiology (2020) 45:2726–2735 https://doi.org/10.1007/s00261-020-02599-z Abdominal Radiology (2020) 45:2726–2735 https://doi.org/10.1007/s00261-020-02599-z HEPATOBILIARY Portosystemic shunt surgery in the era of TIPS: imaging‑based planning of the surgical approach Uli Fehrenbach1 · Safak Gül‑Klein2 · Miguel de Sousa Mendes1 · Ingo Steffen1 · Julienne Stern1,3 · Dominik Geisel1 · Gero Puhl2,4 · Timm Denecke1,5 Received: 27 March 2020 / Revised: 22 May 2020 / Accepted: 26 May 2020 © The Author(s) 2020, corrected publication 2021 * Uli Fehrenbach uli.fehrenbach@charite.de Abstract Purpose With the spread of transjugular intrahepatic portosystemic shunts (TIPS), portosystemic shunt surgery (PSSS) has decreased and leaves more complex patients with great demands for accurate preoperative planning. The aim was to evaluate the role of imaging for predicting the most suitable PSSS approach. Material and methods Forty-four patients who underwent PSSS (2002 to 2013) were examined by contrast-enhanced CT (n = 33) and/or MRI (n = 15) prior to surgery. Imaging was analyzed independently by two observers (O1 and O2) with different levels of experience (O1 > O2). They recommended two shunting techniques (vessels and anastomotic variant) for each patient and ranked them according to their appropriateness and complexity. Findings were compared with the actually performed shunt procedure and its outcome.i Results The first two choices taken together covered the performed PSSS regarding vessels in 88%/100% (CT/MRI, O1) and 76%/73% (O2); and vessels + anastomosis in 79%/73% (O1) and 67%/60% (O2). The prediction of complex surgi- cal procedures (resection of interposing structures, additional thrombectomy, use of a collateral vessel, and use of a graft interposition) was confirmed in 87%, resulting in 80% sensitivity and 96% specificity. Larger shunt vessel distances were associated with therapy failure (p = 0.030) and a vessel distance of ≥ 20 mm was identified as optimal cutoff, in which a graft interposition was used. There was no significant difference between MRI and CT in predicting the intraoperative deci- sions (p = 0.294 to 1.000). (p ) Conclusion Preoperative imaging and an experienced radiologist can guide surgeons in PSSS. CT and MRI provide the information necessary to identify technically feasible variants and complicating factors. Keywords Portosystemic shunt surgery · Magnetic resonance imaging · Computed tomography · Portal hypertension Keywords Portosystemic shunt surgery · Magnetic resonance imaging · Computed tomography · Por systemic shunt surgery · Magnetic resonance imaging · Computed tomography · Portal hypertension Abbreviations IVC Inferior vena cava O1 Observer one (more experienced) O2 Observer two (less experienced) PHT Portal hypertension PSSS Portosystemic shunt surgery PV Portal vein The study was in part presented at the Annual Conference of the European Society of Gastrointestinal and Abdominal Radiology (ESGAR) 2019 in Rome, Italy. Uli Fehrenbach and Safak Gül-Klein contributed equally to this work. Patient population Sixty-five patients who underwent PSSS between March 2002 and September 2013 were retrospectively identified from the surgical database of our hospital. Surgical reports, histology reports, cross-sectional imaging datasets, labora- tory results, and medical records were obtained to generate the endpoint parameter. The patients underwent PSSS after having been classified as unsuitable for TIPS procedure in an interdisciplinary discussion. The main reason for not choos- ing TIPS was prehepatic PHT with chronic extrahepatic por- tal vein occlusion in 31 patients (70%). In seven pediatric patients with patent portal venous flow, PSSS was preferred due to the still incomplete body growth. In two patients, PSSS was indicated because of chronic TIPS occlusion. One patient was treated with PSSS during a hemicolectomy for cecum carcinoma to avoid a further intervention. Another patient was unsuitable for TIPS because of a previously per- formed right hepatic trisectionectomy because of a Klat- skin’s tumor. In the other two patients, PSSS was preferred because of vascular anomalies (intrahepatic portal vein dys- plasia and a mesenteric arteriovenous malformation). Nine- teen of the initially identified patients were excluded because of incomplete medical records or lack of preoperative imag- ing. Two patients underwent an atypical shunt procedure, which was not part of the retrospective imaging-based evalu- ation. These two patients also had to be excluded. Forty-four patients were finally included (Fig. 1). Due to the widespread use of TIPS, PSSS is becoming less common and experience with this operation is dimin- ishing. There are many surgical portosystemic (PS) shunt options to consider in the preoperative planning (e.g., portacaval, mesocaval, splenorenal). The different PSSS techniques modify hepatopetal blood flow in different ways and can be categorized into nonselective, partially selec- tive, and selective types. Nonselective PS shunt techniques establish complete drainage of portal as well as mesenterial blood flow into the inferior vena cava. Partially selective shunt techniques maintain the PS pressure gradient and thus ensure residual blood flow in the portal vein. Selective tech- niques separate the downgradient via esophageal collater- als from the portal hypertensive system and therefore have the smallest effects on portal perfusion. Interestingly, there are no long-term differences in terms of recurrent bleeding, hepatic encephalopathy, or overall survival between the dif- ferent shunt techniques [15, 16]. This is in part attributable to a loss of selectivity over the years [17]. SMV Superior mesenteric vein TIPS Transjugular intrahepatic portosystemic shunt bypass rather than a true PS shunt. Meso-Rex shunts are of special interest in children and younger patients with extra- hepatic portal occlusion. They maintain liver function and hepatopetal flow, therefore mostly avoiding hypersplenism, coagulopathies, and hyperammonemia while at the same time allowing normal neurological development to continue [18–21]. Abstract * Uli Fehrenbach uli.fehrenbach@charite.de 1 Department of Radiology, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin 13353, Germany 2 Department of Surgery, Campus Charité Mitte \| Campus Virchow-Klinikum, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 3 Department of Radiology, Krankenhaus Nordwest, Frankfurt am Main, Germany 4 Department of Surgery, Asklepios Klnik Altona, Hamburg, Germany 5 Department of Diagnostic and Interventional Radiology, Universitätsklinikum Leipzig, Leipzig, Germany Abbreviations IVC Inferior vena cava O1 Observer one (more experienced) O2 Observer two (less experienced) PHT Portal hypertension PSSS Portosystemic shunt surgery PV Portal vein The study was in part presented at the Annual Conference of the European Society of Gastrointestinal and Abdominal Radiology (ESGAR) 2019 in Rome, Italy. Uli Fehrenbach and Safak Gül-Klein contributed equally to this work. * Uli Fehrenbach uli.fehrenbach@charite.de 2 Department of Surgery, Campus Charité Mitte \| Campu Virchow-Klinikum, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany .(123456789 3 2727 Abdominal Radiology (2020) 45:2726–2735 Introduction Transjugular intrahepatic portosystemic shunt (TIPS) has become the most common intervention used today to treat intrahepatic portal hypertension, PHT [1]. Before even being proved to be better than portosystemic shunt surgery (PSSS), TIPS quickly spread worldwide [2]. Moreover, there are various conditions in which TIPS is not feasible or even recommendable [3]. TIPS is usually not used in the treat- ment of pre- and posthepatic PHT [4]. Specifically, TIPS is not suitable for patients with extrahepatic veno-occlusive disease, in which the vascular connection between splenic and portal circulation is interrupted, or for patients with advanced hepatic cirrhosis, because of the high risk of hepatic encephalopathy [5]. Contraindications to the place- ment of a TIPS include severe cardiac disease (e.g., conges- tive heart failure), multiple hepatic cysts, systemic infection/ sepsis, or unrelieved biliary obstruction [6]. Nevertheless, these patients need to be treated, and PSSS is indicated to prevent variceal bleeding, spontaneous bacterial peritoni- tis, hepatorenal syndrome, and progressive liver failure [3, 7–14]. Nonetheless, PSSS is invasive and is associated with the intra- and perioperative risks of major abdominal vascular surgery—its major disadvantage in comparison to TIPS. Therefore, precise preoperative imaging and planning are of utmost importance. The purpose of this study was to evaluate whether CT and MRI are sufficiently accurate and reliable for the pre- operative planning of PSSS in patients who are not suitable for a TIPS procedure. With decreasing surgeon experience caused by the widespread of TIPS, a radiologist may be in an important position to assist the surgical team with pre- surgical planning to help guide patient management. Planning of portosystemic shunt surgery Preoperative images of all 44 patients—contrast-enhanced CT (n = 33) and/or MRI datasets (n = 15; n = 4 patients with both CT and MRI)—were analyzed on a dedicated PACS viewing workstation (Centricity, GE Healthcare, General Electric, Milwaukee, USA) by 2 radiologists with different levels of experience [Observer 1 (O1; T.D.): 12 years and Observer 2 (O2; J.S.): 4 years of experience in abdominal imaging]. Both observers were blinded to the surgical technique and outcomes. Fig. 1 Flowchart of retrospective enrollment consent (Application Number EA1/148/14) because of the retrospective study design. consent (Application Number EA1/148/14) because of the retrospective study design. The two observers evaluated patency and diameters of the portal venous system. Maximum diameters of the possible shunt vessels were measured at the suggested connection (down- and upstream) sites by the radiologists. The short- est distances between the possible shunt vessels [inferior vena cava (IVC), portal vein (PV), left and right portal vein branches, superior mesenteric vein (SMV), splenic vein (SV), and left renal vein (RV)] were measured. Patient population Since there is no “standard” shunt, the anatomy of the individual must be evaluated in each case to select the most appropriate shunt- ing procedure. Careful planning, which shunting vessels and anastomosis (e.g., end-to-side, side-to-side) to choose, is necessary to avoid shunt failure or hepatic encephalopathy caused by (too) high shunt volume. Another shunt technique besides the more common shunt variants already mentioned is the mesoportal (Meso-Rex) shunt, which actually is a i Our Institutional Review Board approved the study proto- col including adult and pediatric patients, waiving informed 1 3 1 Abdominal Radiology (2020) 45:2726–2735 2728 Fig. 1 Flowchart of retrospective enrollment Table 1 Institutional surgical algorithm that represents a synopsis of local surgeon preferences Indication Preferred shunt Acute esophageal bleeding Portacaval (end-to-side) Extrahepatic portal thrombosis Mesocaval, splenorenal (distal or side-to-side) Extrahepatic portal thrombosis in children Meso-Rex Budd–Chiari syndrome Portacaval or mesocaval Ascites Splenorenal (not distal) Possible liver transplantation candidate Portacaval or mesocaval (Gadovist®-Bayer Schering Pharma) at 1.5 T (Siemens Magnetom Avanto, Siemens Healthcare, Erlangen, Ger- many) using an eight-channel body phased-array surface coil. Due to the long observation period, the MRI proto- cols vary within the examined collective. All MRI pro- tocols included multiphase dynamic contrast-enhanced T1-weighted sequences with fat saturation in axial (arterial, portal venous, venous, and delayed phase) and coronal ori- entation (venous phase). 3 Imaging (CT and MRI) Multiphasic contrast-enhanced CT [with iopromide (Ultra- vist 370®, Bayer Schering Pharma) or with iobitridol (Xenetic 350®-Guerbet GmbH)] was performed on a 16- or 64-slice CT scanner (Light Speed Power 16 or VCT 64; GE Medical Solutions, Fairfield, CT) using a triple-phase acqui- sition technique with arterial (~ 15-s delay), portal venous (~ 40-s delay), and equilibrium phase (~ 80-s delay) acquisi- tion. The primary slice thickness was 0.625 mm. Tube volt- age was 120 kV, tube current was modulated automatically based on a noise index of 15 and a maximally allowed cur- rent of 350 mA. Maximum intensity projections (MIPs) were reconstructed for each contrast phase. Structures intervening between possible shunting ves- sels were identified in consensus by O1 and O2 as factors increasing surgical complexity (need for tissue resection before shunt creation). Other factors contributing to com- plexity included large vessel distance (> 20 mm) (necessity of graft interposition), thrombosis adjacent to connecting vessels (requires additional thrombectomy), and unavoidable use of a collateral vein instead of (occluded) major vein for establishing the shunt. Contrast-enhanced MRI was performed with gadoter- ate meglumine (Dotarem®-Guerbet GmbH) or gadobutrol An algorithm for deciding about the surgical shunt tech- nique based on clinical factors (e.g., age, indication, urgency 1 3 2729 Abdominal Radiology (2020) 45:2726–2735 of vascular decompression, vascular beds to relieve) has been proposed by the surgical team and is shown in Table 1. This algorithm takes the physiological properties of each shunt into account and was used in our retrospective analysis as a guideline rather than an absolute standard in assessing the recommendations made by our two observers. of vascular decompression, vascular beds to relieve) has been proposed by the surgical team and is shown in Table 1. This algorithm takes the physiological properties of each shunt into account and was used in our retrospective analysis as a guideline rather than an absolute standard in assessing the recommendations made by our two observers. diameterdistal shunt vessel∕diameterproximal shunt vessel diameterdistal shunt vessel∕diameterproximal shunt vessel The shunt vessel ratio, diameter of the smaller shunt ves- sel, and distance of the connected vessels were correlated with PSSS outcome. (4) Meso-Rex shunt. (4) Meso-Rex shunt. Each technique proposed in our retrospective analysis was classified as “standard” or “complex”. If any factor of com- plexity was present, the procedure was classified as complex. Each technique proposed in our retrospective analysis was classified as “standard” or “complex”. If any factor of com- plexity was present, the procedure was classified as complex. During the decision process, the two observers were blinded to the later chosen surgical procedure. The imaging-based recommendations made by the two observers were compared with the shunt procedures actually accomplished by the sur- geons treating the patients included in our analysis. i During the decision process, the two observers were blinded to the later chosen surgical procedure. The imaging-based recommendations made by the two observers were compared with the shunt procedures actually accomplished by the sur- geons treating the patients included in our analysis. a Hepatorenal syndrome, hepatic encephalopathy, spontaneous bacte- rial peritonitis The success of PSSS was evaluated during postopera- tive hospitalization until discharge (< 30 days). PSSS failure was defined as early shunt occlusion or occurrence of major complications (rebleeding, organ failure, death). Imaging (CT and MRI) Table 2 Characteristics of the study patients PHT portal hypertension, PSSS portosystemic shunt surgery a Hepatorenal syndrome, hepatic encephalopathy, spontaneous bacte- rial peritonitis n % Major underlying causes of PHT Extrahepatic portal vein thrombosis 31 70 Associated with liver cirrhosis 7 Other causes (e.g., clotting disorder) 24 Liver cirrhosis 16 36 Metabolic/toxic 13 Viral hepatitis 2 Autoimmune hepatitis 1 Wilson’s disease 1 2 Rendu–Osler–Weber disease 1 2 Budd–Chiari syndrome 1 2 Post-hemihepatectomy lymph fistula 1 2 Signs and clinical findings in PHT Esophagogastric varices 40 91 Splenomegaly 31 70 Ascites 19 43 Advanced stage symptomsa 3 7 Complications of PHT and indication for PSSS Varices with previous bleeding episode 25 57 Acute variceal bleeding 2 Splenomegaly and secondary thrombocytopenia 19 43 Excessive ascites 5 11 Based on the information available in this retrospective analysis, each of the two observers made a ranked recom- mendation (first and second choice) of two shunt techniques including the most appropriate anastomosis technique: (1) Splenorenal • Anastomosis proximal end-to-side (Linton), distal end-to-side (Warren) or side-to-side (Cooley). (2) Portacaval • Anastomosis end-to-side or side-to-side. (3) Mesocaval • Anastomosis side-to-side. Results The collective consisted of 19 (43%) pediatric patients (0–21 years) with 3 infants (< 2 years), 8 children (2 to 12 years), and 8 adolescents (12 to 21 years) [22]. The mean age in adult patients (n = 25) was 44 years. Both genders were equally represented (22 each) in the study population. Table 2 summarizes diagnoses underlying PHT, signs and clinical findings on admission, and major indications for sur- gery. The average MELD score (applicable to patients ≥ 12 years) was 12 (range 7 to 24). For patients below 12 years of age, the average PELD score was 1.7 (range 0 to 10.9). In the overall collective, the average serum albumin level was 3.42 g/dl (range 2.0–5.1) [23]. To correlate the outcome of PSSS with vessel diameters, we calculated the shunt vessel ratio using the following formula: Statistical analysis Statistical analysis was performed using SPSS Statistics Ver- sion 22 (IBM, Armonk, NY, USA). The χ2 test was used to assess differences in the accuracy of CT and MRI in pre- operative planning. Cohen’s κ was used to evaluate inter- rater reliability. Continuous variables from two independent samples were evaluated using the Mann–Whitney U test. Receiver operating characteristic curve analysis was used to investigate binary classifiers. p values < 0.05 were con- sidered statistically significant. Eighteen patients had portacaval PSSS (Fig. 2), in which end-to-side was the most commonly used anastomosis. Splenorenal (Fig. 3) and mesocaval (Fig. 4) PSSS were per- formed in 15 and 11 cases, respectively (Table 3). A Meso- Rex shunt was not chosen by the surgeons in our evaluated cases. However, as it was part of the available options to the radiologists reading, it is still mentioned in Table 3. All PSSS procedures included in this study were performed or at least supervised by the same surgeon in charge. 3 Abdominal Radiology (2020) 45:2726–2735 2730 Fig. 2 A 17-year-old female PHT patient with Wilson’s disease and recurrent variceal bleeding—PSSS procedure: portacaval side-to- side; a preoperative MRI, post-contrast T1-w, b postoperative MRI, post-contrast T1-w and c postoperative MRI, T2w. Small arrow: IVC; arrowhead: portal vein; bold arrow: portacaval anastomosis Fig. 2 A 17-year-old female PHT patient with Wilson’s disease and recurrent variceal bleeding—PSSS procedure: portacaval side-to- side; a preoperative MRI, post-contrast T1-w, b postoperative MRI, post-contrast T1-w and c postoperative MRI, T2w. Small arrow: IVC; arrowhead: portal vein; bold arrow: portacaval anastomosis Fig. 3 A 71-year-old male PHT patient with excessive ascites after extended right hemihepa- tectomy (diagnosis: intrahepatic cholangiocarcinoma)—PSSS: splenorenal side-to-side; a preoperative CT, oblique MIP reconstruction and b postopera- tive CT, oblique MIP recon- struction. Small arrow: left renal vein; arrowhead: splenic vein; bold arrow: splenorenal anastomosis Fig. 4 A 49-year-old male PHT patient with liver cirrhosis, extrahe- patic portal vein thrombosis, and advanced symptoms—PSSS proce- dure: mesocaval; a preoperative CT, b postoperative CT, axial MIP reconstruction and c postoperative CT, sagittal MIP reconstruction. Small arrow: IVC; arrowhead: SMV; bold arrow: mesocaval anasto- mosis Fig. 3 A 71-year-old male PHT patient with excessive ascites after extended right hemihepa- tectomy (diagnosis: intrahepatic cholangiocarcinoma)—PSSS: splenorenal side-to-side; a preoperative CT, oblique MIP reconstruction and b postopera- tive CT, oblique MIP recon- struction. Small arrow: left renal vein; arrowhead: splenic vein; bold arrow: splenorenal anastomosis Fig. Statistical analysis PSSS procedures and radiological recommenda- tions for each patient are displayed in Supplementary Data. Surgeons actually chose a vessel connection which was not included among the first 2 choices by O1 in 5 cases (11%) and by O2 in 12 cases (26%). In 15 patients, surgeons chose a PSSS technique that was classified as complex by the radiologist. In 13 (87%) of these patients, complexity of the procedure was confirmed by the operative report. The retrospective imaging-based evalu- ation had 80% sensitivity and 96% specificity in predict- ing complex surgical procedures identified in consensus by the two radiologists. In two patients, resection of interpos- ing structures was deemed necessary by the radiologists. In one of these cases, segmental liver resection (segment 1 according to the Couinaud classification) was performed (Fig. 5a). In the second case, embolization of an intervening arteriovenous malformation was performed in preparation for PSSS. In a third patient, a segmental liver resection (seg- ment 1) that was not suggested by the radiology observers was performed. Partial thrombosis of the connecting ves- sels was identified by the radiologists in 4 patients, and thrombectomy was performed in 3 (75%) of these patients (Fig. 5b). Use of a collateral vessel was proposed and also performed in one patient (100%). In 9 patients, the distance between the connecting vessels was > 20 mm, and graft interposition was recommended by the observers. 8 (89%) of these patients actually received a graft (Fig. 5c, d). Table 4 Accuracy in predicting shunt procedure including anasto- motic variants Table 4 Accuracy in predicting shunt procedure including anasto- motic variants O1 O2 Accuracy of shunt procedures recommended on the basis of CT and/or MRI Table 4 Accuracy in predicting shunt procedure including anasto- motic variants O1 O2 Table 4 Accuracy in predicting shunt procedure including anasto- motic variants O1 O2 Accuracy of shunt procedures recommended on the basis of CT and/or MRI 1st option CT 73% 52% MRI 80% 60% p value 0.728 0.756 1st + 2nd option CT 88% 76% MRI 100% 73% p value 0.294 1.000 Accuracy of suggested shunts and proposed anastomotic variant 1st option CT 64% 36% MRI 53% 33% p value 0.538 1.000 1st + 2nd option CT 79% 67% MRI 73% 60% p value 0.720 0.749 Overall, graft interposition (splenorenal n = 3; portacaval n = 2; mesocaval n = 5) was performed in 10 patients of our study population. Statistical analysis 4 A 49-year-old male PHT patient with liver cirrhosis, extrahe- ti t l i th b i d d d t PSSS reconstruction and c S ll IVC Fig. 4 A 49-year-old male PHT patient with liver cirrhosis, extrahe- patic portal vein thrombosis, and advanced symptoms—PSSS proce- dure: mesocaval; a preoperative CT, b postoperative CT, axial MIP reconstruction and c postoperative CT, sagittal MIP reco Small arrow: IVC; arrowhead: SMV; bold arrow: mesocav mosis Fig. 4 A 49-year-old male PHT patient with liver cirrhosis, extrahe- patic portal vein thrombosis, and advanced symptoms—PSSS proce- dure: mesocaval; a preoperative CT, b postoperative CT, axial MIP reconstruction and c postoperative CT, sagittal MIP reconstruction. Small arrow: IVC; arrowhead: SMV; bold arrow: mesocaval anasto- mosis 1 3 3 Abdominal Radiology (2020) 45:2726–2735 2731 In terms of the vessels (splenorenal, portacaval, meso- caval, and Meso-Rex) used for PSSS, accuracy of the two observers was 73%/80% (CT/MRI) for O1 and 52%/60% for O2 if l th i fi t h i t k i t id ti Table 3 Types of portosystemic shunts performed in the study popu- lation Shunt n % Splenorenal 15 34 Side-to-side (Cooley) 10 Distal (Warren) 3 Proximal (Linton) 2 Portacaval 18 41 End-to-side 12 Side-to-side 6 Mesocaval 11 25 Side-to-side 11 Meso-Rex 0 0 Table 4 Accuracy in predicting shunt procedure including anasto- motic variants O1 O2 Accuracy of shunt procedures recommended on the basis of CT and/or MRI 1st option CT 73% 52% MRI 80% 60% p value 0.728 0.756 1st + 2nd option CT 88% 76% MRI 100% 73% p value 0.294 1.000 Accuracy of suggested shunts and proposed anastomotic variant 1st option CT 64% 36% MRI 53% 33% p value 0.538 1.000 1st + 2nd option CT 79% 67% MRI 73% 60% p value 0.720 0.749 Table 3 Types of portosystemic shunts performed in the study popu- lation Shunt n % Splenorenal 15 34 Side-to-side (Cooley) 10 Distal (Warren) 3 Proximal (Linton) 2 Portacaval 18 41 End-to-side 12 Side-to-side 6 Mesocaval 11 25 Side-to-side 11 Meso-Rex 0 0 first choices of vessels and anastomosis was fair (Cohen’s κ = 0.257, p < 0.001). If the shunt vessel and anastomosis technique matched the proposed first two choices, accuracy increased to 79%/72% (O1) and 67%/60% (O2). There was no significant difference in accuracy between MRI- and CT-based recommendations for either observer (p > 0.05, Table 4). Statistical analysis b Axial CT with partial throm- bosis of the extrahepatic portal vein (PSSS procedure: portacaval side-to-side after thrombectomy). c Oblique coronal CT MIP shows a large distance of 29 mm between IVC (small arrow) and superior mesenteric vein (arrowhead). In this patient, an allograft was inter- posed as seen in d (PSSS procedure: mesocaval with graft interposi- tion). d Postoperative oblique coronal CT MIP reconstruction shows the interposed graft (bold arrow) connecting the SMV (arrowhead) and IVC (small arrow) Fig. 5 Factors of complexity. a Oblique axial CT (fused portal venous phase and venous phase) shows an intervening caudate lobe (PSSS procedure: portacaval end-to-side with subsegmental liver resection); bold arrow: caudate lobe; arrowhead: portal vein; small arrow: hepatic artery; asterisk: IVC. b Axial CT with partial throm- bosis of the extrahepatic portal vein (PSSS procedure: portacaval side-to-side after thrombectomy). c Oblique coronal CT MIP shows a large distance of 29 mm between IVC (small arrow) and superior mesenteric vein (arrowhead). In this patient, an allograft was inter- posed as seen in d (PSSS procedure: mesocaval with graft interposi- tion). d Postoperative oblique coronal CT MIP reconstruction shows the interposed graft (bold arrow) connecting the SMV (arrowhead) and IVC (small arrow) Fig. 6 ROC analysis—distance of connected vessels and need for graft interposition; AUC 0.950 (p < 0.001); max. Youden index 0.771 at 20 mm suggest that preoperative diagnostic workup should focus on vascular anatomy [15]. This is the rationale for the study presented here, which, to the best of our knowledge, is the first study focusing on the role of preoperative imaging for predicting the most suitable approach of PSSS. Our analysis shows that contrast-enhanced CT and MRI are suitable for the imaging-based planning of surgical shunt creation and at the same time allow identification of complicating cir- cumstances. The results also show that interpretation of the complex imaging findings and the consecutive planning of the surgical approach require an experienced radiologist and may also require a multidisciplinary discussion to choose the most appropriate surgical management. With its higher periprocedural morbidity due to advanced liver disease, PSSS requires adequate preoperative planning to avoid extensive surgical exploration [24]. With the high- resolution cross-sectional imaging modalities now avail- able, adequate planning of the most suitable shunt should be possible even in patients with advanced PHT. Statistical analysis ROC analysis of vessel distances and graft use showed an AUC of 0.950 (p < 0.001, Fig. 6). According to the Youden index, the optimal cutoff was 20 mm (Youden index 0.771). Only one patient with a distance of > 20 mm did not receive a graft interposition, and this patient devel- oped early shunt occlusion. In terms of the vessels (splenorenal, portacaval, meso- caval, and Meso-Rex) used for PSSS, accuracy of the two observers was 73%/80% (CT/MRI) for O1 and 52%/60% for O2 if only their first choices were taken into consideration. Inter-rater reliability for the first choices was fair (Cohen’s κ = 0.271, p = 0.006). If the shunt technique actually used matched the first or second choice, accuracy increased to 88%/100% (O1) and 76%/73% (O2). Evaluation of recom- mended shunt vessels and the way they should be anastomo- sed (e.g., end-to-side, side-to-side, proximal, distal) showed an accuracy of 64%/53% (O1) and 36%/33% (O2) if only the first choice was considered. Inter-rater reliability for the PSSS was successful in 38 patients (82%), and their shunts were perfused until discharge. Eight patients (18%) showed therapy failure/early shunt occlusion (< 30 days after shunt surgery). Three of these patients (shunt indica- tion: acute variceal bleeding) died during hospitalization.i i Inter-rater reliability for the first choices was fair (Cohen’s κ = 0.271, p = 0.006). If the shunt technique actually used matched the first or second choice, accuracy increased to 88%/100% (O1) and 76%/73% (O2). Evaluation of recom- mended shunt vessels and the way they should be anastomo- sed (e.g., end-to-side, side-to-side, proximal, distal) showed an accuracy of 64%/53% (O1) and 36%/33% (O2) if only the first choice was considered. Inter-rater reliability for the Analysis of PSSS outcome revealed a significant corre- lation between shunt vessel distance and early occlusion. The larger the distance, the higher the risk of early shunt occlusion (p = 0.030). There was no significant correlation between successful PSSS and the diameter of smaller con- necting vessels or the shunt vessel ratio (p > 0.05, Table 5). 1 3 Abdominal Radiology (2020) 45:2726–2735 2732 Fig. 5 Factors of complexity. a Oblique axial CT (fused portal venous phase and venous phase) shows an intervening caudate lobe (PSSS procedure: portacaval end-to-side with subsegmental liver resection); bold arrow: caudate lobe; arrowhead: portal vein; small arrow: hepatic artery; asterisk: IVC. Statistical analysis Adequate evaluation of portal venous anatomy for reliable imaging- based surgical planning has been shown in patients under- going liver transplantation and pancreatic surgery [25, 26]. While it has been shown that precise puncture guidance in TIPS procedures is possible with contrast-enhanced CT and the option of 3D reconstruction [27, 28], similar data for the imaging-based planning of PSSS are not available, and our study was conducted to fill this gap. Besides choos- ing between TIPS and PSSS, the combination of both is a feasible alternative for complex cases with extrahepatic disease and should be kept in mind in the pre-interventional planning [29]. In our collective, the main reason to prefer Fig. 6 ROC analysis—distance of connected vessels and need for graft interposition; AUC 0.950 (p < 0.001); max. Youden index 0.771 at 20 mm Discussion The observed incidence of surgical failure/shunt occlusion (17%) in our patients is attributable to the wide range of shunt indications and a high percent- age of advanced and acute cases with poor hepatic reserve compared to other studies [34, 35]. PSSS to TIPS in adults was a chronic extrahepatic portal vein occlusion. However, TIPS as a possibility should not be excluded per se in these patients, since TIPS can also help in the treatment of extrahepatic portal vein occlusion [30]. in the treatment of extrahepatic portal vein occlusion [30]. Our results show that an experienced reader can (ret- rospectively) predict which surgical technique was going to be used by an experienced surgeon with high accuracy in both CT (88%) and MRI (100%). A less-experienced reader achieves lower accuracies of about 75% with both modalities, which is still adequate, though, the fair inter-rater reliability of their first choices underlines the need for an experienced radiologist in the interpretation of the complex preoperative imaging. Moreover, our findings suggest that there are no significant differences in the preoperative accu- racy of MRI and CT. These findings support an earlier study reporting the good performance of CT venography in the evaluation of portosystemic collateral vessels [31]. In only 10% of the patients included in our retrospective analysis did the surgeon choose a vessel connection not recommended by the experienced reader. The high incidence of therapy failure (40%) in these cases indicates that deviation from preop- erative imaging findings could lead to higher rates of shunt failure and consecutively increased morbidity. Similarly, it has been shown that preoperative CT assessment improves intraoperative management of portosystemic shunts during liver transplantation [32]. Our study has some limitations. Despite the retrospective nature and the small sample size, only patients who under- went surgery were included in our analysis. This is attrib- utable to the fact that PSSS is increasingly being replaced by TIPS. The surgeons performing PSSS were also aware of the preoperative imaging findings; however, there was no documentation how the imaging findings influenced the surgeons’ choice of shunt procedure. Practice patterns and surgeons’ experience are likely variable between institutions, so that the results of the study may not be applicable to other sides. There are various confound factors that could influence the prediction. Discussion Despite the success story of TIPS [2], PSSS can have bene- fits in the treatment of advanced PHT [5]. We retrospectively analyzed 44 patients who underwent PSSS at our center because TIPS was no adequate treatment option. Longer- term results available for the different shunting techniques 3 2733 Abdominal Radiology (2020) 45:2726–2735 Outcome of PSSS during hospitalization (<30 days) Success (n = 36) Failure (n = 8) Significance Mean SD Range Mean SD Range Distance between vessels (mm) 12.67 11.69 56.00 20.38 10.87 33.00 p = 0.030 Small shunt vessel diameter (mm) 10.17 4.39 21.00 10.25 3.01 9.00 p = 0.709 Shunt vessel ratio 0.734 0.567 2.742 0.500 0.156 0.475 p = 0.482 Table 5 Analysis of correlation of shunt vessel diameter and distance with early success and failure/shunt occlusion (< 30 days) Outcome of PSSS during hospitalization (<30 days) PSSS to TIPS in adults was a chronic extrahepatic portal vein occlusion. However, TIPS as a possibility should not be excluded per se in these patients, since TIPS can also help in the treatment of extrahepatic portal vein occlusion [30]. Our results show that an experienced reader can (ret- rospectively) predict which surgical technique was going to be used by an experienced surgeon with high accuracy in both CT (88%) and MRI (100%). A less-experienced reader achieves lower accuracies of about 75% with both modalities, which is still adequate, though, the fair inter-rater reliability of their first choices underlines the need for an experienced radiologist in the interpretation of the complex preoperative imaging. Moreover, our findings suggest that there are no significant differences in the preoperative accu- racy of MRI and CT. These findings support an earlier study reporting the good performance of CT venography in the evaluation of portosystemic collateral vessels [31]. In only 10% of the patients included in our retrospective analysis did the surgeon choose a vessel connection not recommended by the experienced reader. The high incidence of therapy failure (40%) in these cases indicates that deviation from preop- erative imaging findings could lead to higher rates of shunt failure and consecutively increased morbidity. Similarly, it has been shown that preoperative CT assessment improves intraoperative management of portosystemic shunts during liver transplantation [32]. In addition to providing information on vascular anat- failure/occlusion in our cohort. In contrast, the diameters of the veins connected for shunt creation did not correlate with early shunt occlusion. References 1. Knechtle SJ, D’Alessandro AM, Armbrust MJ, Musat A, Kalayo- glu M (1999) Surgical portosystemic shunts for treatment of por- tal hypertensive bleeding: outcome and effect on liver function. Surgery 126 (4):708–711; discussion 711–703 19. Superina R, Bambini DA, Lokar J, Rigsby C, Whitington PF (2006) Correction of extrahepatic portal vein thrombosis by the mesenteric to left portal vein bypass. 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To minimize these effects, we evaluated the first two radiological choices instead of only using a single recommendation. Our results could pave the way for a prospective study, which is needed, to answer the question if pre-surgical imaging provides adequate and important information and impacts patient outcomes. In addition to providing information on vascular anat- omy, MRI and CT identify complicating factors that require additional surgical techniques. Caudate lobe enlargement is common in cirrhosis of different etiologies and is fre- quently found in patients with PHT requiring PSSS [33]. Therefore, a hypertrophic caudate lobe has to be considered as a complicating factor in the planning of potential porta- caval shunt surgery. Our study shows that imaging reliably identifies interfering structures, which can thus be consid- ered in preoperative planning. Large distances between con- necting vessels sometimes require the use of interposition grafts. General recommendations regarding the vein distance above which graft interposition should be performed do not exist. Our analysis identified a vessel distance of 20 mm as the optimal cutoff for use of an interposition graft by experienced surgeons. In addition to its use as a criterion for deciding about the need for an interposition graft, vessel distance was identified as the only risk factor for early shunt In conclusion, preoperative cross-sectional imaging and an experienced radiologist can guide the surgeon in PSSS. Imaging is not only inevitable to provide accurate planning in general but rather to reliably exclude unfavorable shunt variants. Optimized CT and MRI examinations provide the information necessary to identify technically feasible alter- natives and complicating factors (which may require an interposition graft, resection of intervening structures, or prior thrombectomy). 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https://openalex.org/W2970489265	http://seps.unsrat.ac.id/journals/index.php/joseps/article/download/13/12	English	null	Ship-to-Shore Wireless Communication for Asynchronous Data Delivery to the Remote Islands	Journal of Sustainable Engineering. Proceedings Series	2,019	cc-by	2,065	Alwin M. Sambul1, Sherwin R.U.A. Sompie2, Daniel F. Sengkey3, Agustinus Jacobus4, Alicia A.E. Sinsuw5 Department of Electrical Engineering, Faculty of Engineering, Universitas Sam Ratulangi, Jln. Kampus Unsrat, Bahu - Manado 95115, INDONESIA Department of Electrical Engineering, Faculty of Engineering, Universitas Sam Ratulangi, Jln. Kampus Unsrat, Bahu - Manado 95115, INDONESIA Department of Electrical Engineering, Faculty of Engineering, Universitas Sam Ratulangi, Jln. Kampus Unsrat, Bahu - Manado 95115, INDONESIA E-mail: 1asambul@unsrat.ac.id, 2aldo@unsrat.ac.id, 3danielsengkey@unsrat.ac.id, 4a.jacobus@unsrat.ac.id, 5alicia.sinsuw@unsrat.ac.id Abstract. Nowadays, many people who live in remote islands of Indonesia are still facing difficulties in terms of access to information. In the locations where end-to-end communication is not available, the asynchronous approach can be utilized to send information in the form of digital data. In some areas, we could utilize passenger ships or ferries as physical carriers to deliver digital data to the people in the remote islands which are located at a particular range of distance from the ship’s passing routes. This paper reports the channel performance of long- range WiFi connection oversea at 5 GHz using the real ship’s route at the North Sulawesi province‘s water in Indonesia as a sample scenario. The measurement results showed that the most stable ship-to-shore communication can be achieved in ±15 minutes at the maximum distance between the ship and shore of about 4 km. The maximum channel capacity was 120 Mbps for upload (from ship to shore) and 53 Mbps for download (from shore to ship), which is enough to deliver gigabytes of information to the people at the islands every time the ship passes by. The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 Penerbit Fakultas Teknik Universitas Sam Ratulangi doi:10.35793/joseps.v1i1.13 Penerbit Fakultas Teknik Universitas Sam Ratulangi doi:10.35793/joseps.v1i1.13 This paper and its contents may be used under the terms of Creative Commons Attribution 4.0 license. Any further distribution of this paper must maintain attribution to the author(s), title, journal citation and DOI. Published under license by Penerbit Fakultas Teknik Universitas Sam Ratulangi, Manado. Ship-to-Shore Wireless Communication for Asynchronous Data Delivery to the Remote Islands Alwin M. Sambul1, Sherwin R.U.A. Sompie2, Daniel F. Sengkey3, Agustinus Jacobus4, Alicia A.E. Sinsuw5 Alwin M. Sambul1, Sherwin R.U.A. Sompie2, Daniel F. Sengkey3, Agustinus Jacobus4, Alicia A.E. Sinsuw5 This paper and its contents may be used under the terms of Creative Commons Attribution 4.0 license. Any furthe distribution of this paper must maintain attribution to the author(s), title, journal citation and DOI. Published under license by Penerbit Fakultas Teknik Universitas Sam Ratulangi, Manado. 1. Introduction Although according to the recent data two-third of the world population is now enjoying mobile phone connection [1], yet many people, especially at the small and remote islands remain completely cut off. These islands generally have a little population and low income, hence making them economically challenging for building out telecommunication infrastructure [2]. This situation brings difficulties for the inhabitants to communicate and to access information from the outside worlds. In such locations where the end-to-end communication is not available, the asynchronous approach can be utilized to send information in the form of digital data. This approach usually uses vehicles that physically carry a computer with a storage device and a limited telecommunication module (usually WiFi) between remote areas in order to effectively create a data communication link [3, 4]. In some areas, such as in North Sulawesi province of Indonesia, we could utilize passenger ships or ferries as physical carriers to deliver digital data to the people in the remote islands which are located at a particular range of distance from the ship’s passing routes. As the ships usually have regular routes and schedules, we could also expect that the data can be delivered in a timely manner. This paper reports the channel performance of long-range WiFi connection oversea at 5 GHz using the real ship’s route at the North Sulawesi province‘s water in Indonesia as a sample scenario. 103 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 Penerbit Fakultas Teknik Universitas Sam Ratulangi doi:10.35793/joseps.v1i1.13 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 2.1. Sample scenario & experiment p p As the solution proposed in this paper utilizes passenger ships as physical carriers to deliver digital data to the remote islands, it is necessary to determine a sample scenario in which the solution will be tested. To do this, a continuous GPS tracking from the deck of a passenger ferry traveling its typical route from Manado to Tahuna was conducted. The data showed that the ship was cruising at an average speed of 13.32 knot and passing by a small island of Makalehi (coordinate 2°43'38.2"N 125°10'38.4") at about 2.17 km of minimum distance to its shore. This island has a population of 1,287 in an area of 4.2 km2 and situated at the border of Indonesia’s sea territory. Makalehi island was chosen as the sample of the scenario as it is categorized as one of the small and outer islands by the Government of the Republic of Indonesia, and currently has no reliable telecommunication infrastructure to provide any data connectivity. Figure 1: Configuration of equipment Figure 1: Configuration of equipment To simulate the sample scenario, the experiment was focused to measure the channel capacity of long- range WiFi connectivity between a moving station on a chartered boat cruising at the same speed of passenger ship mentioned earlier (±13.32 knot), and passing by perpendicularly to a base station at the shore at least 2.17-km distance. The experiment was done at the Wori waters near Siladen island, while the base station was situated at a fishing dock of Bawoho village at the north of Manado city (coordinate 1°34'58.1"N 124°49'03.1"E). 3. Result and Discussions Figure 2 shows the GPS tracking and timing of the ship’s location while measuring the channel performance. The shore’s base station is located at the mainland and showed as a white asterisk. The boat was cruising from north to southwest toward the channel between Tongkaina Cape and Bunaken Island. Figure 2: Ship's location GPS tracking Figure 2: Ship's location GPS tracking The measurement was done in the duration of 52 minutes and the ship’s distance to the base station is shown in Figure 3. As can be seen from this figure, the ship’s closest position occurred 39 minutes after the measurement began (Figure 4). Clearly, the signal levels changed proportional to the distance between the ship and the base station, as shown by Figure 5. Only for comparison, in Figure 5 we also included signal level’s data from a directional antenna that was included in the experiment. The measurement was done in the duration of 52 minutes and the ship’s distance to the base station is shown in Figure 3. As can be seen from this figure, the ship’s closest position occurred 39 minutes after the measurement began (Figure 4). Clearly, the signal levels changed proportional to the distance between the ship and the base station, as shown by Figure 5. Only for comparison, in Figure 5 we also included signal level’s data from a directional antenna that was included in the experiment. With a more focusing on the results from the omnidirectional antenna, the data of wireless channel capacity, which is the maximum amount of traffic or signal that can move over the communication channel [5], are shown in Figure 6 and 7. Tx channel capacity defines the capacity of data transmission from ship to the shore, while Rx channel capacity means the capacity from shore to ship. As shown from these data, the channel capacity of both Tx and Rx were increased significantly between 11:00 to 11:15 of the measurement time. The maximum Tx channel capacity was 120 Mbps and the maximum Rx channel capacity was 53 Mbps. With a more focusing on the results from the omnidirectional antenna, the data of wireless channel capacity, which is the maximum amount of traffic or signal that can move over the communication channel [5], are shown in Figure 6 and 7. 2.2. Long-range WiFi antennas g g The equipment of the long-range WiFi antenna system was separated into two subsystems: a) ship side, and b) shore side. The ship side used a dual polarity omnidirectional antenna (airMAX® Omni® AMO- 5G13 from Ubiquiti Networks) with 13 dBi gain at 5 GHz, while the shore side used a sectoral antenna airMAX® AM-5G19-120 from Ubiquiti Networks) with 19 dBi gain at 5 GHz. Both antennas were elevated at 2 meters from the floor using iron tripods. Each of these antennas was connected to a wireless access point (Rocket®M AirMAX® Base Station from Ubiquiti), monitored remotely using a laptop connected to the access point using UTP cable. Due to the lack of power source at the boat and at the shore, all equipment at each side was powered by a deep-cycle 12V 100Ah battery (Luminous®) with an inverter. Figure 1 shows the configuration of the equipment. 104 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 3. Result and Discussions Tx channel capacity defines the capacity of data transmission from ship to the shore, while Rx channel capacity means the capacity from shore to ship. As shown from these data, the channel capacity of both Tx and Rx were increased significantly between 11:00 to 11:15 of the measurement time. The maximum Tx channel capacity was 120 Mbps and the maximum Rx channel capacity was 53 Mbps. Figure 3: Distance to signal strength Figure 3: Distance to signal strength Figure 3: Distance to signal strength 105 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi doi:10.35793/joseps.v1i1.13 Figure 4: Distance between the ship and the base station Figure 4: Distance between the ship and the base station Figure 5: Upload Channel Capacity. Figure 5: Upload Channel Capacity. Figure 6: Download channel capacity Figure 6: Download channel capacity Figure 6: Download channel capacity 4. Conclusions The measurement results showed that the most stable ship-to-shore communication can be achieved in a duration of ±15 minutes at the maximum distance between the ship and shore of about 4 km. The maximum channel capacity was 120 Mbps for upload (from ship to shore) and 53 Mbps for download (from shore to ship), which is enough to deliver gigabytes of information to the people at the islands every time the ship passes by. This shows that this asynchronous approach using ship-to-shore wireless communication can be used effectively for data delivery to the remote islands. 106 Penerbit Fakultas Teknik Universitas Sam Ratulangi doi:10.35793/joseps.v1i1.13 The 1st International Conference on Sustainable Engineering Practices (IConSEP) Penerbit Fakultas Teknik Universitas Sam Ratulangi Journal of Sustainable Engineering: Proceedings Series 1(1) 2019 doi:10.35793/joseps.v1i1.13 Acknowledgments Acknowledgments The authors acknowledge the support of DPRM Dikti, the Ministry of Research, Technology and Higher Education, the Republic of Indonesia. References [1] GSMA Intelligence, “Definitive data and analysis for the mobile industry”, 2017. [Online] Available: https://www.gsmaintelligence.com [2] Mauro Margalho Coutinho, Alon Efrat, Thienne Johnson, Andrea Richa, and Mengxue Liu, “Healthcare Supported by Data Mule Networks in Remote Communities of the Amazon Region,” International Scholarly Research Notices, vol. 2014, Article ID 730760, 8 pages, 2014. doi:10.1155/2014/730760. [2] Mauro Margalho Coutinho, Alon Efrat, Thienne Johnson, Andrea Richa, and Mengxue Liu, “Healthcare Supported by Data Mule Networks in Remote Communities of the Amazon Region,” International Scholarly Research Notices, vol. 2014, Article ID 730760, 8 pages, 2014. doi:10.1155/2014/730760. [3] R. C. Shah, S.Roy, S.Jain, and W.Brunette, “DataMULEs: modeling and analysis of a three-tier architecture for sparse sensor networks,” Ad Hoc Networks, vol.1, no. 2-3, pp.215–233, 2003. [4] Shyam, “Connecting Remote Islands: GSM and Broadband Networks for Indonesia,” Vihaan Networks Limited, 2015. [Online] Available: http://www.vnl.in/media/4989/16- vnl_br_connecting- remote-island_r2.pdf [5] Techopedia, “Channel Capacity,” [Online] Available: https://www.techopedia.com/definition/248 73/channel-capacity 107 107
W4283073212.txt	https://ehjournal.biomedcentral.com/counter/pdf/10.1186/s12940-022-00869-5	en		Pleural mesothelioma risk by industry and occupation: results from the Multicentre Italian Study on the Etiology of Mesothelioma (MISEM)	Environmental health	2,022	cc-by	8,940	(2022) 21:60 Migliore et al. Environmental Health https://doi.org/10.1186/s12940-022-00869-5 Open Access RESEARCH Pleural mesothelioma risk by industry and occupation: results from the Multicentre Italian Study on the Etiology of Mesothelioma (MISEM) Enrica Migliore1,2* , Dario Consonni3, Susan Peters4, Roel C. H. Vermeulen4, Hans Kromhout4, Antonio Baldassarre5, Domenica Cavone6, Elisabetta Chellini7, Corrado Magnani2,8, Carolina Mensi3, Enzo Merler9, Marina Musti6, Alessandro Marinaccio10 and Dario Mirabelli1,2 Abstract Background: The Italian mesothelioma registry (ReNaM) estimates mesothelioma incidence and addresses its etiology by assessing cases’ exposures but cannot provide relative risk estimates. Objectives: i) To estimate pleural mesothelioma relative risk by industry and occupation and by ReNaM categories of asbestos exposure; and ii) to provide quantitative estimates of the exposure–response relationship. Methods: A population-based mesothelioma case–control study was conducted in 2012–2014 in five Italian regions. Cases and age and gender frequency-matched controls were interviewed using a standard ReNaM questionnaire. Experts coded work histories according to international standard classifications of industries/occupations and assigned asbestos exposure according to ReNaM categories. Job codes were further linked to SYN-JEM, a quantitative job-exposure matrix. Cumulative exposure (CE, f/mL-years) was computed by summing individual exposures over lifetime work history. Unconditional logistic regression analyses adjusted by gender, centre and age were fitted to calculate odds ratios (OR) and 95% confidence intervals (CI). Results: Among men we observed increased risks of mesothelioma in many industries and associated occupations, including: asbestos-cement (OR = 3.43), manufacture of railroad equipment (OR = 8.07), shipbuilding and repairing (OR = 2.34), iron and steel mills (OR = 2.15), and construction (OR = 1.94). ORs by ReNaM exposure categories were as follows: definite/probable occupational exposure (OR = 15.8, men; OR = 8.80, women), possible occupational (OR = 2.82, men; OR = 3.70, women), sharing home with an exposed worker (OR = 2.55, men; OR = 10.3, women), residential (OR = 2.14, men; OR = 3.24, women). Based on SYN-JEM, mesothelioma risk increased by almost 30% per f/ mL-year (OR = 1.28, CI 1.16–1.42). Conclusions: Out study involved five regions with historically different types and levels of industrial development, encompassing one third of the Italian population and half of Italian mesothelioma cases. As expected, we found increased pleural mesothelioma risk in the asbestos industry and in trades with large consumption of asbestos *Correspondence: enrica.migliore@cpo.it 1 Cancer Epidemiology Unit, CPO Piemonte and University of Turin, Turin, Italy Full list of author information is available at the end of the article © 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. Migliore et al. Environmental Health (2022) 21:60 Page 2 of 13 materials. Clear associations were found using both qualitative (ReNaM classifications) and quantitative estimates (using SYN-JEM) of past asbestos exposure, with clear evidence of an exposure–response relationship. Keywords: Pleural mesothelioma, Asbestos, Occupational exposure, Exposure assessment, Case–control study Background All uses of asbestos have been banned since 1999 in the European Union, and in some member states the ban was issued much earlier [1]. The incidence of malignant mesothelioma (MM) – the neoplasm most strongly associated with asbestos – showed a decline in Sweden, where the first ban had been issued in 1975 for crocidolite [2]. In the other European countries MM incidence was still increasing at the turn of century, although less steeply than in the past [1]; a recent decreasing trend in mortality has been observed in some Northern and Western European countries like Sweden and The Netherlands, but not yet in Italy [3]. Furthermore, it is widely acknowledged that asbestos remaining in place may result in exposure for workers engaged in maintenance and renovation of buildings and industrial plants, as well as for members of the general population due to the presence of weathering asbestos-containing materials. The epidemiological surveillance of MM remains, therefore, of interest. In Italy use of asbestos was forbidden in 1992 and a National Mesothelioma Registry (ReNaM) was started in 1993. Registration is accomplished by its regional operating centres (centres, from now on). ReNaM centres identify incident MM cases in their regional populations, systematically collect information about their occupational and non-occupational exposures to asbestos, carry out the exposure assessment and feed data to ReNaM. Statistics are periodically published describing the proportion of cases with recognized occupational exposures and their distribution by industry and occupation [4]. Interestingly, it was found that about 10% of all Italian cases had no recognized exposure at work but had been exposed to asbestos in non-occupational settings [5]. The ability to describe etiology characterizes ReNaM and differentiates it from general cancer registries. However, exposure data being restricted to cases, ReNaM cannot provide risk estimates by industry and occupation. Furthermore, previously unrecognized opportunities and circumstances of exposure have been identified, but only when they gave rise to obvious clusters of cases in specific factories, industries or small areas [6–10]. To overcome such limitations a population-based case–control design would be instrumental, following the example of the French Programme National de Surveillance des Mésothéliomes (PNSM) [11]. To assess its feasibility and evaluate its potential results we conducted a populationbased case–control study. The specific aims of this study were to i) estimate pleural mesothelioma relative risk by industry and occupation and by ReNaM categories of asbestos exposure; and ii) provide quantitative estimates of the exposure–response relationship. Methods The study included pleural MM cases with histological confirmation of diagnosis. Cases were enrolled into the study as soon as they were identified. Given the constraints imposed by the funding body on the timeextension of the study (two years and six months, from October 2012 to March 2015), cases occurring during the recruitment period but registered and, thus, interviewed later were excluded. Only centers that could take advantage of a rapid alert system for the identification of incident cases were, therefore, involved. Five centers, Piedmont, Lombardy, Veneto, Tuscany and Apulia, participated in the study; in Piedmont the source population was limited to residents in the province of Turin and the local health district of Casale Monferrato, whereas in Veneto recruitment was restricted to residents in the provinces of Venice and Padua (Table 1). Population controls were randomly selected from the regional rosters of citizens registered with the National Health Service. Such lists largely coincide with residents, as they are based on data from the municipal registrar offices. The average update lag is about six months, so control selection was carried out at mid-year of the recruitment year (in Piedmont and Veneto of the first recruitment year) of cases. Controls were frequency matched to the expected gender- and age-distribution of cases. Personal interviews were carried out by trained interviewers who were blind to the case/control status of the study subjects. In Lombardy and Tuscany organizational and administrative constraints led, however, to conduct separate series of interviews for cases and controls: whereas those of cases were performed by occupational health officials of the Local Health Authorities, those of controls were carried out by ad hoc interviewers. Occupational and non-occupational circumstances that could have entailed exposure to asbestos were investigated using the standardized ReNaM questionnaire, administered by trained interviewers to all study subjects. A next of kin was interviewed in case of participants’ death or when their conditions prevented direct interviews [5]. Lifetime occupational histories were collected Migliore et al. Environmental Health (2022) 21:60 Page 3 of 13 Table 1 Characteristics of cases and controls, the MISEM study, 2012–2015, Italy Men Women Cases Total Controls Cases Controls N (%) N (%) N (%) N (%) 463 (48.6) 490 (51.4) 163 (41.7) 228 (58.3) Age (years): < 50 50–54 9 (1.9) 24 (4.9) 4 (2.5) 18 (7.9) 17 (3.7) 30 (6.1) 6 (3.7) 7 (3.1) 55–59 30 (6.5) 45 (9.2) 10 (6.1) 17 (7.5) 60–64 42 (9.1) 54 (11.0) 12 (7.4) 24 (10.5) 65–69 90 (19.4) 117 (23.9) 16 (9.8) 43 (18.9) 70–74 111 (24.0) 102 (20.8) 46 (28.2) 51 (22.4) 75–79 92 (19.9) 62 (12.7) 30 (18.4) 25 (11.0) 80–84 57 (12.3) 38 (7.8) 29 (17.8) 25 (11.0) 85 + 15 (3.2) 18 (3.7) 10 (6.1) 18 (7.9) Centre: Piedmont 167 (36.1) 159 (32.5) 67 (41.4) 108 (47.4) Lombardy 146 (31.5) 141 (28.8) 59 (36.2) 62 (27.2) Veneto 55 (11.9) 106 (21.6) 19 (11.7) 37 (16.2) Tuscany 65 (14.0) 26 (5.3) 14 (8.6) 7 (3.1) Apulia 30 (6.5) 58 (11.8) 4 (2.5) 14 (6.1) Interview: Direct 353 (76.2) 454 (92.7) 97 (59.5) 217 (95.2) Next of kin 110 (23.8) 36 (7.4) 66 (40.5) 11 (4.8) Blue collar jobs: Ever 403 (87.0) 361 (74.0) 112 (68.7) 130 (57.0) Never 60 (13.0) 127 (25.9) 50 (30.7) 98 (43.0) Always 238 (51.4) 164 (33.5) 79 (48.5) 80 (35.1) Number of jobs: Mean 3.86 3.47 2.48 2.42 Std deviation 2.14 2.05 1.68 1.66 for cases and controls, supplemented by job-specific modules allowing the description of the working environment, the tasks carried out by the interviewees or in their presence, the general ventilation and local exhaust systems. Full residential histories were also reconstructed, including residential addresses, the presence of industrial premises including iron and steel foundries, chemical plants, power plants, and asbestos-cement industries in proximity to residences were collected, as well as details on the characteristics of residential buildings, including the presence of prefabricated structures, asbestoscement structures (walls and roofs), insulating materials. Lastly, the occupational histories of family members were collected. Work histories were coded by Regional Operating Centre (COR) experts blind to the case–control status of study subjects. For the purposes of this study, industries and job titles were coded according to the International Standard Industrial Classification of Economic Activities (ISIC), second revision 1971 [12] and the International Standard Classification of Occupations (ISCO), second revision 1968 [13]. Job histories were truncated to the year of diagnosis (for cases) and enrolment (for controls). Industries were coded at the finest possible detail: the four- and five-digit level in ISIC and, respectively, ISCO classifications. Three-digit level ISCO codes to be used in data analysis were then generated by truncation of fivedigit codes. Quantitative indices of exposure to asbestos were obtained by merging the list of coded employment periods with the estimates of exposure provided by SYN-JEM [14]. SYN-JEM is a quantitative job-exposure matrix for five occupational respiratory carcinogens developed in the framework of the SYNERGY study, coordinated Migliore et al. Environmental Health (2022) 21:60 by the International Agency for Research on Cancer (IARC) [15]. In SYN-JEM exposure to asbestos has been estimated in fibre per millilitre units (f/mL) by country, region, historical period and job, where jobs have been classified according to ISCO 1968 [13]. Merging the job history with SYN-JEM provided yearly quantitative estimates of exposure, allowing the calculation of: (i) cumulative exposure (CE, in f/mL-y), (ii) duration of exposure, and (iii) average exposure intensity, by dividing CE by duration. Unlagged and lagged (10-, 20- and 30-year lag) indices were calculated. Cases and controls also underwent the ReNaM standard assessment of the exposure source and probability [5]. Local experts, usually industrial hygienists or occupational health physicians with specific knowledge of the local past uses and natural occurrence of asbestos, performed such assessment. Every job was assessed based on all the information available to the experts, such as interview data, previous interviews to possible earlier cases observed in the same workplaces, direct knowledge of industries and workplaces. Exposure probability was classified as definite (use of asbestos described at interview or already known to experts), probable (asbestos certainly used in the plant, but use by the interviewee unknown), possible (recognized asbestos use in the job or industrial activity, but unknown whether in the plant), unlikely (use of asbestos not described at interview and unknown to experts) or unknown (information inadequate to classify the job into any of the previous categories). The possibility of para-occupational exposure (familial exposure in the ReNaM classification) was evaluated by examining the occupational histories of relatives (parents, siblings, spouses etc.) during the periods when they shared home with study subjects. Exposure was classified as “familial” (living with an occupationally exposed person), unlikely or unknown. All residences, including those held habitually during holidays, were assessed for residential proximity to industrial or natural asbestos sources, based on their address and the spatial distribution of known sources. Exposure was classified as “residential” (residence in proximity – based on raters’ judgement – to one or more identified sources of asbestos pollution), unlikely or unknown. Other non-occupational exposures may have occurred, such as the presence of asbestos-containing materials in the home environment or at school, the use of asbestos or the intervention on asbestos-materials during home maintenance and repairs or leisure-time activities, all of which were investigated in specific sections of the questionnaire. Exposure was assessed as “other non-occupational” (when any of such circumstances has occurred), unlikely or unknown. Page 4 of 13 According to the ReNaM guide-lines for exposure assessment [16], when multiple circumstances and routes of exposure are present, as is often the case, the individual overall classification is determined by the most severe exposure category ever experienced by a study subject, which is conventionally established according to the gradient (from most to less severe): occupational, familial, residential, other non-occupational exposures. Odds ratios (OR) and 95% confidence intervals (CI) by industry and job – ever vs. never employment – were calculated by unconditional logistic regression adjusting by centre (as in Table 1) and 5-year age class (from < 50 to ≥ 85 years) for men and women separately. We used full, four-digit ISIC codes for industries and three-digit ISCO codes for occupations. ORs and CIs were also calculated: (i) by gender and ReNaM exposure category (individual overall classification, as above), adjusted by centre, age-class (as above) and type of interview (direct or proxy), and (ii) by gender and CE, adjusted by centre, age-class, type of interview and binary indicator variables for ever-exposure in nonoccupational settings (i.e.: familial, residential or other non-occupational circumstances). In the analyses by ReNaM exposure category, we used as reference the combination of unlikely and unknown exposures. The unlikely exposure category could have been a better choice, but it could not be used because of the relatively small number of cases and control so classified. We also combined definite and probable occupational exposures, as the number of cases and controls classified as probably exposed was small, especially among women. CE was modeled both as a categorical and a continuous variable. In the first case, CE categories were built based on the CE distribution among exposed controls, considering as cut-off values the median (exposed below and above the median) and tertiles (first, second and third CE tertile). In the latter case, either the untransformed or the natural-log transformed variables were used. Unlagged and lagged (at 10-, 20- and 30-years lag) analyses were carried out. All analyses were performed also by combining men and women, adding gender to the model. Analyses by ReNaM exposure category were replicated also by using as reference only unlikely exposures, and by lumping together all occupational exposures or, on the opposite, by separating definite, probable and possible exposures. Models were compared by calculating the Akaike information criterion (AIC). We additionally fit a cubic spline model (with the same adjustment variables) allowing the slope of the function to change at predefined bending points (five knots at 10/25/50/75/90 percentiles), to better capture and describe the features of the exposure–response Migliore et al. Environmental Health (2022) 21:60 association, using untransformed and natural-log transformed CE, for men and women separately and considering the above specified lags (0, 10, 20 and 30 years). Sensitivity analyses were carried out by replicating all models based on the ReNaM exposure indices and CE after (a) leaving out centres one at a time, (b) additional adjustment for the condition of blue-collar (BC) worker, and (c) restriction to BC workers. To this purpose, the study subjects were classified as ever vs. never holding a BC job. All occupations in the ISCO classification associated with a code equal to 5.10.00 or higher were considered BC jobs and included working proprietors, farmers and manual workers in industry and services. All analyses were carried out with Stata 16 (Stata Corp. 2019, College Station, TX, USA). The study was approved by the Internal Review Board of the coordinating centre (Cancer Epidemiology, Turin). Centre participation was approved by their respective Internal Review Boards. Participants gave their written, informed consent before interview. Results In Table 1 the main characteristics of cases and controls are described. Supplementary Table S1 shows the distribution of cases and controls by centre, along with the size of target populations and recruitment periods. Direct interviews were obtained for 450 of the 626 cases eligible for the study (71.8%), and for 671 of the 718 controls (93.5%). For the remaining cases and controls, information was obtained from relatives (mainly from spouses, sons or daughters). Seventy-four percent of cases were men, with a mean age of 70.6 years (sd = 9.0). Cases and controls were very similar regarding the number of reported jobs (3.86 vs. 3.47, respectively, in men and 2.48 vs. 2.42 in women). Selected results for men and women by industry (ordered by ISIC 4-digit codes) and job (ordered by ISCO 3-digit codes) are plotted in Figs. 1 and 2. The full set of results for industries and occupations with at least three exposed cases is provided in Supplementary Tables S2 and S3. No study subject reported employment in asbestos mining or in associated occupations. Asbestos transformation activities are lumped together in the ISIC classification under the rubric “Manufacture of non-metallic mineral products not elsewhere classified”, ISIC code 3699, for which we found a more than three-fold increase in pleural MM risk, among men; in women there were six exposed cases and no exposed controls. Employment in various industries known to have entailed extensive use of insulation materials was associated with high ORs, namely: manufacture of railroad equipment (ISIC code 3842), ship building and repairing Page 5 of 13 (ISIC code 3841), chemical industry (in particular: manufacture of basic industrial chemicals, ISIC code 3511), oil refineries (ISIC code 3530), iron and steel mills (ISIC code 3710), glass industry (ISIC code 3620). We found excess risk also in jobs specifically associated with some of these industries, such as blacksmiths (ISCO code 839), metal furnacemen (ISCO code 721), rolling mill workers (ISCO code 722), metal casters (ISCO code 724), metal processors not elsewhere classified (ISCO code 729), as well as glass formers and cutters (ISCO code 891). Construction industry was associated with an approximately two-fold MM risk in men and in women (albeit with a rather large CI). Among men this was by far the largest exposure group with 119 exposed cases. In addition, many occupations in the construction industry were also associated with increased ORs: electrical fitters (ISCO code 851), plumbers and pipe-fitters (ISCO code 871), roofers (ISCO code 953), general construction workers (ISCO 959) and construction painters (ISCO code 931). The manufacture of special industrial machinery and equipment was also associated with an almost two-fold increase in MM risk among men (ISIC code 3824), and even higher risks were entailed by related jobs like welders and flame cutters (ISCO code 872), sheet metal workers (ISCO code 873) and structural metal preparers and erectors (ISCO code 874) as well as plumbers and pipe-fitters (ISCO code 871), who may be employed also in this trade. Interestingly, MM risk in men was high after employment in freight transport by road (ISIC code 7114) and in water transport (OR = 3.31; CI 1.12–9.84, not shown in Fig. 1 and in Supplementary table S2 as this trade corresponds to a three-digit ISIC code: 712), including supporting services to water transport (ISIC code 7123). Correspondingly, ship’s engine room personnel (ISCO code 982) and railway engine drivers (ISCO code 983) had increased ORs. MM risk was elevated among men also in manufacture of textiles not elsewhere classified (ISIC code 3219), which mainly included production of felts and mattresses, in the pulp and paper industry (ISIC code 3411) and in the poorly defined group of manufacturing industries not elsewhere classified (ISIC code 3909). As to occupations, paper makers in men (ISCO code 734) and spinners/winders and weavers in men and women (ISCO codes 752, 754) had increased ORs – with wide CIs. Stock clerks (ISCO code 391) and occupational conditions not corresponding to any ISCO item – such as military service and unemployment – were at high risk among men, in addition to the jobs previously mentioned in relation with their corresponding industries. Thirtyfive male cases and 16 controls reported, respectively, 39 and 21 employment periods as stock clerks, out of which Migliore et al. Environmental Health (2022) 21:60 Page 6 of 13 Fig. 1 Odds ratios and 95% confidence intervals for pleural mesothelioma by gender and industry, according to the International Standard Industry Classification (ISIC, 4-digit codes), 1971 – Industries with at least 20 exposed cases and controls—the MISEM study, 2012–2015, Italy 30 and 12 were spent in industrial settings where the use of asbestos-containing materials was possible or even likely, such as the chemical, rubber and plastics industry, steel mills and the construction of industrial machinery and equipment. Among women, the only trade associated with a clearcut increase in MM OR was that of business services not elsewhere classified (ISIC code 8329). This is a large group, which may include businesses as different as cleaning services and marketing. The ten women cases in our study reported 20 employment periods in ISIC 8329, 19 of which were spent in cleaning services, while the three controls had 3 employment periods, one of which in cleaning services. It is worth mentioning that such cleaning services had been often described at interview as industrial cleaning services, and in at least two cases in work-settings well known for having entailed exposure to asbestos. Consistently with these results, charworkers, cleaners and related workers (ISCO code 552) was the female job code with the largest MM risk. ReNaM exposure indices for occupational and nonoccupational exposures were strongly associated with increased risk of pleural MM in both genders (Table 2). Overall, for definite/probable occupational exposure, we found an OR of about 15 while possible occupational exposures also corresponded to a three-fold elevated risk. Elevated ORs were also found for familial exposures, especially in women. The results of the analyses in which occupational exposures had been either grouped (definite, probable and possible) or considered as distinct categories are reported, respectively, in Supplementary Tables S4 and S5. Comparing the AIC values for the corresponding, gender-specific models from Table 2 and Supplementary Tables S4 and S5 suggests Migliore et al. Environmental Health (2022) 21:60 Page 7 of 13 Fig. 2 Odds ratios and 95% confidence intervals for pleural mesothelioma by gender and occupation, according to the International Standard Code of Occupations (ISCO, 3-digit codes), 1968 – Occupations with at least 20 exposed cases and controls—the MISEM study, 2012–2015, Italy Table 2 Number of cases and controls, odds ratio (OR) and 95% confidence intervals (CI) by modality of exposure, according to the Italian National Mesothelioma Register (ReNaM) classification. Reference category: unlikely/unknown exposure, lag 10 years – the MISEM study, 2012–2015, Italy ReNaM exposure Men and women Men Cases Ctrls ORa 122 382 Occupational, definite or probable 343 Unlikely/unknown exposure 110 CI 1.00 (ref ) 14.8 Women Cases Ctrls ORb 67 (10.3–21.2) 306 248 96 CI 1.00 (ref ) 15.8 Cases Ctrls ORb 55 (10.5–23.8) 37 CI 134 1.00 (ref ) 14 8.80 (3.66–21.2) Occupational, possible 61 80 3.02 (1.93–4.75) 48 67 2.82 (1.68–4.72) 13 13 Familial 40 45 4.63 (2.67–8.02) 14 28 2.55 (1.15–5.62) 26 17 Residential 44 75 2.39 (1.42–4.03) 20 42 2.14 (1.07–4.28) 24 33 3.24 (1.33–7.86) Other non-occupational 13 23 2.58 (1.17–5.72) 7 8 4.67 (1.49–14.6) 6 15 2.16 (0.64–7.29) 0.07 P-Wald test gender interaction AIC 3.70 (1.34–10.3) 10.3 1409.71 AIC:Akaike information criterion a OR adjusted by centre, gender, age, and type of interview b OR adjusted by centre, age, and type of interview 1002.86 416.62 (4.10–26.1) Migliore et al. Environmental Health (2022) 21:60 Page 8 of 13 that those from Table 2 fitted data better. Furthermore, in Supplementary Table S6 we report our findings from analyses using only unlikely exposures as the reference category, rather than combining them with unknown exposures: all ORs for the occupational and non-occupational exposure categories corresponding to those from Table 2 were slightly higher, and indeed the unknown exposure category itself was associated with an increased OR among men. All results in Table 2 and Supplementary Tables S4-S6 were obtained after allowing for a 10-years lag in the analyses. Unlagged and 20-years lagged results as well as the AIC values for corresponding models were very similar, whereas introducing a 30-year lag slightly reduced the OR point estimates, increasing the AIC values (results not shown). In Table 3 we describe the exposure–response relationship between MM risk and quantitative estimates of cumulative (occupational) exposure to asbestos. Exposed subjects had an approximately double risk of developing pleural MM. A positive linear trend (P-value < 0.001) for increasing OR was found in categorical analyses by CE. Spline modelling confirmed the association between CE and pleural MM, with the OR increasing steeply up to CE values around 1 f/mL-y and more slowly thereafter (Fig. 3). Results remained substantially unchanged in lagged analyses; they are shown in Table 3 alongside un-lagged findings: at 20-years lag the AIC was minimized, while widening the lag to 30 years increased it. At lag 20 the OR increased from 1.45 (CI 0.96–2.21) at < 0.34 f/mL-y Table 3 Number of cases and controls, odds ratio (OR) and 95% confidence interval (CI) by exposure (ever vs never) and cumulative exposure from SYN-JEM, unlagged and at lag 10, 20 and 30 years, men and women – the MISEM study, 2012–2015, Italy Lag 0 SYN-JEM exposure Cases Ctrls OR a CI Lag 10 Lag 20 a a OR CI OR Lag 30 CI ORa CI Ever/never Unexposed 278 446 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) Exposed 333 262 2.22 (1.70–2.91) 2.21 (1.69–2.90) 2.29 (1.74–3.00) 2.15 (1.64–2.82) P-Wald test gender interaction AIC 0.27 0.28 0.28 0.59 1597.49 1595.26 1591.05 1609.33 Below/above median cumulative exposure (in f/mL-y) Unexposed < 0.86 ≥ 0.86 278 446 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) 120 131 1.71 (1.21–2.42) 1.71 (1.21–2.42) 1.84 (1.30–2.59) 1.74 (1.24–2.46) 213 131 2.66 (1.95–3.64) 2.65 (1.94–3.61) 2.68 (1.96–3.66) 2.52 (1.84–3.45) P-trend P-Wald test gender interaction AIC < 0.001 < 0.001 < 0.001 0.46 0.48 0.47 < 0.001 0.76 1593.89 1591.87 1589.08 1607.56 Tertiles of cumulative exposure (in f/mL-y) Unexposed < 0.34 0.34–1.62 > 1.62 278 446 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) 64 87 1.35 (0.88–2.05) 1.34 (0.88–2.04) 1.45 (0.96–2.21) 1.21 (0.79–1.85) 118 87 2.52 (1.75–3.62) 2.53 (1.76–3.64) 2.59 (1.80–3.73) 2.48 (1.72–3.57) 151 88 2.65 (1.85–3.77) 2.62 (1.84–3.72) 2.69 (1.88–3.83) 2.64 (1.85–3.77) P-trend P-Wald test gender interaction AIC < 0.001 < 0.001 < 0.001 0.39 0.36 0.30 < 0.001 0.58 1591.96 1589.78 1587.16 1601.36 Cumulative exposure, continuos Unexposed 278 446 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) Unit exposure: 1 f/mL-y 333 262 1.28 (1.16–1.42) 1.28 (1.16–1.41) 1.28 (1.16–1.42) 1.31 (1.18–1.46) AIC 1603.43 1601.83 1600.27 1612.64 Log-cumulative exposure, continuos Unexposed 278 446 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) 1.00 (ref ) Unit exposure: 1 log(f/mL-y + 1) 333 262 2.06 (1.62–2.61) 2.05 (1.61–2.60) 2.06 (1.62–2.61) 2.12 (1.65–2.72) AIC AIC Akaike information criterion a OR adjusted by centre, gender, age and type of interview 1594.23 1592.57 1590.98 1603.91 Migliore et al. Environmental Health (2022) 21:60 Page 9 of 13 Fig. 3 Odds ratios and 95% confidence intervals for pleural mesothelioma by cumulative exposure to asbestos (natural log-scale) modelled as a restricted cubic spline with five knots at 10/25/50/75/90 percentiles, men, lag10, 20 and 30 years – the MISEM study, 2012–2015, Italy to 2.59 (CI 1.80–3.73) at 0.34–1.62 f/mL-y and 2.69 (CI 1.88–3.83) at > 1,62 f/mL-y, while the OR per 1 f/mL-y of CE was 1.28 (CI 95% 1.16–1.42). Figure 3 shows that applying different lags did not modify the shape of the exposure–response relationship. Gender-specific analyses were also carried out and results for men are shown in Supplementary Table S7: they were very similar to those in Table 3, with more evident trends in categorical analyses and almost identical results for those treating CE as a continuous variable. Results from sensitivity analyses, replicating the same models as in Tables 2 and 3 are shown in Supplementary Tables S8 and S9 (by ReNaM exposure category and, respectively, by CE after additional adjustment for blue-collar status), and S10 and S11 (as before, restricted to blue-collar workers). Additional adjustment for bluecollar status brought little if any changes in results and model fit for the ReNaM exposure categories; ORs by CE category and unit exposure were slightly reduced. After restriction to ever blue-collar workers, the ORs associated with ReNaM exposure categories slightly decreased in men (but not in women) and confidence intervals were widened. ORs by CE category and by f/mL-year were reduced, but there remained a positive trend with increasing CE category and the risk did increase by f/ mL-year. Discussion In this population-based study, using different approaches, we found clear associations between MM risk and i) selected industries and occupations; ii) occupational and non-occupational exposures, classified according to ReNaM; and iii) occupational exposure indices calculated with a quantitative job-exposure matrix, SYN-JEM. MISEM allowed for the first time the calculation of relative risk estimates by industry and occupation, providing evidence of increased risk for various trades which entailed widespread use of asbestos-containing materials and had relatively large prevalence in the general population. It also put to test the standard ReNaM exposure assessment, by computing risk estimates by ReNaM exposure index, showing that all ReNaM exposure categories were associated with substantially increased MM risk and that their conventional ranking broadly parallels MM risk. Lastly, linkage with SYN-JEM provided quantitative estimates of occupational exposures and exposure–response analyses showed a sub-linear relationship between CE and MM risk. The Italian asbestos industry included all main types of asbestos products manufacturers. The first asbestos textiles factory opened around 1870 in the outskirts of Turin and several plants, mainly in Piedmont and Migliore et al. Environmental Health (2022) 21:60 Lombardy, were active up to the late 1980s. The oldest asbestos-cement plant was started in Casale Monferrato in 1907; asbestos-cement production peaked during the early 1970s, sustained by about 40 factories scattered all over Italy, a few of which continued activity until the asbestos ban in 1992. These industries, as well as the production of asbestos insulation boards, asbestos cardboard and asbestos brake and clutch linings, were included for the analysis in the “Manufacture of non-metallic mineral products not elsewhere classified” ISIC group (code 3699), which was associated with a clear-cut increase in MM risk—notwithstanding a most likely underestimation of the true risk for asbestos workers, due to the lack of specificity of this ISIC group, which comprises also non-asbestos industries. A further limitation is that, in 2012–2014, when MISEM cases and controls were recruited, a large number of former Italian asbestos workers had already died: asbestos-cement production was the single most important branch, estimated to use some 85% of all raw asbestos [17], and a pooled study of Italian cohorts with 13,076 workers from 21 asbestos-cement plants had registered 6626 decedents (52.7%) by the end of followup in 2012 [18]. The progressive shrinking of the pool of former asbestos workers made their exposure in the general population even rarer than originally; the limited number of exposed cases and controls in the study dataset accounts for the wide confidence interval of the risk estimate for men, and the absence of exposure controls among women. Asbestos-containing materials and products (ACMs) found large industrial use and we observed increased MM risk in the trades where such ACMs had been mostly employed and in the associated occupations. Manufacture of railroad equipment, ship building and repairing, chemical industry, oil refineries, iron and steel mills and the glass production industry are well known for the extensive use of insulation materials such as insulation blocks, asbestos felts and mattresses, and sprayed asbestos, whose fragility may entail substantial exposures during application and maintenance; indeed, when incompletely confined or during maintenance also production workers may get indirectly exposed. Such industries as well as various related jobs were associated with high ORs, and the same exposure patterns may be the explanation for similarly increased ORs in the manufacture of special industrial machinery and equipment and among welders and flame cutters, sheet metal workers and structural metal preparers and erectors. We found elevated MM risk in the construction industry and in several occupations within it. This is in agreement with previous studies [19–24]. Notably, in this sector exposure continued (and is still continuing) after Page 10 of 13 the asbestos ban due to presence of large quantities of still unremoved ACMs. Indeed, the majority of occupational exposures among male MM cases in Italy (15.5%) were estimated to have occurred in the construction industry [4]. Freight transport by road and water are two further economic activities for which exposure data from ReNaM had given a warning, as they accounted for about 6% of all asbestos occupational exposures [4]. Moreover, engine-room personnel were the most frequently exposure associated occupation. Our findings confirmed an increased MM risk. Most occupational exposures for MM cases in women in Italy have been recorded in the textile industry and were assessed as due to the use of brake and clutch linings and structural fire-proofing and insulating materials [25]. Among men, stock clerks were at high MM risk. This apparently surprising result may be explained by their predominant employment in industrial settings where the use of asbestos-containing materials was likely (see results). Also, in women, the increased OR for charworkers, cleaners and related workers may be due to their engagement mainly in industrial cleaning services. Risk estimates by ReNaM exposure category showed that ReNaM exposure indices for both occupational and non-occupational exposures were strongly associated with MM risk in men as well as in women. Occupational exposures (definite or probable) entailed the highest MM risk in men – as expected, as exposures at work are known to reach on average higher levels. However, this did not seem to be the case for women: even if their risk estimates have wide and overlapping confidence intervals, familial exposures were associated with the most elevated OR. This finding is consistent with other studies, such as the observed mortality from pleural malignancies (as a proxy for MM) among the wives of Casale Monferrato asbestos-cement workers: whereas these women had never been employed at the local asbestos-cement factory, they experienced an about 18-fold increase in mortality compared with the general female population. Such increase had been considered to be due to exposures at home, while accomplishing tasks such as cleaning workers’ clothes [26]. The results by ReNaM categories were confirmed when the analyses were repeated by changing the reference category: rather than combining unlikely and unknown exposures, only unlikely exposures were then used as reference. Point estimates were slightly increased, and the unknown exposure category itself was associated with a two-fold risk increase in men; this result suggests the unknown exposure category had been enriched by exposed individuals, which could be explained if experts Migliore et al. Environmental Health (2022) 21:60 engaged in exposure assessment had been conservative in their judgment, assigning exposures to this category when in doubt. An additional result from MISEM is the assessment of the exposure–response relationship between asbestos and MM, which was made possible by associating the occupational histories of cases and controls with SYNJEM, the job-exposure matrix developed for SYNERGY, a pooled analysis of case–control studies on lung cancer and occupation coordinated by IARC [14]. SYN-JEM had several features we deemed desirable. It estimated asbestos exposure by occupation according to the ISCO classification, which we also used in MISEM. Its estimates were data-driven, and Italian data were included in the ExpoSYN dataset from which SYN-JEM was modeled [27]. Furthermore, such estimates were: (i) region- even if not country-specific, so that we applied to MISEM the SYN-JEM estimates for Southern-Western European countries; and (ii) calendar-period-specific, accounting, thus, for exposure changes over the considerable time spanned by work histories. We used CE as the simplest summary metric of lifelong occupational exposures and assessed its association with MM risk using various modelling approaches: CE was positively associated with risk in categorical analyses as well as in those treating exposure as a continuous variable. Spline models showed that the OR increased steeply up to about 1 f/mL-y and more slowly at higher CE values. Non-linearity of the exposure–response relationship for pleural MM has been already suggested [28]. Our findings, however, seem to point to a stronger deviation from linearity than previously suggested. A contributory factor in our study may have been represented by type of distortion in the true shape of a linear dose–response described, among the others, by Steenland and coworkers [29]: they showed that, when CE is the metric of interest, random errors associated with assigning individual exposure levels based on group measurements or estimates – which of course happens when JEMs are applied – the shape is biased upwards in the middle range of CE and downwards at the highest CE values. A further limitation of our analysis of the exposure– response relationship is that we could obtain quantitative estimates only for occupational exposures. We did, however, adjust our models for non-occupational exposures. Finally, we want to mention several limitations associated with interview and response differences between cases and controls. The first one is that interviewers could not be blinded to the case/control status in Lombardy and Tuscany, as here cases are always interviewed by occupational health officials, based on agreements between these centres and Local Health Authorities. Indeed, differences in health and mental status between Page 11 of 13 cases and controls are often obvious – even more so if we consider that interviews to respondents were relatively common among cases but not among population controls. We adjusted, therefore, by centre and type of interview (direct vs proxy) all our analyses. We also conducted a set of sensitivity analyses by leaving out each centre one at a time. A second point is the larger proportion of proxy respondents among cases, associated with patients’ rapidly declining health. As proxies are expected to provide less detailed descriptions of the work and living environment, we adjusted our analyses by type of respondent. Residual confounding, if any, may have biased our findings towards the null. Finally, we obtained a lower response rate among controls. We observed declining response rates among population controls in two studies conducted in the Casale Monferrato area in respectively, 1987–1993 and 2001–2006: responding controls dropped from 83 to 63% of eligible subjects [30, 31]. As response rate depends on education and socio-economic status, its difference between cases and controls may have biased our findings. Findings by Ferrante et al. [30] were confirmed by a re-analysis where education had been used as a proxy to socio-economic status [32]. We addressed the potential non-response bias by using ever employment as a blue-collar worker as a proxy: we first repeated our set of analyses by introducing it as a covariate and then by restricting analyses to blue collar workers. Both sets of results confirmed the OR increase by CE observed in our main analyses, even if the OR point estimates appeared to be slightly reduced and confidence intervals widened. In conclusion, we conducted a population-based case–control study with nation-wide population basis. Having involved five centers serving regional populations ranging from Northern to Southern Italy, we included areas with historically different types and levels of industrial development. We took advantage of the ReNaM network and experience, in particular of its detailed questionnaire and, additionally, carried out a quantitative exposure assessment by linkage with SYNJEM, a JEM developed using data (among the others) from Italy relative to a time span overlapping the work histories of cases and controls. We observed increased MM risk in the asbestos industry, manufacture of railroad equipment, ship building and repairing, chemical industry, oil refineries, iron and steel mills, glass production industry, constructions and freight transport by road and water, as well as in associated occupations. Non-occupational exposures to asbestos, as identified by the ReNaM categories for residential, familial and other non-occupational exposures, also entailed a Migliore et al. Environmental Health (2022) 21:60 remarkable increase in MM risk. Finally, we observed that MM risk was sub-linearly proportional to CE. Abbreviations MISEM: Multicentre Italian Study on the Etiology of Mesothelioma; MM: Malignant mesothelioma; ReNaM: National Mesothelioma Registry; COR: Registry Operating Centre; ISIC: International Standard Industrial Classification of Economic Activities; ISCO: International Standard Classification of Occupations; IARC: International Agency for Research on Cancer; CE: cumulative exposure; OR: Odds Ratios; CI: Confidence Intervals; AIC: Akaike information criterion; BC: Blue-collar. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12940-022-00869-5. Additional file 1: Supplementary Table S1. Characteristics of the study. Supplementary Table S2. Pleural mesothelioma in men and women: odds ratio (OR) and 95% confidence intervals (CI) by industry according to the International Standard Industry Classification (ISIC, 4-digit codes), 1971 – Industries with at least 3 exposed cases. Supplementary Table S3. Pleural mesothelioma in men and women: odds ratio (OR) and 95% confidence intervals (CI) by occupation, according to the International Standard Code of Occupations (ISCO, 3-digit codes), 1968 – Occupations with at least 3 exposed cases. Supplementary Table S4. Number of cases and controls, odds ratio (OR) and 95% confidence intervals (CI) by modality of exposure according to the Italian National Mesothelioma Register (ReNaM) classification. Reference category: unlikely/unknown exposures. All occupational exposure categories (definite, probable and possible) combined, lag 10 years. Supplementary Table S5. Number of cases and controls, odds ratio (OR) and 95% confidence intervals (CI) by modality of exposure according to the Italian National Mesothelioma Register (ReNaM) classification. Reference category: unlikely/unknown exposures. Distinct occupational categories (definite, probable and possible), lag 10 years. Supplementary Table S6. Number of cases and controls, odds ratio (OR) and 95% confidence intervals (CI) by modality of exposure according to the Italian National Mesothelioma Register (ReNaM) classification. Reference category: unlikely exposures, lag 10 years. Supplementary Table S7. Number of cases and controls, odds ratio (OR) and 95% confidence interval (CI) by exposure (ever vs never) and cumulative exposure from SYN-JEM, unlagged and at lag 10, 20 and 30 years, men. Supplementary Table S8. Number of cases and controls, odds ratio (OR) and 95% confidence intervals (CI) by modality of exposure, according to the Italian National Mesothelioma Register (ReNaM) classification. Reference category: unlikely/unknown exposure, lag 10 years. Analyses adjusted also by blue-collar status, as a proxy for socio-economic status. Supplementary Table S9. Number of cases and controls, odds ratio (OR) and 95% confidence interval (CI) by exposure (ever vs never) and cumulative exposure from SYN-JEM, unlagged and at lag 10, 20 and 30 years, men and women. Analyses adjusted also by blue-collar status, as a proxy for socio-economic status. Supplementary Table S10. Number of cases and controls, odds ratio (OR) and 95% confidence intervals (CI) by modality of exposure, according to the Italian National Mesothelioma Register (ReNaM) classification. Reference category: unlikely/unknown exposure, lag 10 years. Analyses restricted to blue-collar cases and controls. Supplementary Table S11. Number of cases and controls, odds ratio (OR) and 95% confidence interval (CI) by exposure (ever vs never) and cumulative exposure from SYN-JEM, unlagged and at lag 10, 20 and 30 years, men and women. Analyses restricted to blue-collar cases and controls. Acknowledgements We thank Lützen Portengen who carried out the linkage of occupational histories with SYN-JEM. We are indebted with patients and their care givers, and with population controls. This study would not have been possible without the cooperation of the chest surgeons, pneumologists, oncologists and pathologists who participate to our regional rapid alert networks. Page 12 of 13 Authors’ contributions EM (Enrica Migliore) and DM conceptualized and designed the study, coordinated and supervised data collection, carried out the analyses, drafted the initial manuscript, and reviewed and revised the manuscript; DC (Dario Consonni) contributed to the planning and design of the study, supervised and performed data analysis and critically reviewed and revised the manuscript; SP, RCHV and HK carried out the job-exposure matrix-based exposure assessment, and reviewed manuscript; AB, DC (Domenica Cavone), EC, CM (Corrado Magnani), CM (Carolina Mensi), EM (Enzo Merler), MM collected data, carried out expert-based exposure assessment, and reviewed the manuscript. AM contributed to the planning and design of the study and reviewed and revised the manuscript. All authors read and approved the final manuscript. Funding MISEM was funded by a grant (to DM) from the Centro Controllo Malattie (Centre for Disease Control and Prevention) of the Italian Ministry of Health, 2012 research program. Data management and analysis was further financially supported by a grant (to DM) from the Istituto Nazionale per l’Assicurazione contro gli Infortuni sul Lavoro (INAIL—Italian Insurance Institute against Accidents at Work and Occupational Diseases), internal research plan 2016–2018, ID 55. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate. The study was approved by the Internal Review Board of the coordinating centre (Cancer Epidemiology, Turin). Centre participation was approved by their respective Internal Review Boards. Participants gave their written, informed consent. Competing interests CM (Corrado Magnani), DM, EM (Enzo Merler) and AB served as expert witness for the Public Prosecution Office in court cases of asbestos-related neoplasms due to occupational or environmental exposures. All other authors declare they have nothing to disclose. Author details 1 Cancer Epidemiology Unit, CPO Piemonte and University of Turin, Turin, Italy. 2 Interdepartmental Centre G. Scansetti for Studies On Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy. 3 Occupational Health Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. 4 Department of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands. 5 Occupational Medicine Unit, Careggi University Hospital, Florence, Italy. 6 Interdisciplinary Department of Medicine, Section of Occupational Medicine “B. Ramazzini”, University of Bari, Bari, Italy. 7 Unit of Environmental and Occupational Epidemiology, Cancer Prevention and Research Institute, Florence, Italy. 8 Department of Translational Medicine, Unit of Medical Statistics and Cancer Epidemiology, University of Eastern Piedmont and CPO Piemonte, Novara, Italy. 9 Occupational Health Unit, Department of Prevention, Padua, Italy. 10 Unit of Occupational and Environmental Epidemiology ‑ Italian Mesothelioma Register, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, Italian Workers’ Compensation Authority (INAIL), Rome, Italy. Received: 3 January 2022 Accepted: 31 May 2022 References 1. Montanaro F, Bray F, Gennaro V, Merler E, Tyczynski JE, Parkin DM, et al. Pleural mesothelioma incidence in Europe: evidence of some deceleration in the increasing trends. Cancer Causes Control. 2003;14:791–803. 2. Ji J, Sundquist J, Sundquist K. 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https://openalex.org/W2131398998	https://www.omicsonline.org/opiorphin-secretion-pattern-in-healthy-volunteers-gender-difference-and-organ-specificity-2161-1009.1000136.pdf	English	null	Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity	Biochemistry and analytical biochemistry :	2,013	cc-by	11,997	Abstract Background: Opiorphin is an endogenous human peptide regulator of enkephalin bioavailability. It inhibits enkephalin-inactivating ectopeptidases to produce analgesia and antidepressant-like effects in standard rodent models via activation of µ and/or δ opioid pathways. Our aim was to establish the quantitative profile of secretion and distribution of this regulator in young adult volunteers. Methods: We developed a specific ELISA-based method, in tandem with RP-HPLC chromatography, to determine opiorphin levels in blood, urine, semen and milk of healthy male and female volunteers. We also investigated the presence of the mature opiorphin in tears and saliva because it was previously reported that PROL1 gene, encoding opiorphin precursor, is primarily expressed in human lachrymal and salivary glands. Results: Opiorphin circulates as an endocrine messenger in the human bloodstream at 0.3-1.1 ng/ml median ranges. Its physiological concentration under basal conditions is higher in males than in females while it is significantly lower in sixth month pregnant compared to non-pregnant volunteers. It is eliminated in the urine with a significant higher rate in men compared to women. Opiorphin is distributed, at rates 10 times higher than in plasma, in the semen of normozoospermic donors and in the milk of lactating women as both free molecular and cation mineral-binding forms. Opiorphin is secreted in tears and saliva at the highest physiological concentrations, demonstrating that salivary and lachrymal glands are the tissues of major expression and secretion of PROL1 gene mature products. Discussion: Our data, associated with database searches from human transcriptome and our previous functional findings, provide evidence that opiorphin exerts organ-specific and gender-specific functions in human physiological systems through neuroendocrine, paracrine/autocrine and/or exocrine mechanisms. Keywords: Opiorphin; Inhibitor of enkephalin; Inactivating peptidases; ELISA based assay; Human biofluids; PROL1 gene Keywords: Opiorphin; Inhibitor of enkephalin; Inactivating peptidases; ELISA based assay; Human biofluids; PROL1 gene [15] and whose local and systemic secretion is stimulated under adrenergic-mediated response to environmental stress in male rats [16,17]. The physiological range of circulating sialorphin in the plasma of conscious adult male rats is 1.9 ± 0.2 ng/ml under resting conditions and 7-12 ng/ml under adrenergic or acute stress stimulated conditions [16]. Under adrenergic stimulated conditions of anesthetized adult male rats the maximal values in saliva may attain 1 µg peptide /ml saliva over the 30 min period of saliva collection [18,19]. In addition, we also identified the presence of sialorphin in urine that is excreted through renal glomerular filtration [16]. Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity Dufour E1, Villard-Saussine S1,2, Mellon V3, Leandri R4, Jouannet P4, Ungeheuer MN3 and Rougeot C1* Dufour E1, Villard-Saussine S1,2, Mellon V3, Leandri R4, Jouannet P4, Ungeheuer MN3 and Rougeot C1* 1Pasteur Institute, Laboratory of Pharmacology of Pain, Department of Structural Biology and Chemistry, Paris cedex, France 2Bio-Rad, 9 Avenue du Canada 91959 Courtaboeuf Cedex, France 3Pasteur Institute, Platform Clinical Investigation and Access to Biological Resources (platform ICAReB), Paris cedex, France 4Cochin Hospital, Laboratory of Reproductive Biology - CECOS, Paris cedex, France Corresponding author: Catherine Rougeot, Pasteur Institute, Laboratory of Pharmacology of Pain, Department of Structural Biology and Chemistry, France, Tel: 33 (0)1 40 61 34 45; Fax: 33 (0)1 45 68 83 99; E-mail: catherine.rougeot@pasteur.fr Received June 07, 2013; Accepted June 22, 2013; Published June 25, 2013 Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Copyright: © 2013 Dufour E, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract In female rats, sialorphin is detected in the mammary glands and the placenta and also in breast milk [11,16] (and unpublished observations by Rougeot C). Received June 07, 2013; Accepted June 22, 2013; Published June 25, 2013 Biochemistry & Analytical Biochemistry Biochemistry & Analytical Biochemistry Dufour et al., Biochem Anal Biochem 2013, 2:3 DOI: 10.4172/2161-1009.1000136 Open Access Research Article Introduction Opiorphin, a QRFSR pentapeptide, is an endogenous human peptide regulator that was discovered using a functional biochemical approach [1,2]. It is a physiological dual inhibitor of Zn-ectopeptidases, neutral endopeptidase (NEP EC3.4.21.11) and aminopeptidase N (AP-N EC3.4.11.2), which are implicated in the rapid inactivation of endogenous circulating opioid agonists, namely the enkephalins. Opiorphin protects enkephalins from inactivation by ectoenkephalinases and thus improves the specific binding and affinity of enkephalin-related peptides to membrane opioid receptors [3]. Enkephalins are opioid neuropeptides, which play key roles in the control of nociceptive transmission and in the modulation of the activity of cerebral structures governing motivation and the adaptive balance of emotional states [4,5]. By increasing the half-life of circulating enkephalins, opiorphin, at systemically or centrally active doses (1-2 mg/kg, iv or 5-10 µg/kg icv), produces analgesia in various rodent models of pain and also antidepressant-like effects in standard rat or mouse models of depression, via endogenous enkephalin-related activation of µ and/or δ opioid pathways [1,6-9]. The exciting discovery of human opiorphin followed from the characterization of rat sialorphin but opiorphin has revealed its own specificity notably in terms of molecular structure as it shares only 40% sequence similarity with sialorphin peptide. Human opiorphin is the mature peptide product of the PRL1 precursor polypeptide of Rat sialorphin (QHNPR peptide) was the first identified natural enkephalin catabolizing inhibitor that induces potent antinociception in mammals [10,11]. It is an endogenous androgen-regulated peptide and also modulates, at doses equivalent to physiological circulating levels, male sexual behavior [12-14]. We also demonstrated that sialorphin is an exocrine and endocrine peptide messenger whose major expression in rat salivary and prostate glands is activated by androgen regulation Copyright: © 2013 Dufour E, 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. Volume 2 • Issue 3 • 1000136 Page 2 of 11 Written informed consent was obtained from each subject following a detailed explanation of the purpose of the study, the procedures and the possible risks. This biomedical research study was performed at Institut Pasteur and the associated investigational sites and the study protocol was approved by the French Ethical Committee (so-called Comité de Protection des Personnes) of Cochin Hospital (Paris, France): accession n°. Am3485-2-2308 on 25th April 2007. Study subjects We studied 3 groups of healthy young adult volunteers of 26 ± 6 yr mean age. Group 1: opiorphin was measured in the blood, in the salivary and lachrymal secretions and in urines, among male and female healthy volunteers. Group 2: opiorphin production was studied among pregnant women, during the first sixth month of pregnancy, in the blood and saliva and also in their breastfeeding milk during the second to third weeks after delivery. Group 3: analyses were conducted on the sperm of normozoospermic donors (2.9 ± 0.4 ml of ejaculate, mean ± SEM for 11 subjects) after 4 ± 2 days of sexual abstinence and also of the blood, urines and salivary secretions. Opiorphin was also measured in the seminal fluid of patients (1.0 ± 0.2 ml of ejaculate, mean ± SEM for 5 subjects) with congenital bilateral absence of the vas deferens (CBAVD), an abnormal anatomy of the male genital tract inducing azoospermia and the ejaculation of semen devoid of the testis, epididymis and seminal vesicles secretions. Sample preparation: Sample preparation is a critical step to quantify small molecules in complex biological media and its quality can greatly influence the overall sensitivity and specificity of the subsequent analyses. Indeed, the highly abundant house-keeping proteins present in most biological fluids, in particular in plasma, semen, milk and saliva can mask detection of the less abundant bioactive peptides such as opiorphin. After de-frosting at 4°C, the biological samples were treated according to the following conditions: Acid-methanol extraction: To one volume sample four volumes of 0.1% trifluoroacetic acid (TFA, Sigma-France) in methanol were added at 4°C, and the mixture immediately processed by vigorous stirring. This first step results in the precipitation and the elimination of high molecular weight proteins that are denatured in acid-methanol solutions. The soluble low molecular weight molecules (in particular opiorphin), found in the methanol phase, were separated from the precipitate by centrifugation at 4700 rpm for 30 min at 4°C and were lyophilized at -110°C for 48 h. Preliminary examination of all subjects was carried out to check for concomitant or severe disorders, clinically significant disease or recent intervention in the oral cavity and for negativity for HIV, HCV or HBV serology. The subjects received no medication (anti- inflammatory, analgesic, antidepressant and/or anti-hypertensive) for at least 7 days prior to sampling. They were self-described as nondependent on addictive drugs (alcohol, tobacco). Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Introduction The blood, saliva and urine samples of healthy volunteers were provided by the clinical research investigator and associate of the ICAReB platform of Institut Pasteur (Paris, France). The milk samples were obtained from healthy pregnant women from the Unit of Obstetrics of Necker- Enfants Malades hospital and the semen samples from healthy donors and infertile patients from the Unit of Biology of the Reproduction of Cochin hospital (Paris, France). 248 amino acids. The PROL1 gene encodes the secreted preproprotein that contains, in the N-terminal region, the peptide QRFSR. It is intra- cellularly processed during granule maturation by selective cleavage at consensus sites, i.e., the recognition sites for secretion signal peptidase and for paired basic amino acid convertases (http://www.genecards. org/cgi-bin/carddisp.pl?gene=PROL1). The gene was identified by Dickinson et al in 1996, but was only detected at that time in human lachrymal and salivary glands [20]. The discovery of opiorphin is the first demonstration of the existence of a physiological regulator of enkephalin bioavailability in humans. When the extent of the functions mediated by the endogenous enkephalinergic pathways are considered, the characterization of this upstream modulator of opioid pathways in humans is of major interest from both physiological and physiopathological points of views. Indeed, endogenous human opiorphin appears to intervene in the process of adaptation mediated by enkephalins, which are associated with nociception and emotion-related behaviors. As a consequence, opiorphin could be a biological marker with a potential diagnostic role in various pathological situations, such as hyperalgesic syndromes, depressive states and socio-relational behavioral imbalances. Study protocol Sample collection: Ex vivo, pharmacokinetic and metabolic data reveal that opiorphin, similarly to all peptide mediators of the neuroendocrine system, presents a short metabolic half-life, i.e., about 5 min in human plasma (unpublished observations by Rougeot C). The mixture of peptidase inhibitors previously used for sialorphin detection [16] protected opiorphin from degradation by circulating human peptidases. Thus the biological samples were taken and collected (i.e., 2-5 ml of saliva, 30 ml of whole blood, 20 ml of urine per subject) into previously cooled polypropylene tubes (4, 15 or 50 ml, Nunc-VWR, France) containing a mixture of peptidase inhibitors: Péfabloc 0.4 mM, Aprotinin 1000 KIU/ml, EDTA 1 mM, bestatine 150 µM, leupeptin 1 µM and pepstatin 1 µM (Sigma-Aldrich, France). For the semen samples the peptidase inhibitors were limited to 1000 KIU/ ml Aprotinin and 0.4 mM Pefabloc, according to previous validation tests and allowing the examination of spermatozoa concentration and motility in parallel with standard markers. Saliva was collected under two conditions, basal and stimulated: a drop of diluted lemon was applied to the mouth floor to stimulate saliva production. Tear samples were collected after local administration of one drop of saline containing diluted lemon, applied to the inner corner of the eye, to induce the production of tears. Blood samples were centrifuged for 15 min at 300×g and 4°C to separate plasma from whole blood and the resulting plasma supernatants were carefully collected. All samples were immediately stored at -80°C, they were transported in a dry ice container to the laboratory. Here, our objective was to determine the human biological fluids in which opiorphin circulates and to establish its quantitative profile of secretion and distribution in male and female healthy volunteers, in order to then establish an investigative program in patients suffering different pathologies of interest. Based on our previous knowledge of sialorphin expression, samples were collected from the blood, saliva, tears and urine of adult healthy volunteers (male and female, mean age 26 ± 6 yr) and from the milk of lactating healthy women (2-3 wk after delivery) as well as from the semen of healthy donors and of men with congenital bilateral absence of vas deferens (CBAVD). Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 method The opiorphin derivative selected for the coating on the micro- titration plate (96-well-ImmunoPlate, Nunc-VWR) has the following sequence: Y-[CH2]12-QRFSR peptide. Indeed, from previous tests it appears that the presence of the 12-polyethylene linker introduced between the tyrosine residue and the opiorphin peptide sequence facilitates the coating and the accessibility of the immobilized peptide to the antibody. Reverse phase C18-HPLC Chromatography: The resuspended dried-extracts, obtained during the procedures described above, were applied (150 µl) to the top of the C18/RP-HPLC analytical column (150×4.5 mm Luna 5 µ Phenomenex-France or ACE 5 µ AIT-France) under TFA 0.1%-water solvent equilibrium conditions. The various components were eluted and isolated according to their hydrophobic characteristics, in a 30-min linear gradient from 0% to 80% acetonitrile, containing 0.1% TFA (Lichrosol, Merck) at a 1 ml/ min flow rate (Surveyor HPLC system, ThermoScientific-France). The entire HPLC system was thermo-regulated at 12°C. Each fraction (1 ml) was collected and lyophilized at -110°C for 48 h. The determination of plasma opiorphin levels was achieved by analyzing each HPLC- collected fraction, from 15 to 26 min, using the opiorphin ELISA. This HPLC purification step, that was critical in the case of the plasma samples, was also applied to the milk, semen and tear samples in order to determine the authentic presence of opiorphin molecule. Optimized assay conditions are summarized as follows. For the coating, 40 ng of the Y-[CH2]12-QRFSR peptide per 200 µl coating buffer were added to individual wells (central 60 wells) on a 96- well micro-titration plate and incubated overnight at 4°C with light shaking. In parallel, 100 µl of standard (from 500 ng/ml to 1.9 ng/ml) or samples that were serially diluted 2-fold with incubation buffer were pre-incubated in Matrix 0.75 ml ScreenMates tubes (ThermoScientific) overnight at 10°C in the presence of 100 µl anti-opiorphin antibody diluted at 1/80 000. The following day, after washing 5 times (microplate washer, BioTech-France) with washing buffer, 250 µl of saturation buffer were added to the individual coated-wells and incubated at least 1 h at 20°C. Then, after 5-times washing, 100 µl of the pre-incubated immunological reaction (anti-opiorphin antibody plus sample or reference peptide) were transferred on the coated and saturated micro- titration plates and incubated 1.30 h at 10°C under humid atmosphere. After washing 5 times, 100 µl of the anti-rabbit IgG conjugated to HRP, diluted at 1/3 000, were added to each well and incubated 1 h at 20°C. Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Development of the ELISA - Opiorphin The respective incubation buffers were the following: Coating buffer: potassium phosphate buffer at 100 mM and pH 7.1; Saturation buffer: Tris buffer at 20 mM pH 7.5+150 mM NaCl+0.1% Tween 20+0.5% gelatin; First incubation buffer (anti-opiorphin antibody+sample or reference-peptide): Tris buffer at 200 mM and pH 7.5+150 mM NaCl+0.1% Tween 20+0.1% bovine serum albumine (BSA); Second incubation buffer (anti-rabbit IgG antibody conjugated to HRP): Tris buffer at 20 mM and pH 7.5+150 mM NaCl+0.1% Tween 20+0.1% At the beginning of this study, none of the opiorphin detection system was commercially available. Thus, our expertise in the field of bioactive peptide isolation and quantification allowed us to develop and validate our own competitive ELISA system [18,21]. Study subjects Regarding group 2, women exhibiting significant pathology during pregnancy, including gestational diabetes and hypertension, were excluded. Volume 2 • Issue 3 • 1000136 Page 3 of 11 Saliva, tears and milk dried-extracts were reconstituted in 1 volume of pyrolyzed pure water corresponding to the initial volume of samples, processed by centrifuge at 4700 rpm for 30 min at 4°C; the resulting supernatants were conserved at -80°C until subsequent procedures. In the case of sperm samples, dried-extracts were reconstituted in 0.2 times the initial volume to attain a 5X final concentration. In the case of urine samples, dried-extracts were reconstituted in 0.4 times the initial volume to achieve a 2.5X final concentration. type immuno-assay is applicable for the specific quantification of the peptide: competition between the measured free antigen and the coated or labelled antigen towards the anti-antigen antibodies [22]. Opiorphin QRFSR-peptide is not immunogenic therefore production of the anti- opiorphin antibodies in the animal requires the injection of the peptide conjugated in a covalent manner to a carrier molecule. Anti-opiorphin antibody was generated after administration of the ovalbumin-[CO- NH]-YQRFSR immunogen (Genosphere Laboratories, France) to rabbits and a precise follow-up of the specific immune response (CovalAb biotechnologies, France). The polyclonal antibody, selected on the basis of its affinity for opiorphin, is referred to as 3RBF-SAB. C18 Solid-phase Extraction: The acidified (HCl 0.1 N final concentration) and clarified biological samples were applied to C18-SepPak cartridges (Waters, France) preconditioned with three successive cycles of methanol (Lichrosolv, Merck, France) and pure water and ultimately maintained in 0.1% TFA-water. After washing with 0.1% TFA-water (5 ml), the analytes were eluted according to a multi-step gradient of methanol-water containing 0.1% TFA: 5%, -20%, -40%, -60% and 100% (5 ml each). The successive fractions were collected at 4°C, frozen at -80°C and then lyophilized at -110 °C for 48h. This extraction-purification step was critical in the case of plasma and milk. The dried-extracts of the fractions, 20 and 40% methanol, were reconstituted in pyrolyzed pure water and pooled in order to concentrate 50 fold the initial volume of plasma samples or 2.5-fold the initial volume of milk samples. The specific immunological reaction was revealed using a universal system supplied by Pierce (ThermoScientific-France), namely, purified IgG immunoglobulin antibodies against rabbit IgG immunoglobulin conjugated to the horseradish peroxydase enzyme (HRP) followed by final addition of the chromogenic tetramethylbenzidine specific HRP substrate (Step UltraTMB-ELISA). Recovery Recovery was determined by adding tritiated or standard opiorphin to the biological samples in the same amounts as pre-established concentrations and processed as described above. The mean recovery was expressed as measured opiorphin concentration/added opiorphin concentration X 100%. Recovery of the marker opiorphin-peptide alone (QR [3H] FSR) in SepPak fractions, eluted in 20% and 40% methanol, was 95%. Recovery of the marker opiorphin-peptide alone (QR [3H] FSR) in eluting RP-HPLC fractions was 68%. For HPLC, recovery was done every 7-10 sample injections after applying a stringent washing column procedure. method After incubation an ultimate wash was performed and 100 µl of the HRP chromogenic substrate were added and incubated 30-45 min at 20°C. Finally, the reaction was stopped, according to the manufacture’s protocol, by adding 100 µl 4N H2SO4. Plates were read at 450 mm with a micro-plate spectrophotometer (Infinite M200, Tecan-France) and the results were successively analyzed with Magellan (Tecan), Excel Microsoft and Prism GraphPad (La Jolia, USA) softwares. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Characteristics of the Elisa-Opiorphin The competitive-ELISA immunoassay for opiorphin is a reproducible and specific assay. The IC50 was determined at 30 ± 9 ng/ml (mean ± SD) (50 pmol/ml) for n=9 independent inter-day assays, each B/B0, % B 90 80 70 60 50 40 30 20 10 1 10 100 1000 [Opiorphin QRFSR-peptide], ng/ml (log scale) Standard QRFSR-peptide 1-a 11, HPLC fraction: 20 min 1/16 1/8 1/4 1/2 IR-Opiorphin, ng/ml B/B0, % A B 90 80 70 60 50 40 30 20 10 60 50 40 30 20 10 0 -10 12 14 16 18 20 22 24 26 28 30 1 10 100 1000 Fractions, min [Opiorphin QRFSR-peptide], ng/ml (log scale) Standard QRFSR-peptide 1-a 11, HPLC fraction: 20 min 1/16 1/8 1/4 1/2 Figure 1: Analyses of human plasma by RP-HPLC and ELISA-Opiorphin. A. HPLC chromatography in tandem with ELISA-opiorphin detection of individual plasma extract. The plasma sample was extracted using a C18 solid-phase extraction procedure before loading onto a C18-ACE-HPLC column. After lyophilization each 1 min fraction was analyzed by ELISA-Opiorphin and the results expressed as concentration of immunoreactive (IR) opiorphin in ng/ml (Y axis) by HPLC- fraction in min (X axis). B. Analysis of individual purified plasma sample using ELISA-Opiorphin method. Standard curve (open squares) relating QRFSR-peptide concentration in ng/ml (log scale, X axis) to the percentage of binding in the presence (B) of standard peptide / binding in the absence (B0) of the standard peptide (linear scale, Y axis). Sample curve (open circles) relating serially diluted (2-fold) individual purified plasma sample to B/B0, % response. Note that there is a parallel between the dose-response standard curve and the serial dilutions of the sample. Each point represents the mean ± SD of duplicates. IR-Opiorphin, ng/ml A 60 50 40 30 20 10 0 -10 12 14 16 18 20 22 24 26 28 30 Fractions, min Standard QRFSR-peptide Figure 1: Analyses of human plasma by RP-HPLC and ELISA-Opiorphin. A. HPLC chromatography in tandem with ELISA-opiorphin detection of individual plasma extract. The plasma sample was extracted using a C18 solid-phase extraction procedure before loading onto a C18-ACE-HPLC column. After lyophilization each 1 min fraction was analyzed by ELISA-Opiorphin and the results expressed as concentration of immunoreactive (IR) opiorphin in ng/ml (Y axis) by HPLC- fraction in min (X axis). B. Analysis of individual purified plasma sample using ELISA-Opiorphin method. principe and reagents Due to the small size of the opiorphin (693 Da), only a competitive Volume 2 • Issue 3 • 1000136 Page 4 of 11 BSA and; Washing buffer: 1 tablet PBS-Sigma in 200 ml pure pyrolyzed water+0.1% tween 20. with 9 points of concentration (from 500 to 1.9 ng/ml) in duplicate. The detection limit at IC85 is 2 ng/ml (3 pmol/ml) of opiorphin QRFSR- peptide. The immunoassay is specific for opiorphin: the functionally related sialorphin QHNPR-peptide, as well as the QRFS, RFSR and FSR opiorphin-related fragments were not significantly recognized by the antibody (cross-reaction ≤ 1%). Furthermore, a structural selectivity at the C-terminal part of the QRFSR-peptide for its binding affinity to the antibody was observed: substitution of the L-Arg5 by the stereoisomer D-Arg5 resulted in the loss of antibody recognition. Similarly, substitution at the N-terminal part of the peptide by a pyroGlu1 instead of Gln1, also inhibited antibody recognition. In contrast, the addition at the N-terminal position of the QRFSR peptide of amino acids such as Cys or Tyr did not alter antibody recognition. Results and Discussion We previously demonstrated that the functional homologue of human opiorphin, rat sialorphin, is an endocrine signal messenger of intercellular communication [10,16,17,19]. To determine whether opiorphin exhibits a similar secretory behavior we investigated and quantified its presence in the bloodstream of healthy male and female Statistical Analysis A standard curve (8-9 concentration points in duplicate) was generated and fitted for each micro-titration plate and the opiorphin concentration of samples was determined using the regression equation obtained from each corresponding calibration curve. The individual values were expressed as the mean ± SD for at least 2 determination points each in duplicate. Given the small number of subjects (n<30) non- parametric statistical analyzes were used and the results among each analyzed group expressed as the median concentration of opiorphin in ng/ml, the range and mean ± SEM were also indicated in brackets. The significance of differences among groups was assessed by the Kruskal-Wallis one-way analysis of variance (KWT, a non-parametric method) to compare repeated measures in each group between several independent variables across the experimental conditions. When a significant difference among the groups was obtained, the Mann- Whitney post hoc test (MWT) was applied to compare each paired group. For all statistical evaluations, the level of significance was set at P ≤ 0.05. All statistical analyses were carried out using the software StatView®5 statistical package (SAS, Institute, Inc., USA). A standard curve was generated for each intra-assay and the concentration of each sample was determined using the regression equation obtained from each corresponding calibration curve. Given the small number of subjects (n < 30) the significance of differences among studied groups was assessed by non-parametric statistical analyzes. The results among analyzed groups were expressed as follows: median concentration of opiorphin (range, mean ± SEM) in ng/ml. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Characteristics of the Elisa-Opiorphin Standard curve (open squares) relating QRFSR-peptide concentration in ng/ml (log scale, X axis) to the percentage of binding in the presence (B) of standard peptide / binding in the absence (B0) of the standard peptide (linear scale, Y axis). Sample curve (open circles) relating serially diluted (2-fold) individual purified plasma sample to B/B0, % response. Note that there is a parallel between the dose-response standard curve and the serial dilutions of the sample. Each point represents the mean ± SD of duplicates. Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000136 ISSN:2161-1009 Biochem, an open access journal Page 5 of 11 Page 5 of 11 Page 5 of 11 renal glomerular filtration (unpublished observation by Rougeot C). Thus the significant difference between the rates of opiorphin urinary secretion in men compared to women could be attributed to a gender difference in its circulating systemic concentrations; however, as seen above, we failed to observe clear statistical differences in bloodstream opiorphin levels under our experimental conditions. subjects. All plasma samples were submitted to a C18-solid phase extraction, according to the procedure described in Methods. Under such conditions, recovery of the marker QR[3H]FSR-opiorphin added to plasma samples was 68% ± 2%, n=4. Plasma extracts then underwent a purification step using RP-HPLC chromatography. The retention time of the reference opiorphin-peptide in the chromatographic system, using a C18-ACE column, is 20 ± 0.5 min and the ELISA gave a 70 ± 7% (n=10) recovery of the opiorphin QRFSR-peptide. subjects. All plasma samples were submitted to a C18-solid phase extraction, according to the procedure described in Methods. Under such conditions, recovery of the marker QR[3H]FSR-opiorphin added to plasma samples was 68% ± 2%, n=4. Plasma extracts then underwent a purification step using RP-HPLC chromatography. The retention time of the reference opiorphin-peptide in the chromatographic system, using a C18-ACE column, is 20 ± 0.5 min and the ELISA gave a 70 ± 7% (n=10) recovery of the opiorphin QRFSR-peptide. Taken together, our data suggest that, as in the case of rat sialorphin, human opiorphin expression and secretion is influenced by gender. Secretion of opiorphin in human semen The median concentration of opiorphin in human plasma was 0.96 ng/ml (range 0.4-2.7 ng/ml, mean ± SEM 1.07 ± 0.12 ng/ml) for 21 healthy adult male subjects and 0.55 ng/ml (range 0.2-3.4 ng/ml, mean ± SEM 0.99 ± 0.24 ng/ml) for 16 healthy adult female subjects and 0.11 ng/ml (range 0.1-1.5 ng/ml, mean ± SEM 0.28 ± 0.18 ng/ml) for 8 healthy pregnant women. The Kruskal-Wallis one-way analysis of variance (KWT) showed an overall heterogeneity in opiorphin plasma levels among the three groups, H=13.9, P=0.001 (Figure 2). Although the median concentration of opiorphin appeared higher in male plasma, there was no significant difference between male and non-pregnant women paired groups. Only a tendency toward lower concentration in non-pregnant women emerged with a P value of 0.148 using the Mann Whitney U-Test (MWT). This is probably due to the larger range in the distribution of values among the female group. Despite this heterogeneity, a statistical significance emerges when comparing the 2 female groups, pregnant and non-pregnant women (P=0.002 by MWT). The physiological range of circulating opiorphin in young adult men (26 yr mean age) is similar to circulating sialorphin in conscious young adult male rats (8 wk old) established by competitive RIA at 1-2 ng/ml under basal conditions [16]. We propose that, in analogy with its functional homologue in rat, human opiorphin is secreted as an endocrine messenger and circulates in the bloodstream to be delivered to a variety of distant NEP and APN-target tissues, where it induces a response. We previously demonstrated that the functional homologue of human opiorphin, rat sialorphin, is expressed in prostate gland [15,18]. Therefore, in the present study, we analyzed the concentration of opiorphin in human semen obtained from healthy donors. In order to measure the biological parameters of the seminal fluid of donors, in particular spermatozoa motility and vitality, the seminal fluids were collected in tubes containing a limited cocktail of peptidase. A comparative study was then conducted to establish the best conditions for opiorphin extraction. As shown in Figure 3a, treatment * Opiorphin plasma levels, ng/ml 3.5 3 2.5 2 1.5 1 .5 0 Men Women Pregnant Women (21) (16) (8) Figure 2: Box plot representations of plasma opiorphin levels in healthy young men (n=21), women (n=16) and pregnant women (n=8). Single individual values below the 10th or above the 90th percentile are represented as open circles. Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Characteristics of the Elisa-Opiorphin Furthermore, the convergent data, notably the high heterogeneity in opiorphin plasma levels among females -which may be related to differences in the menstrual cycle-, the low rates of circulating opiorphin levels in the blood of pregnant women and the low rates of urinary opiorphin in females compared to males, suggest that the secretion of opiorphin in the female systemic compartment responds to its own physiological hormonal status, notably during menstrual cycle and gestation. By this way, it is of interest that, the NEP-cell surface target for opiorphin exhibits different expression patterns during menstruation and progesterone induces transcription of the NEP gene by the coordinated action of multiple androgen responsive elements [23,24]. Unfortunately, for our study, the specific phase in the menstrual cycle was not allocated when blood collection. As shown in Figure 1a, HPLC elution profile of the human plasma extract, referenced 1a-11, demonstrates that immunoreactive opiorphin is detected at 20-21 min retention time (Rt), identical to that of the reference peptide. This purification step separates opiorphin from the ELISA-interfering plasma molecular population (22 min Rt) which inhibits the competitive assay by nonspecifically enhancing the B0 values (Figure 1a). The immunoreactive curve obtained from successive dilutions of the HPLC fraction 20 min (1a-11, open circles, Figure 1b) is parallel to the dose-response curve corresponding to the reference QRFSR-peptide (open squares). This indicates that the antibody recognizes the natural opiorphin-peptide contained in human plasma extract with the same affinity as the synthetic pure peptide. The plasma level of opiorphin for this sample was established at 1.4 ± 0.2 ng/ml for n=4 points of determination (Figure 1b). Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Secretion of opiorphin in human semen The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. P<0.01 by MWT; (n) number of subjects. Opiorphin plasma levels, ng/ml 3.5 3 2.5 2 1.5 1 .5 0 Men Women Pregnant Women The physiological median concentration of opiorphin in human urine samples was 7 ng/ml (range 4-27 ng/ml, mean ± SEM 8.3 ± 1.5 ng/ml) for n=15 male subjects and 4 ng/ml (range 1-6, mean ± SEM 3.9 ± 0.4 ng/ml) for n=15 female subjects. Statistical analyses revealed that urinary excretion of opiorphin in males is significantly greater than in females (P<0.001 vs. women by MWT). Four (± 2)- day sexual abstinence was requested before sampling, therefore, the possibility that part of the male urinary opiorphin comes from urethral semen contamination may be excluded (see the next chapter). Pharmacokinetic analyses in rats following iv administration of radiolabeled opiorphin (QR[3H]FSR-peptide) revealed that the native peptide, as well as its metabolites, are rapidly excreted, as soon as 1 min post-injection, from the blood compartment into the urine via (21) Figure 2: Box plot representations of plasma opiorphin levels in healthy young men (n=21), women (n=16) and pregnant women (n=8). Single individual values below the 10th or above the 90th percentile are represented as open circles. The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. P<0.01 by MWT; (n) number of subjects. Figure 2: Box plot representations of plasma opiorphin levels in healthy young men (n=21), women (n=16) and pregnant women (n=8). Single individual values below the 10th or above the 90th percentile are represented as open circles. The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. P<0.01 by MWT; (n) number of subjects. Volume 2 • Issue 3 • 1000136 Page 6 of 11 The physiological median concentration of opiorphin in semen from normozoospermic healthy human donors was 12.0 ng/ml for 11 subjects (range 4.5 to 30.9, mean ± SEM 12.9 ± 2.4 ng/ml or 43.6 ± 10.2 ng/ejaculate volumes, Figures 3a and 4). Interestingly, opiorphin was also detected in the semen of patients with congenital bilateral absence of the vas deferens inducing infertility, although testicular spermatogenesis is normal. Secretion of opiorphin in human semen The opiorphin levels (median at 3.5 ng/ ml, range from 3 to 8.5 ng/ml, mean ± SEM 4.7 ± 1.0 ng/ml, 5.8 ± 1.6 ng/ejaculate volume, n=5, Figures 3b and 4) of these patients were lower than those of normozoospermic donors. However, the presence of opiorphin in the semen of CBAVD patients demonstrates that about 15% of the peptide secretion in the seminal fluid originates from the prostate glands. of human semen samples with EDTA (1mM final concentration) prior to acid-methanol extraction is a crucial step for opiorphin quantification, with values 6-10 times higher than those in the absence of the chelating agent (P=0.002 by MWT). This suggests that opiorphin in seminal fluid is mainly associated in a molecular complex, involving a cation-mineral element, potentially Zn++ ion, a prostate biochemical marker that is found in semen at 55-420 mg/ml concentration range in fertile men [25]. Under these extraction conditions, recovery of the opiorphin-peptide marker, added to the sample before acid-methanol extraction, was 70%. of human semen samples with EDTA (1mM final concentration) prior to acid-methanol extraction is a crucial step for opiorphin quantification, with values 6-10 times higher than those in the absence of the chelating agent (P=0.002 by MWT). This suggests that opiorphin in seminal fluid is mainly associated in a molecular complex, involving a cation-mineral element, potentially Zn++ ion, a prostate biochemical marker that is found in semen at 55-420 mg/ml concentration range in fertile men [25]. Under these extraction conditions, recovery of the opiorphin-peptide marker, added to the sample before acid-methanol extraction, was 70%. Using the RP-HPLC chromatographic system with a C18-Luna column, the retention time of the reference opiorphin-peptide is 22-23 min. Fractionation coupled to ELISA analysis of two semen extracts, referenced 3a-13 and 3b-02 (Figure 3b), revealed that seminal immuno-reactive opiorphin is primarily eluted at 22-23 min retention time (Rt). This peak is also active as it inhibited, by at least 50%, membrane-bound human NEP activity (method described in ref. 53). A second hydrophobic immunoreactive population (Rt=27 min) was also observed that can represent up to 50% of the immunoreactive opiorphin. This molecular population could correspond to an opiorphin oligomer complexed via a metal cation such as seminal Zn++ ion that was not completely dissociated during the treatment, prior to extraction procedure, by the EDTA chelating agent. Secretion of opiorphin in human semen Interestingly, there was a quantitative difference between the chromatographic profiles of the two samples; sample 3a-13 is an extract of whole semen from the fertile group while the extract 3b-02 consists only of prostatic secretions belonging to the infertile group. Database searches of the human transcriptome reveal that the PROL1 gene is expressed in the male reproductive system, in particular in testis and prostate glands as well as in epididymis. These data, provided by affymetrix GeneChips technology, should explain the higher opiorphin values obtained in healthy donors compared to CBAVD patients. Furthermore, it has been reported that the level of expression of the PROL1 gene in corpora cavernosa tissue samples of men with erectile dysfunction (ED, associated with diabetic or non- diabetic aetiology) is severely down-regulated compared to normal volunteers [26]. Testes contain a great variety of different bioactive peptides that play a role in the local control of gonadal development and also function through autocrine/paracrine mechanisms [27]. All the components of the endogenous opioid system, which includes opiorphin, are Sample treatment before extraction Sample EDTA 1mM HCl 0.1N 10 12 14 16 18 20 22 24 26 28 30 9 8 7 6 5 4 3 2 Sample 3 a-13 Sample 3 b-02 B A (11) + - - + + + + - + (6) (6) 35 30 25 20 15 10 5 0 Opiorphin seminal levels, ng/ml IR-Opiorphin, ng/ml Fractions, min Figure 3: Analyses of healthy and CBAVD human semen by RP-HPLC and ELISA-Opiorphin. A. Box plot representations of sperm opiorphin levels in healthy young men under different extraction conditions. Single individual values below the 10th or above the 90th percentile are represented as open circles. The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. P<0.01 by MWT; (n) number of subjects. B. HPLC chromatography–tandem ELISA-opiorphin detection of two semen extracts. Both samples, 3a-13 (healthy donor) and 3b-02 (CBAVD patient) were extracted by an acid-methanol procedure prior to loading onto a C18-Luna-HPLC column. After lyophilization each 1 min fraction was analyzed by ELISA-Opiorphin and the results expressed as concentration of immunoreactive (IR) opiorphin in ng/ml (Y axis) by HPLC-fraction in min (X axis). Figure 3: Analyses of healthy and CBAVD human semen by RP-HPLC and ELISA-Opiorphin. A. Box plot representations of sperm opiorphin levels in healthy young men under different extraction conditions. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Secretion of opiorphin in human milk The physiological median concentration of opiorphin in milk was 8.3 ng/ml for a range of 3 to 23 ng/ml and mean ± SEM of 10.7 ± 2.4 ng/ ml, for 7 subjects (Figure 6). Interestingly, we observed that opiorphin QRFSR-peptide circulates in milk, as in the seminal fluid, as a soluble complex that interacts with metal cation. Human opiorphin binding to metal cations has been reported by Kotynia et al. [32] and also reported by us for rat sialorphin [16]. Thus, opiorphin is distributed in milk under 2 forms: a free form and a major mineral-binding molecular species which help prevent peptide degradation and/or facilitate transport or it may function as carrier for different minerals, in particular calcium. Rat sialorphin is expressed in the mammary glands of gestational female rats, is secreted into the milk of lactating rats and is present in the stomach of newborn rats (unpublished observations by Rougeot C). In order to determine whether opiorphin is secreted in human milk we analyzed its distribution in samples from healthy lactating women (n=7) at 3-5 wk post-delivery period. A C18-solid phase (SepPak cartridge) extraction procedure was applied for each sample to eliminate the ELISA-interfering milk components. As in the case of the seminal fluid, previous treatment of milk samples by EDTA (1 mM final concentration) before extraction is a crucial step to obtain quantifiable milk opiorphin, with values at least 2-3 times higher than those in absence of the chelating agent. Using these extraction conditions, recovery of the opiorphin peptide marker was 97%. As shown in Figure 5a, the immunoreactive curve obtained from successive dilutions of the milk sample extract, referenced 2-06, is parallel to the dose-response curve corresponding to the reference QRFSR-peptide. The antibody, therefore, recognizes Milk is a major source of biologically active peptides with opioid or peptidase inhibitory activities that function as modulators of various regulatory processes in neonatal development, ranging from gastrointestinal functions to centrally and/or peripherally mediated neuroendocrine functions after crossing the gut-blood barrier [33-36]. Affymetrix GeneChips technology provided tissue expression data for the PROL1 gene, encoding opiorphin precursor, which in the female reproductive system is expressed in addition to mammary gland in ovary, uterus and also amniotic fluid. Secretion of opiorphin in human milk NEP and AP-N membrane- bound cell surface peptidases, which are targets for opiorphin, are also expressed by different cell populations of the human breast, ovarian granulosa, endometrial and chorionic placental cells, in addition to fetal membranes [28,37-39]. These findings suggest that opiorphin may play a local physiological regulatory role in follicular growth, ovulation, embryo implantation processes and maternal-fetal interface by controlling the local concentration of NEP and/or AP-N- sensitive functionally active regulatory peptides. Further studies will be necessary to support our hypothesis that opiorphin participates in the regulation of reproductive physiology, operating at multi-states as a local exocrine and paracrine/autocrine regulator and a distal endocrine regulator in female reproduction, pregnancy and fetal development as well as in parturition and the post-natal lactation period for newborn development. (11) (5) Healthy Agenesis Donors Patients Opiorphin seminal levels, n/ml 35 30 25 20 15 10 5 0 Figure 4: Dot plot representations of semen opiorphin levels in healthy donors and CBAVD patients. Each individual opiorphin value is represented by an open circle for healthy donors (n=11) and by a black square for CBAVD patients (n=7). The mean ± SEM for both groups are represented. Secretion of opiorphin in human semen Single individual values below the 10th or above the 90th percentile are represented as open circles. The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. P<0.01 by MWT; (n) number of subjects. B. HPLC chromatography–tandem ELISA-opiorphin detection of two semen extracts. Both samples, 3a-13 (healthy donor) and 3b-02 (CBAVD patient) were extracted by an acid-methanol procedure prior to loading onto a C18-Luna-HPLC column. After lyophilization each 1 min fraction was analyzed by ELISA-Opiorphin and the results expressed as concentration of immunoreactive (IR) opiorphin in ng/ml (Y axis) by HPLC-fraction in min (X axis). Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000136 Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Page 7 of 11 the milk peptide with the same affinity as the synthetic pure peptide. RP-HPLC fractionation using a C18-ACE column and ELISA analysis of the same milk extract showed that immunoreactive opiorphin is detected at 20–21 min retention time (Rt), which coincides with the reference peptide. As in the case of the semen samples, an additional major immuno-reactive peak at 25–26 Rt was also observed. This hydrophobic molecular population corresponds to a metal cation- binding opiorphin oligomer, because treatment by a strong chelating agent, DTPA (diethylenetriaminepenta-acetic acid, Sigma) at 16 mM final concentration, prior to HPLC chromatography, completely restored the native opiorphin product that is eluted at 20-21 min Rt (Figure 5b). present in the male genital tract and are implicated in the control of sperm motility-regulating levels of enkephalin endogenous opioid peptides in seminal fluid [28-31]. The presence of both membrane- bound targets of opiorphin (AP-N and NEP ectoenkephalinases) and of enkephalins (opioid receptors) in sperm cells [29-31] is consistent with the important level of opiorphin and enkephalins in the seminal fluid and support the idea that the inhibition of enkephalin-degrading ectopeptidases by opiorphin could help maintain sperm motility by increasing enkephalin bioavailability. The data taken together suggest that the distribution of opiorphin in the human reproductive tract mediates male sexual function, as previously demonstrated for sialorphin in rats [12-14]. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Secretion of opiorphin in human tears In 1996, Dickinson et al described the expression of the PROL1 gene precursor in human lachrymal glands [20]. Here we investigated the presence of opiorphin, the mature secretory peptide product of the PROL1 gene, in the tears of male and female healthy volunteers. RP-HPLC fractionation in tandem with ELISA analysis of the female tear extracts showed that immunoreactive opiorphin is detected at the retention time of the reference QRFSR-peptide, confirming that opiorphin circulates in tears. Analyses of all the human tear samples collected from healthy females showed that the median physiological concentration of opiorphin is 220 ng/ml (range 2 to 1109 ng/ml, mean ± SEM 296 ± 77 ng/ml, n=15), while that of healthy men ranges from 2 to 183 ng/ml (n=9). Opiorphin levels of male tears were, for most individuals, inferior to the detection limit (6 out of 9 subjects ≤ 2 ng/ml) Figure 4: Dot plot representations of semen opiorphin levels in healthy donors and CBAVD patients. Each individual opiorphin value is represented by an open circle for healthy donors (n=11) and by a black square for CBAVD patients (n=7). The mean ± SEM for both groups are represented. Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000136 ISSN:2161-1009 Biochem, an open access journal Page 8 of 11 Page 8 of 11 young adult males of 26 ± 6 yr mean age, showed that the median concentration of opiorphin is 53 ng/ml (range 7-196 ng/ml, mean ± SEM 69 ± 11 ng/ml) for 23 subjects under basal conditions. Under conditions of chemical stimulation of salivation, by applying a drop of diluted lemon to the mouth floor, the median opiorphin concentration was 163 ng/ml (range 80-1057 ng/ml, mean ± SEM 335 ± 83 ng/ml) for 18 subjects. Salivary secretion of opiorphin in males is significantly increased under conditions of salivation stimulation by citric acid ( =0.0001 vs. basal by MWT, Figure 7). Saliva samples collected from healthy females showed a median concentration of opiorphin of 30 ng/ ml (range 6-219 ng/ml, mean ± SEM 61 ± 23 ng/ml) for 10 subjects under basal conditions, and of 158 ng/ml (range 24-1091 ng/ml, mean ± SEM 213 ± 76 ng/ml) for 13 subjects under saliva stimulating conditions. Statistical analyses demonstrated that salivary secretion of opiorphin in women is significantly increased under conditions of citric acid stimulation (P=0.006, Figure 7) compared to resting conditions. Secretion of opiorphin in human tears Thus, at baseline and under chemical stimulated conditions, the levels of opiorphin in male compared with female saliva were not significantly different. and the tear volume collected was in general very low. So, opiorphin is secreted in tears at the highest physiological rates, demonstrating that lachrymal glands are the tissue of major expression and secretion of PROL1 gene products. and the tear volume collected was in general very low. So, opiorphin is secreted in tears at the highest physiological rates, demonstrating that lachrymal glands are the tissue of major expression and secretion of PROL1 gene products. Tears, a critical body fluid of the eye, contain a large variety of molecules including peptides, which are primarily secreted by the lachrymal glands. Components of tear fluid contribute to the defense system of the eye and the functional ocular surface-central nervous system-lachrymal gland neural network is crucial in maintaining ocular surface health and homeostasis. Proenkephalin A-derived peptides, Met5- and Leu5-enkephalins, and their cognitive receptors are present and the physiological significance of endogenous enkephalins in lachrymal function, as an inhibitor of lachrymal secretion, is reported [40-42]. The present study demonstrates the presence of lachrymal tissue-specific expression and secretion of opiorphin in tear fluid from healthy volunteers. A role for opiorphin in modulating lachrymal fluid homeostasis by increasing enkephalin bioavailability in the case, for example, of certain causes of epiphora could be evoked. We propose a paracrine and/or autocrine role of opiophin in the lachrymal system and at the ocular surface. Further studies will provide new clues for a molecular understanding of lachrymal gland function and regulation. It is interesting to note that the rate of rat sialorphin salivary secretion under stimulated conditions of the sympathetic nervous system that innervates the salivary glands is similar to human opiorphin salivary secretion under conditions of local chemical stimulation of the mouth nerve endings. However, the secretion rate of salivary sialorphin in male rats is significantly higher than that of female rats [17,18], while the significant increase in opiorphin following chemical stimulation is not sex-linked (P=0.289 by MWT). Strikingly, the level of salivary opiorphin for one healthy female volunteer was high, both in basal (1072 ng/ml) and in stimulated conditions (1637 ng/ml). The clinical data associated with this sample revealed that the subject was treated Secretion of opiorphin in human saliva Opiorphin from human saliva has been well characterized at both molecular and functional levels [1]. Here, in a comparative study, we examined the secretory levels of opiorphin in salivary fluids of healthy young adult men and women, including pregnant women. Statistical analysis of human saliva samples collected from healthy IR-Opiorphin, ng/ml B/B0, % A B 100 90 80 70 60 50 40 30 20 10 30 25 20 15 10 5 0 -5 12 14 16 18 20 22 24 26 28 30 1 10 100 1000 Fractions, min [Opiorphin QRFSR-peptide], ng/ml (log scale) Standard QRFSR-peptide 2-06 milk extract Figure 5: Analyses of human milk by RP-HPLC and ELISA-Opiorphin. A. Analysis of individual milk samples using the ELISA-Opiorphin method. Standard curve (open squares) relating opiorphin concentration in ng/ml (log scale, X axis) to the B/B0 (the percentage of binding in the presence (B) of standard peptide / binding in the absence (B0) of the standard peptide: linear scale, Y axis) and sample curve (open circles) relating serially diluted (2-fold) individual purified milk sample (2-06) to the B/B0, % response. Note that there is a parallel between the dose-response standard curve and serial dilutions of the sample. Each point represents the mean ± SD of duplicates. B. HPLC chromatography in tandem with ELISA-opiorphin detection of individual milk extracts. The milk sample (2-06) was extracted by the C18 solid-phase extraction procedure and the dried extract was treated with DTPA, 16 mM final concentration, prior to loading onto the C18-ACE-HPLC column. After lyophilization each 1 min fraction was analyzed by ELISA-Opiorphin and the results expressed as concentration of immunoreactive (IR) opiorphin in ng/ml (Y axis) by HPLC-fraction in min (X axis). Figure 5: Analyses of human milk by RP-HPLC and ELISA-Opiorphin. A. Analysis of individual milk samples using the ELISA-Opiorphin method. Standard curve (open squares) relating opiorphin concentration in ng/ml (log scale, X axis) to the B/B0 (the percentage of binding in the presence (B) of standard peptide / binding in the absence (B0) of the standard peptide: linear scale, Y axis) and sample curve (open circles) relating serially diluted (2-fold) individual purified milk sample (2-06) to the B/B0, % response. Note that there is a parallel between the dose-response standard curve and serial dilutions of the sample. Each point represents the mean ± SD of duplicates. B. HPLC chromatography in tandem with ELISA-opiorphin detection of individual milk extracts. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Secretion of opiorphin in human saliva The milk sample (2-06) was extracted by the C18 solid-phase extraction procedure and the dried extract was treated with DTPA, 16 mM final concentration, prior to loading onto the C18-ACE-HPLC column. After lyophilization each 1 min fraction was analyzed by ELISA-Opiorphin and the results expressed as concentration of immunoreactive (IR) opiorphin in ng/ml (Y axis) by HPLC-fraction in min (X axis). Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000136 Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Page 9 of 11 Page 9 of 11 Page 9 of 11 with thyroid hormones. Consequently, the sample was excluded from statistical analyses. Opiorphin milk levels, ng/ml Healthy lactating women 3-5 wk post-delivery (7) 35 30 25 20 15 10 5 0 Figure 6: Dot plot representations of milk opiorphin levels in healthy lactating women at 3-5 wk post-delivery. Each individual opiorphin value is represented by open circle for 7 samples. The mean ± SEM is represented. The saliva samples collected from sixth month pregnant women showed a median concentration of opiorphin of 78 ng/ml (range 20-237, mean ± SEM 99 ± 29 ng/ml) for 8 subjects under resting conditions, which is not significantly different from that obtained among non-pregnant women of the same age. In contrast, under stimulated conditions the median level of salivary opiorphin was 60 ng/ ml (range 34-205 ng/ml, mean ± SEM 77 ± 16 ng/ml) for 10 subjects. Thus, the chemical stimulation of saliva secretion actually causes a slight decrease in opiorphin levels in the pregnant female population. In contrast then to the non-pregnant female population studied, the secretory opiorphin rates in stimulated saliva is significantly lower (P=0.028 vs. non-pregnant women by MWT, Figure 7). Opiorphin quantification in human saliva using liquid chromatography in tandem with mass spectrometry (MS) was recently reported by Brkljačić et al. [43]. In their report, opiorphin concentration in the whole saliva of 14 young healthy individuals (23 ± 3 yr) is 9.4 ± 5.4 ng/ml (n=8 females) and 7.2 ± 4.6 ng/ml (n=6 males), which is 7 times lower than our measured basal values. Several possibilities could explain such important differences: - the methodology employed: mass spectrometry vs. Secretion of opiorphin in human saliva immunoassay; - the conditions of saliva collection: absence of peptidase inhibitors for LC-MS/MS analyses and - the conditions of sample preparation: absence of extraction procedure for LC-MS/MS analyses. Moreover, using SELDI-TOF MS analysis we previously noted that in the presence of TFA, the reference synthetic QRFSR-peptide, as well as purified opiorphin from human saliva, present two molecular masses, 690 and 769. Furthermore, QRFSR- peptide was found in the saliva in two active forms Gln1- and Glp1- RFSR with molecular masses of 690 and 665 respectively [1]. All these active opiorphin-related derivatives are invisible to MS/MS detection because of their different masses. Figure 6: Dot plot representations of milk opiorphin levels in healthy lactating women at 3-5 wk post-delivery. Each individual opiorphin value is represented by open circle for 7 samples. The mean ± SEM is represented. Opiorphin saliva levels, ng/ml Conditions Women Men Pregnant Women (18) (10) (13) (23) (8) (10) B S B S B S 1400 1200 1000 800 600 400 200 0 * Opiorphin saliva levels, ng/ml Conditions Women Men Pregnant Women (18) (10) (13) (23) (8) (10) B S B S B S 1400 1200 1000 800 600 400 200 0 * Figure 7: Box plot representations of salivary opiorphin levels in healthy young men, women and pregnant women under basal (B) and stimulated (S) salivary secretion conditions. Single individual values below the 10th or above the 90th percentile are represented as open circles. The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. P<0.01; P<0.001 by MWT; (n) number of subjects, B basal conditions, S stimulated conditions. Affymetrix GeneChips technology provided tissue expression data for the PROL1 gene encoding opiorphin precursor, which in the oral cavity is expressed in parotid, submandibular and sublingual gland substructures only in adult stages, including early adulthood (from 19 yr). These developmental stage data coincide perfectly with those obtained previously for the SMR1RAT1 gene encoding sialorphin precursor. However, sialorphin expression in rat salivary glands is limited to the submandibular glands, which does not seem the case in humans, the contribution of each gland in the production of saliva opiorphin has yet to be determined. Secretion of opiorphin in human saliva Salivary opiorphin may act on its membrane-bound target sites, i.e., NEP and/or AP-N ectopeptidases, that are present in oral sites as well as in the lower segments of the digestive axis and control salivary concentrations of circulating NEP- and/or AP-N-sensitive regulatory peptides [44-46] and/or modulate colonic motility via an indirect action on enkephalin-dependent opioid receptors [8]. Salivary opiorphin may also be absorbed and enter the circulation to mediate endocrine and behavioral effects [1]. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Acknowledgement system, notably in the salivary glands as well as in the ocular system, notably in the lachrymal glands. In these tissues, opiorphin precursor is selectively processed, in a manner similar to the classic maturation pathway of peptide-hormone precursors, to give rise to a mature peptide product, the opiorphin pentapeptide. Mature opiorphin peptide can then be secreted from intracellular stores into the extracellular space, in response to specific stimuli and transported within local fluids, such as semen, milk, tears and saliva and within the bloodstream to exert paracrine, endocrine and/or neuroendocrine actions on local and distal target cells. system, notably in the salivary glands as well as in the ocular system, notably in the lachrymal glands. In these tissues, opiorphin precursor is selectively processed, in a manner similar to the classic maturation pathway of peptide-hormone precursors, to give rise to a mature peptide product, the opiorphin pentapeptide. Mature opiorphin peptide can then be secreted from intracellular stores into the extracellular space, in response to specific stimuli and transported within local fluids, such as semen, milk, tears and saliva and within the bloodstream to exert paracrine, endocrine and/or neuroendocrine actions on local and distal target cells. We would like to thank the contributions to the present project of Pr. Y. Dumez and collaborators (Centre of Obstetrics of Necker-Enfants Malades hospital, Paris) for their inputs on establishing the clinical research program and C. Ottone who was in charge of sample processing, cryopreservation and distribution and traceability at all steps. The present address of R. LEANDRI: Groupe d'activité de Médecine de la Reproduction, CHU de Toulouse, 330 Avenue de Grande Bretagne, 31059 Toulouse cedex 9. This work was in large part supported by funding sources from BioRad laboratories and from the “Direction de la Valorisation et des Partenariats Industriels” and “Dons et Mécénat d'entreprises” Institut Pasteur. No conflicts of interest, financial or otherwise, are declared by the author(s). Opiorphin is a natural inhibitor of enkephalin-inactivating ectopeptidases in humans. Based on its known biological functions, endogenous opiorphin could facilitate adaptative responses to stressful or threatening environmental stimuli by potentiating enkephalin- dependent antinociceptive response via activation of endogenous opioid-dependent pathways [1,7]. Here, we presented a physiological biomedical research program and now it is of interest to identify different human pathological states that up-regulate or down-regulate the levels of circulating opiorphin peptide. In this context, we have already established a biomedical research protocol in collaboration with Dr. References 1. Wisner A, Dufour E, Messaoudi M, Nejdi A, Marcel A, et al. (2006) Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. Proc Natl Acad Sci U S A 103: 17979-17984. 2. Rougeot C, Messaoudi M (2007) [Identification of human opiorphin, a natural antinociceptive modulator of opioid-dependent pathways]. Med Sci (Paris) 23: 37-39. 3. Tóth F, Tóth G, Benyhe S, Rougeot C, Wollemann M (2012) Opiorphin highly improves the specific binding and affinity of MERF and MEGY to rat brain opioid receptors. Regul Pept 178: 71-75. 4. König M, Zimmer AM, Steiner H, Holmes PV, Crawley JN, et al. (1996) Pain responses, anxiety and aggression in mice deficient in pre-proenkephalin. Nature 383: 535-538. 5. Nieto MM, Guen SL, Kieffer BL, Roques BP, Noble F (2005) Physiological control of emotion-related behaviors by endogenous enkephalins involves essentially the delta opioid receptors. Neuroscience 135: 305-313. Interestingly, we found that in vivo, in particular in milk and seminal fluids, opiorphin circulates under two forms: a free form and a cation mineral-binding molecular form. In vitro opiorphin binding ability for metal cation has been reported [32]. An opiorphin-mineral complex could prevent peptide degradation and/or facilitate transport or could modify peptide bioactivity. To discriminate between these different hypotheses it will be important to define the metabolic half- life of the complex compared to free opiorphin (metabolic half-life at 4-5 min in human plasma) by ex vivo, pharmacokinetic and metabolic studies as well as by determining its inhibitory potency on human NEP and AP-N metallo-peptidases by in vitro FRET-based enzyme assays [48]. 6. Javelot H, Messaoudi M, Garnier S, Rougeot C (2010) Human opiorphin is a naturally occurring antidepressant acting selectively on enkephalin-dependent delta-opioid pathways. J Physiol Pharmacol 61: 355-362. 7. Rougeot C, Robert F, Menz L, Bisson JF, Messaoudi M (2010) Systemically active human opiorphin is a potent yet non-addictive analgesic without drug tolerance effects. J Physiol Pharmacol 61: 483-490. 8. Tian XZ, Chen J, Xiong W, He T, Chen Q (2009) Effects and underlying mechanisms of human opiorphin on colonic motility and nociception in mice. Peptides 30: 1348-1354. 9. Yang QZ, Lu SS, Tian XZ, Yang AM, Ge WW, et al. (2011) The antidepressant- like effect of human opiorphin via opioid-dependent pathways in mice. Neurosci Lett 489: 131-135. Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Conclusion The quantitative profile of opiorphin secretion in biological fluids of male and female volunteers is summarized in Table 1. Our findings, associated with human transcriptome data, demonstrate that the PROL1 gene encoding opiorphin precursor is expressed and translated in the reproductive system, notably the testis, epididymis and prostate in men and in the mammary glands of women, in the gastrointestinal Figure 7: Box plot representations of salivary opiorphin levels in healthy young men, women and pregnant women under basal (B) and stimulated (S) salivary secretion conditions. Single individual values below the 10th or above the 90th percentile are represented as open circles. The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. P<0.01; **P<0.001 by MWT; (n) number of subjects, B basal conditions, S stimulated conditions. Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000136 Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 Opiorphin levels, ng/ml Median (range) Number of subjects Subjects 26 ± 6 yr mean age Men Non-pregnant women Pregnant women 6 month pregnancy BioFluids Bloodstream 0.96 (0.4-2.7) n=21 0.55 (0.2-3.4) n=16 0.11 (0.1-1.5) n=8 Urines 7 (4-27) n=15 4 (1-6) n=15 Semen Healthy donors 12 (4.5-30.9) n=11 Semen CBAVD 3.5 (3-8.5) n=5 Milk 2-3 week post-delivery 8.3 (3-23) n=7 Tears (<2-183) n=9 220 (2-1109) n=15 Saliva Basal conditions 53 (7-196) n=23 30 (6-219) n=10 78 (20-237) n=8 Saliva Stimulated conditions 163 (80-1057) n=18 158 (24-1091) n=13 60 (34-205) n=10 Table 1: Summary of opiorphin values in biological fluids of male and female volunteers. Table 1: Summary of opiorphin values in biological fluids of male Table 1: Summary of opiorphin values in biological fluids of male and female volunteers. Acknowledgement Yves Boucher to determine the secretion profile of opiorphin in patients suffering from stomatodynia, also called “mouth burning syndrome” which is a chronic oral mucosal pain with uncertain ethio- pathogenesis [47]. product of Vcsa1) relaxes corporal smooth muscle tissue and increases erectile function in the ageing rat. BJU Int 99: 431-435. Dickinson DP, Thiesse M (1996) cDNA cloning of an abundant human lacrimal gland mRNA encoding a novel tear protein. Curr Eye Res 15: 377-386. 42. Zagon IS, Campbell AM, Sassani JW, McLaughlin PJ (2012) Spontaneous episodic decreased tear secretion in rats is related to opioidergic signaling pathways. Invest Ophthalmol Vis Sci 53: 3234-3240. 21. Rougeot C, Tiberghein C, Minary P, Dray F (1991) Basal and PAF-, interleukin 1-, ether stress-induced hypothalamic pituitary adrenal secretion of conscious rat: modulation by PAF antagonists. J Lipid Mediat 4: 45-59. 43. Brkljačić L, Sabalić M, Salarić I, Jerić I, Alajbeg I, et al. (2011) Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of opiorphin in human saliva. J Chromatogr B Analyt Technol Biomed Life Sci 879: 3920-3926. 22. Engvall E (1980) Enzyme immunoassay ELISA and EMIT. Methods Enzymol 70: 419-439. 44. Holzer P, Holzer-Petsche U (1997) Tachykinins in the gut. Part II. Roles in neural excitation, secretion and inflammation. Pharmacol Ther 73: 219-263. 23. Zheng R, Shen R, Goodman OB Jr, Nanus DM (2006) Multiple androgen response elements cooperate in androgen regulated activity of the type 1 neutral endopeptidase promoter. Mol Cell Endocrinol 259: 10-21. 45. Lundy FT, Linden GJ (2004) NEUROPEPTIDES AND NEUROGENIC MECHANISMS IN ORAL AND PERIODONTAL INFLAMMATION. Crit Rev Oral Biol Med 15: 82-98. 24. Iwase A, Ando H, Nagasaka T, Goto M, Harata T, et al. (2007) Distribution of endothelin-converting enzyme-1 and neutral endopeptidase in human endometrium. J Histochem Cytochem 55: 1229-1235. 46. Sato Y, Itoh H, Suzuki Y, Tatsuta R, Takeyama M (2013) Effect of pilocarpine on substance P and calcitonin gene-related peptide releases correlate with salivary secretion in human saliva and plasma. J Clin Pharm Ther 38: 19-23. 25. Chia SE, Ong CN, Chua LH, Ho LM, Tay SK (2000) Comparison of zinc concentrations in blood and seminal plasma and the various sperm parameters between fertile and infertile men. J Androl 21: 53-57. 47. Braud A, Vandenbeuch A, Zerari-Mailly F, Boucher Y (2012) Dental afferents project onto gustatory neurons in the nucleus of the solitary tract. J Dent Res 91: 215-220. 26. Tong Y, Tar M, Melman A, Davies K (2008) The opiorphin gene (ProL1) and its homologues function in erectile physiology. BJU Int 102: 736-740. 27. product of Vcsa1) relaxes corporal smooth muscle tissue and increases erectile function in the ageing rat. BJU Int 99: 431-435. product of Vcsa1) relaxes corporal smooth muscle tissue and increases erectile function in the ageing rat. BJU Int 99: 431-435. 14. Tong Y, Tiplitsky SI, Tar M, Melman A, Davies KP (2008) Transcription of G-protein coupled receptors in corporeal smooth muscle is regulated by the endogenous neutral endopeptidase inhibitor sialorphin. J Urol 180: 760-766. 35. Janecka A, Fichna J, Mirowski M, Janecki T (2002) Structure-activity relationship, conformation and pharmacology studies of morphiceptin analogues--selective mu-opioid receptor ligands. Mini Rev Med Chem 2: 565-572. 15. Rosinski-Chupin I, Huaulmé JF, Rougeot C, Rougeon F (2001) The transcriptional response to androgens of the rat VCSA1 gene is amplified by both binary and graded mechanisms. Endocrinology 142: 4550-4559. 36. Kost NV, Sokolov OY, Kurasova OB, Dmitriev AD, Tarakanova JN, et al. (2009) Beta-casomorphins-7 in infants on different type of feeding and different levels of psychomotor development. Peptides 30: 1854-1860. 37. Atherton AJ, Anbazhagan R, Monaghan P, Bartek J, Gusterson BA (1994) Immunolocalisation of cell surface peptidases in the developing human breast. Differentiation 56: 101-106. 16. Rougeot C, Vienet R, Cardona A, Le Doledec L, Grognet JM, et al. (1997) Targets for SMR1-pentapeptide suggest a link between the circulating peptide and mineral transport. Am J Physiol 273: R1309-1320. 17. Rougeot C, Rosinski-Chupin I, Rougeon F (1998) From the gene for the submandibular rat1 protein (SMR1) precursor to receptor sites for SMR1 mature peptides: Novel genes and hormones in salivary glands. Biomedical Reviews, Bulgarian-American Center, Varna, Bulgaria. 38. Imai K, Kanzaki H, Fujiwara H, Maeda M, Ueda M, et al. (1994) Expression and localization of aminopeptidase N, neutral endopeptidase, and dipeptidyl peptidase IV in the human placenta and fetal membranes. Am J Obstet Gynecol 170: 1163-1168. 18. Rougeot C, Rosinski-Chupin I, Njamkepo E, Rougeon F (1994) Selective processing of submandibular rat 1 protein at dibasic cleavage sites. Salivary and bloodstream secretion products. Eur J Biochem 219: 765-773. 39. Imai K, Kanzaki H, Mori T (1996) Cell surface peptidases in human endometrium. Mol Hum Reprod 2: 425-431. 40. Cripps MM, Bennett DJ (1992) Proenkephalin A derivatives in lacrimal gland: occurrence and regulation of lacrimal function. Exp Eye Res 54: 829-834. 19. Rougeot C, Rosinski-Chupin I, Mathison R, Rougeon F (2000) Rodent submandibular gland peptide hormones and other biologically active peptides. Peptides 21: 443-455. 41. Meneray MA, Fields TY, Bennett DJ (1998) Gi2 and Gi3 couple met-enkephalin to inhibition of lacrimal secretion. Invest Ophthalmol Vis Sci 39: 1339-1345. 20. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161-1009.1000136 References Affymetrix GeneChip technology provided evidence that the PROL1 gene, encoding opiorphin precursor, is expressed in human brain, in particular in a key output structure of the basal ganglia in the globus pallidus that contains dense enkephalinergic fibers. Opiorphin pentapeptide modulation of enkephalinergic pathways in striatopallidal projection neurons may be of functional importance, in particular in the regulation of central pain perception and mood-related states. Therefore, it is of great interest to explore whether opiorphin is present in the cerebrospinal fluid of healthy volunteers and to determine its secretory processing in the central nervous system. 10. Rougeot C, Messaoudi M, Hermitte V, Rigault AG, Blisnick T, et al. (2003) Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity. Proc Natl Acad Sci U S A 100: 8549-8554. 11. Rougeot C (2004) From the gene to candidate drug. The discovery of sialorphin using inverse pharmacology. BIOforum Europe 5: 52-55. 12. Messaoudi M, Desor D, Nejdi A, Rougeot C (2004) The endogenous androgen- regulated sialorphin modulates male rat sexual behavior. Horm Behav 46: 684- 691. 13. Davies KP, Tar M, Rougeot C, Melman A (2007) Sialorphin (the mature peptide Volume 2 • Issue 3 • 1000136 Page 11 of 11 34. Clare DA, Swaisgood HE (2000) Bioactive milk peptides: a prospectus. J Dairy Sci 83: 1187-1195. product of Vcsa1) relaxes corporal smooth muscle tissue and increases erectile function in the ageing rat. BJU Int 99: 431-435. Gnessi L, Fabbri A, Spera G (1997) Gonadal peptides as mediators of development and functional control of the testis: an integrated system with hormones and local environment. Endocr Rev 18: 541-609. 48. Rougeot C (2009) Method for identifying BPLP and opiorphin agonists or antagonists.: In PCT/EP2009/050567 (WO/2009/090265, Ed.). 28. Fujiwara H, Imai K, Inoue T, Maeda M, Fujii S (1999) Membrane-bound cell surface peptidases in reproductive organs. Endocr J 46: 11-25. 29. Agirregoitia E, Valdivia A, Carracedo A, Casis L, Gil J, et al. (2006) Expression and localization of delta-, kappa-, and mu-opioid receptors in human spermatozoa and implications for sperm motility. J Clin Endocrinol Metab 91: 4969-4975. 30. Subirán N, Agirregoitia E, Valdivia A, Ochoa C, Casis L, et al. (2008) Expression of enkephalin-degrading enzymes in human semen and implications for sperm motility. Fertil Steril 89: 1571-1577. 31. Subirán N, Candenas L, Pinto FM, Cejudo-Roman A, Agirregoitia E, et al. (2012) Autocrine regulation of human sperm motility by the met-enkephalin opioid peptide. Fertil Steril 98: 617-625. 32. Kotynia A KE, Czapor H, Brasun J (2010) The synthesis of opiorphin and studies on its binding ability toward Cu(II). Tetrahedron Letters 51: 2486-2488. 33. Meisel H (1997) Biochemical properties of regulatory peptides derived from milk proteins. Biopolymers 43: 119-128. Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161- 1009.1000136 Citation: Dufour E, Villard-Saussine S, Mellon V, Leandri R, Jouannet P, et al. (2013) Opiorphin Secretion Pattern in Healthy Volunteers: Gender Difference and Organ Specificity. Biochem Anal Biochem 2: 136. doi:10.4172/2161- 1009.1000136 Biochem Anal Biochem Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000136 ISSN:2161-1009 Biochem, an open access journal
https://openalex.org/W4289914068	https://www.scielo.br/j/rbsmi/a/shSGy4h6Jj4D8YzZ38WRnzs/?lang=en&format=pdf	English	null	Maternal mortality by COVID-19 in Brazil: updates	Revista Brasileira de Saúde Materno Infantil	2,022	cc-by	1,272	Dear editor, infected by COVID-19. A Spanish study found that cesarean sections are associated with a clinical worsening of asymptomatic or mild symptoms in pregnant women5; 21.6% of the women who underwent cesarean sections presented clinical worsening, compared to 4.9% of those who had a vaginal delivery. Added to this, 13.5% of the first group needed to be taken to ICU after surgery, compared to none of those who underwent vaginal delivery. Considering that Brazil is the second country with the highest proportion of cesarean sections in the world,6 it is essential to reassess the risks and benefits of each type of delivery in pregnant women with COVID-19 in the country, to avoid complications. The content of Souza and Amorim’s article1 has immense relevance. Thus, it is important to update information about the panorama of maternal mortality by COVID-19 in the country. The article provides information about the year 2020, while data from 2021 show an even greater impact on this population. Important new information came from a recently created “Observatório Obstétrico Brasileiro COVID-19” 2: (Brazilian Obstetrics Observatory COVID-19) Pregnant and puerperal women’s average weekly deaths by COVID-19 doubled more than in 2021 when compared to 2020. In 2020, 459 pregnant and puerperal women’s deaths were registered, with a weekly average of 10.6 deaths. While in 2021, until July 2021, 1.153 deaths were registered, with a weekly average of 44.3 deaths.2 Although the average weekly deaths in the country’s general population also increased in 2021, with an increase of 60.5%,3 the increase in pregnant and puerperal women group was even higher, reaching an increase of 151%.2 In addition to the deaths directly caused by COVID-19 infection, the unfavorable outcomes of pregnancy as an indirect consequence of the virus must also be taken in consideration. In a study carried out with 2,753 Brazilian pregnant and puerperal women, one-third responded that they had reduced their antenatal consultations and exams during the pandemic.7 More than half of the pregnant women said they were very concerned about leaving their homes to monitor their pregnancy. Adequate prenatal care is effective and necessary to prevent maternal morbidities and deaths, as it tracks and treats infections and maternal nutritional problems.8,9 In this way, prenatal care performed improperly, due to the pandemic, increases the chances of unfavorable maternal outcomes even for pregnant women who are not infected by the virus. http://dx.doi.org/10.1590/1806-9304202200020014 Rev. Bras. Saúde Mater. Infant., Recife, 22 (2): 443-444 apr-jun., 2022 Authors’ contribution 8. Bhutta ZA, Das JK, Bahl R, Lawn JE, Salam RA, Paul VK, et al. Can available interventions end preventable deaths in mothers, newborn babies, and stillbirths, and at what cost? Lancet. 2014 Jul; 384 (9940): 347-70. All authors participated in the construction of the letter to the editor and approved the final version. The authors declare no conflict of interest. 9. Pacheco AJ, Katz L, Souza AS, Amorim MM. Factors associated with severe maternal morbidity and near miss in the São Francisco Valley, Brazil: a retrospective, cohort study. BMC Pregnancy Childbirth. 2014 Feb; 14: 91. Dear editor, breastfeeding women.10 Despite the lack of consensus, vaccination was subsequently extended to all pregnant or puerperal women within 45 days of pregnancy.11 Thus, the prospect of a reduction in the infection and deaths has brought hope to this population who were so affected by the COVID-19 pandemic. Received on July 21, 2021 Dear editor, In addition, the survey data made by the Observatory states that the main cause of maternal deaths, when infected by the virus, is the lack of access to adequate treatment. In agreement with these statements, Takemoto’s study,4 cited by the article in question,1 reports that 20% of pregnant and puerperal women hospitalized by Sars-CoV-2 did not have access to the Intensive Care Unit (ICU) and 36% were not intubated.4 Thus, the lack of ICU and intubation affected a third of maternal deaths during the pandemic, representing the serious failure of the healthcare in Brazil. In this context, the Ministry of Health published a technical note in May, 2021 in which it recommended pregnant women who first had comorbidities to be vaccinated , as well as puerperal, recently gave birth and In addition, the type of delivery also seems to influence the maternal outcomes of pregnant women This article is published in Open Access under the Creative Commons Attribution license, which allows use, distribution, and reproduction in any medium, without restrictions, as long as the original work is correctly cited. 443 Michels BD et al. Bruna Depieri Michels 1 https://orcid.org/0000-0003-0936-1171 Betine Pinto Moehlecke Iser 2 https://orcid.org/0000-0001-6061-2541 1,2 Universidade do Sul de Santa Catarina. Av. José Acácio Moreira, 787. Dehon. Tubarão, SC, Brazil. CEP: 88.704-070. E-mail: bruna.michelss@hotmail.com Bruna Depieri Michels 1 https://orcid.org/0000-0003-0936-1171 Bruna Depieri Michels 1 Betine Pinto Moehlecke Iser 2 https://orcid.org/0000-0001-6061-2541 Betine Pinto Moehlecke Iser 2 https://orcid.org/0000-0001-6061-2541 Betine Pinto Moehlecke Iser 2 https://orcid.org/0000-0001-6061-2541 1,2 Universidade do Sul de Santa Catarina. Av. José Acácio Moreira, 787. Dehon. Tubarão, SC, Brazil. CEP: 88.704-070. E-mail: bruna.michelss@hotmail.com 1,2 Universidade do Sul de Santa Catarina. Av. José Acácio Moreira, 787. Dehon. Tubarão, SC, Brazil. CEP: 88.704-070. E-mail: bruna.michelss@hotm 7. Instituto Patrícia Galvão (BR). Mulheres grávidas e puérperas diante do coronavírus [Internet]. São Paulo: Instituto Patrícia Galvão; 2020; [access in 2021 Apr 20]. Available from: https://assets-institucional- ipg.sfo2.cdn.digitaloceanspaces.com/2020/10/ INSTITUTOPATRICIAGALVAOLOCOMOTIVA_ RelatorioGravidezeCovidVersaoFinal.pdf breastfeeding women.10 Despite the lack of consensus, vaccination was subsequently extended to all pregnant or puerperal women within 45 days of pregnancy.11 Thus, the prospect of a reduction in the infection and deaths has brought hope to this population who were so affected by the COVID-19 pandemic. Final version presented on August 23, 2021 Final version presented on August 23, 2021 Approved on August 31, 2021 References 1. Souza ASR, Amorim MMR. Mortalidade materna pela COVID-19 no Brasil. Rev Bras Saúde Mater Infant. 2021 Feb; 21 (Suppl 1): 253-6. 10. Ministry of Health (BR). Nota Técnica nº 627/2021-CGPNI/ DEIDT/SVS/MS. 19 de maio de 2021. Orientações referentes à suspensão temporária da vacinação contra a covid-19 com a vacina AstraZeneca/Oxford em gestantes e puérperas; interrupção da vacinação contra a covid-19 em gestantes sem comorbidades e continuidade da vacinação contra a covid-19 em gestantes com comorbidades [Internet]. Brasília (DF): Ministry of Health; 2021; [access in 2021 Jul 01]. Available from: https://portaldeboaspraticas.iff.fiocruz. br/wp-content/uploads/2021/05/Nota-Te%CC%81cnica- 651-2021-CGPNI-DEIDT-SVS-MS.pdf 2. Observatório Obstétrico Brasileiro (OOBr). COVID-19 [Internet]. Espírito Santo: OOBr; 2020; [access in 2021 Apr 21]. Available from: https://observatorioobstetrico. shinyapps.io/covid_gesta_puerp_br/ 2. Observatório Obstétrico Brasileiro (OOBr). COVID-19 [Internet]. Espírito Santo: OOBr; 2020; [access in 2021 Apr 21]. Available from: https://observatorioobstetrico. shinyapps.io/covid_gesta_puerp_br/ 3. Conselho Nacional de Justiça (BR). Portal da Transparência. Óbitos com suspeita ou confirmação de COVID-19 [Internet]. Brasília: Conselho Nacional de Justiça; 2020; [access in 2021 Jul 13]. Available from: https:// transparencia.registrocivil.org.br/especial-covid 11. Ministry of Health (BR). Nota Técnica nº 2/2021-SECO- VID/GAB/SECOVID/MS. Trata-se de atualização das recomendações referentes a vacinação contra a covid-19 em gestantes e puérperas até 45 dias pós-parto [Internet]. Brasília (DF): Ministry of Health; 2021; [access in 2021 Jul 13]. Available from: https://sei.saude.gov.br/sei/controla- dor_externo.php?acao=documento_conferir&codigo_veri- ficador=0021464579&codigo_crc=4863F560&hash_down- load=3cfd43ffbcbac4b08f37ce10fc87697b0116fa8bbb63 303a6110477124d1d99cd053c45d86c748bfe31764f024e- 1f046f2de39d9289b8534bdbbb87ed5c878df&visualiza- cao=1&id_orgao_acesso_externo=0 4. Takemoto MLS, Menezes MO, Andreucci CB, Nakamura‐ Pereira M, Amorim MM, Katz L, et al. The tragedy of COVID‐19 in Brazil: 124 maternal deaths and counting. Int J Gynecol Obstet. 2020 Jul; 15 (1): 154-6. 5. Martínez-Perez O, Vouga M, Melguizo SC, Acebal LF, Panchaud A, Muñoz-Chápuli M, et al. Association between mode of delivery among pregnant women with COVID-19 and maternal and neonatal outcomes in Spain. JAMA. 2020 Jul; 324 (3): 296-9. 6. World Health Organization (WHO). WHO recommendations: intrapartum care for a positive childbirth experience. Geneva; WHO; 2018. Final version presented on August 23, 2021 Approved on August 31, 2021 Approved on August 31, 2021 Rev. Bras. Saúde Mater. Infant., Recife, 22 (2): 443-444 apr-jun., 2022 444
https://openalex.org/W4287603566	https://zenodo.org/records/4255255/files/IJNSS%20V7I4-3%20pp%2016-20.pdf	English	null	Student suicide incidences in Bangladesh: what do the data say?	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	2,566	INTRODUCTION other countries, even though it is a major public health issue in the country. The country still lacks any kind of suicide surveillance system, national suicide database, and national suicide prevention strategies for both general and specific cohorts (Mamun and Griffiths 2019; Shah et al. 2017). Suicide is such an issue that everyone pays attention to after it has been done. In recent years, the number of students committing suicide has surged immensely. In our current education system, students are put under huge pressure to do well in their examinations. This kind of pressure has caused mental health issues for youngsters. As a result, the number of students committing suicide is rising exponentially. On top of that, the people of our country still hold a stigma against suicide and mental health issues. Families try to hide the fact that their child has depression or has tried to commit suicide because they think it is something to be ashamed of (Arafat 2019; Mamun and Griffiths 2020a, b; Mamun et al. 2020b). However, evidence-based suicide data is needed for the implementation of targeted prevention strategies. Therefore, this study investigated student suicide-related factors (e.g., type of academic institutes, year of study, month in which the suicide occurred, specific suicide reasons, method of suicide, etc.) that could be helpful to the public health policymakers in Bangladesh. Corresponding Author Sumaiya Aziz s.aziz.rmc@gmail.com Sumaiya Aziz s.aziz.rmc@gmail.com Article history This paper aims to find out the recent cases of students committing suicide in the last one year as well as analyzing the cases based on age, gender, educational level, and the reason behind the incidents. The research focuses on the suicide cases committed by students in Bangladesh in the last one year, from July 2019 to June 2020. The paper heavily relies on secondary research from different journals and newspapers. The study shows that female students are more prone to committing suicide compared to men. It also shows that younger school-going students are more likely to commit suicide. The suicide incidents are mostly caused by academic stress, depression, financial crisis, family issues, being raped and blackmailed to release intimate pictures. Further research can be conducted based on primary research. Future research can also focus on specific age groups like very young children as we found out that younger children are more prone to danger. Received: 07 September 2020 Accepted:17 October 2020 Received: 07 September 2020 Accepted:17 October 2020 Received: 07 September 2020 Accepted:17 October 2020 Keywords Student suicide, student, student mental health, suicide, suicide in Bangladesh Student suicide, student, student mental health, suicide, suicide in Bangladesh How to cite this article: Rubayet F, Aziz MN, Maliha SR, Autoshi NA, Aziz S and Khatun S (2020). Student suicide incidences in Bangladesh: what do the data say? International Journal of Natural and Social Sciences, 7(4): 16-20. DOI: 10.5281/zenodo.4255255 International Journal of Natural and Social Sciences, 2020, 7(4):16-20 International Journal of Natural and Social Sciences, 2020, 7(4):16-20 ISSN: 2313-4461 & 2617-6637 Farhana Rubayet1, Mustafa Nizamul Aziz2, Syeeda Raisa Maliha3, Nafisa Anum Autoshi4, Sumaiya Aziz5, Shahanara Khatun6 1Anwer Khan Modern Medical College, Dhaka; 2,3,4East West University, Dhaka; 5Rajshahi Medical College, Rajshahi; 6Alimonessa Technical College, Jessore, Bangladesh METHODOLOGY This research relies greatly on secondary data. All of the information has been collected from newspapers and articles from other authors. Newspapers for the past one year have been gone Additionally, Bangladesh appears to have fewer suicide prevention measures in place compared to 17 Rubayet et al, International Journal of Natural and Social Sciences, 2020, 7(4): 16-20 RESULTS AND DISCUSSION admit card Prothom Alo 3-Feb-20 12 Hajiganj, Chandpur Female 15 School NR Prothom Alo 28-Jan-20 13 Sadar, Panchagarh Female 13 School Being raped The Daily Star 6-Jan-20 14 Magura Female 21 University NR Prothom Alo 03-Jan-20 Table 1: Student suicide incidences in Bangladesh Rubayet et al, International Journal of Natural and Social Sciences, 2020, 7(4): 16-20 18 15 Kaunia, Barishal Female 14 School Due to result (J.S.C.) Prothom Alo 31-Dec-19 16 Gosairhat, Shariatpur Female 14 School Due to result (J.S.C.) Dhaka Tribune 31-Dec-19 17 Sarkarpara, Thakurgaon Male 14 School NR Prothom Alo 21-Dec-19 18 Harinarayanpur, Kushtia Female 17 College Due to fail on test examination Prothom Alo 11-Dec-19 19 Shyamoli, Dhaka Male 23 University Financial crisis and frustration Dhaka Tribune 4-Dec-19 20 Satkania, Chattogram Male NR University NR Dhaka Tribune 28-Nov-19 21 Faridganj, Chandpur Female 14 School Family issues Prothom Alo 22-Nov-19 22 Munshibazar, Faridpur Male NR University Mental depression Dhaka Tribune 16-Nov-19 23 Khanka Sharif area, Rajshahi Male NR University NR Dhaka Tribune 28-Oct-19 24 Chhaygharia, Brahmanbaria Male 16 School Family issues and abandonment by family Dhaka Tribune 25-Oct-19 25 Chilmari, Kurigram Female 13 School NR Prothom Alo 13-Oct-19 26 Bishwanath, Sylhet Female NR NR Being raped Prothom Alo 10-Oct-19 27 Lakhai, Habiganj Male NR University Mental depression Prothom Alo 3-Oct-19 28 Azimpur, Dhaka Male 18 College Depression and dissatisfaction The Daily Star 30-Sep-19 29 Bhandaria, Pirojpur Female 19 School After being blackmailed to spread her intimate photographs Dhaka Tribune 31-Aug-19 30 Jaintiapur, Sylhet Female 17 School NR Dhaka Tribune 5-Jul-19 NR - Not reported. Rate and gender of suicidal cases From our findings, it was discovered some important factors regarding student suicide in Bangladesh. First and foremost, it looks like gender has an impact on such tendencies to commit suicide. Out of the 30 cases we have almost 63% of the students committing suicide are female. This could be because of some of the incidences that women are more prone to like rape or blackmail regarding leaking photos. Such results have been backed up by the research of Kumar et al. (2017) and Tsirigotis et al. (2011), where they have stated then females have a higher NR - Not reported. almost 63% of the students committing suicide are female. This could be because of some of the incidences that women are more prone to like rape or blackmail regarding leaking photos. RESULTS AND DISCUSSION Such results have been backed up by the research of Kumar et al. (2017) and Tsirigotis et al. (2011), where they have stated then females have a higher tendency to commit suicide compared to men. RESULTS AND DISCUSSION through by searching keywords like student suicide in Bangladesh. After collecting all 30 cases from the last one year, the cases were individually reviewed and analyzed based on age, gender, education level, and reason for committing suicide. The study observed 30 suicidal cases from July 2019 to June 2010 (one fiscal year). The present study investigated risk factors of Bangladeshi students’ suicide (i.e., gender, study year, place, onset time, and specific suicide reasons) because they are among the most prone to suicide compared to other students worldwide (Arafat and Mamun, 2019). This study has been conducted to find out the cases of students committing suicide in Bangladesh from July 2019 to June 2020. As the data were from secondary sources, so no formal ethical clearance was required. Table 1: Student suicide incidences in Bangladesh Cases Location Gender Age School/ College/ University Reason for suicide Press Media Date 1 Manirampur, Jashore Male 14 Madrasa Family issues Prothom Alo 27-Jun-20 2 Jurain, Dhaka Male NR College NR Daily Bangladesh 26-Jun-20 3 Chittagong Female 19 University After being blackmailed to spread her intimate photographs Dhaka Tribune 14-Jun-20 4 Mohammadpur, Magura Female 16 Madrasa Due to result (S.S.C.) Prothom Alo 1-Jun-20 5 Haripur, Thakurgaon Female 16 School Due to result (S.S.C.) Prothom Alo 1-Jun-20 6 Ullapara, Sirajganj Female 16 School Due to result (S.S.C.) United News of Bangladesh 1-Jun-20 7 Khetlal, Joypurhat Female NR School Family issues The daily Observer 25-Apr-20 8 Belkuchi, Sirajgonj Female 10 NR Financial crisis Kaler Kantho 11-Apr-20 9 Charghat, Rajshahi Female 15 School Being raped The Daily Star 20-Feb-20 10 Laksam, Comilla Male 24 University Depression Dhaka Tribune 15-Feb-20 11 Domar, Nilphamari Female NR School Due to the wrong information on S.S.C. Academic results If the girls felt like nothing would happen even if the pictures were leaked or if they could have gathered enough courage to ask for help, this tragic incident wouldn’t have taken place. 23% of students (7 out of 30) have committed suicide because of their academic results. This gives us some idea about the pressure children are put under for their studies. This result has been supported by other studies as well. Rate and gender of suicidal cases From our findings, it was discovered some important factors regarding student suicide in Bangladesh. First and foremost, it looks like gender has an impact on such tendencies to commit suicide. Out of the 30 cases we have covered, 19 were female and 11 were male. So, International Journal of Natural and Social Sciences, ISSN: 2313-4461; www.ijnss.org Rubayet et al, International Journal of Natural and Social Sciences, 2020, 7(4): 16-20 19 Depression The number of students committing suicide from college or madrasa is comparatively low. It is highly alarming that such young children are taking such a big decision to end their lives. Even if we consider age, 33% (10 out of 30 students) belong to the age group of 10-15, 30% belong to the age group of 15-20 and only 3 students out of 30 belong to the age group of 20-25. The study of Kessler et al. (2007) supports this fact by stating that adolescence is a time where people are most vulnerable to a mental disorder as emotional reactions are heightened at this age. It is sad but true that till now, many families do not believe that depression is a real disease. As a result, they don’t give much attention if their child is behaving weirdly. Rather, sometimes they scold the child for that. From our study, 13% of the students have committed suicide because of depression and frustration. Financial crisis It is so heartbreaking to see that a 10-year-old child committed suicide as she was scolded when she asked for food. Her family had been starving for days as they were going through the extreme financial crisis because of the COVID-19 pandemic. Researchers agree that putting pressure on students to do well in their examinations increases their mental suffering and sometimes end up in suicides (Arafat and Mamun 2019; Jahan et al. 2020; Mamun and Griffiths2020a, b; Mamun et al. 2020a, b). Reasons for suicide There are 2 cases where the female students have been blackmailed by saying that their intimate pictures will be leaked. The victims could not bear the emotional torture and decided to end it. However, this also sheds light on our family and society’s mindset. Now, if we focus on the reasons behind these suicides, 6 reasons have been found out from these 30 cases. However, the reason behind 8 of the cases has not been reported. Age distribution gives us a clear idea of the fact that domestic peace has a huge impact on the mental health of children. So, if there is some sort of hostility in the family where parents are always fighting, it has a very bad impact on the child. Another observation we have here is that 50% of the students committing suicide are school-going children, followed by university students in the second position (27%). Being raped 3 female students have committed suicide after being raped, two of them being 13 and 15 years old. Having to go through such a bad experience at such a young age is so traumatic that they decided not to live anymore. Further research should be conducted to figure out the reason behind such actions taken by children as well as figure out a solution. This is not only for research but also for the sake of the children. Limitation of study Jahan S, Araf K, Griffiths MD, Gozal D and Mamun, MA (2020). Depression and suicidal behaviors among Bangladeshi mothers of children with Autism Spectrum Disorder: A comparative study. Asian Journal of Psychiatry, 51, 101994. Since the data were secondary and relies on online search of the newspaper there might have a chance of not reported cases in the newspaper. Kessler R, Amminger G, Aguilar-Gaxiola S, Alonso J., Lee S and Ustun T (2007). Age of onset of mental disorders: A review of recent literature. Current Opinion in Psychiatry, 20, 359–364. Family issues Another 23-year-old LLB student committed suicide as he felt like he couldn’t fulfill his family’s wishes by earning enough money or being successful in his career. 13% of students have been found out to be committing suicide because of family issues. This International Journal of Natural and Social Sciences, ISSN: 2313-4461; www.ijnss.org Rubayet et al, International Journal of Natural and Social Sciences, 2020, 7(4): 16-20 20 CONCLUSION In a study conducted by Shah et al. (2017), it had been stated that almost 61% of suicides in Bangladesh are committed by people under the age of 30, who are mostly students. In the study that we conducted, we figured out a total of 30 cases of student suicide and figured out 6 major reasons causing the tragedy. Kumar K, Sattar F, Bondade S, Hussain M, and Priyadarshini M (2017).A gender-specific analysis of suicide methods in deliberate self-harm. Indian Journal of Social Psychiatry, 33, 7. Mamun MA and Griffiths MD (2020a). PTSD-related suicide six years after the Rana Plaza collapse in Bangladesh.Psychiatry Research, 287, 112645. The fact that other authors have had similar results in their studies proves the fact that our analysis and interpretation are correct. According to Yozwiak et al (2012), the critical cause of students committing suicide can be academic stress, personal and family-related trouble, financial crisis, drug usage, traumatic events, and other mental health issues. Mamun M and Griffiths M (2020b). A rare case of Bangladeshi student suicide by gunshot due to unusual multiple causalities. Asian Journal of Psychiatry, 49, e101951. Mamun MA, Siddique AB, Sikder Md T and Griffiths MD (2020a). Student Suicide Risk and Gender: A Retrospective Study from Bangladeshi Press Reports. International Journal of Mental Health and Addiction. The only limitation of this study is that it is only based on secondary research. Further research can be conducted based on primary research. Future research can also focus on specific age groups like very young children as we found out that younger children are more prone to danger. Mamun M, Misti J and Griffiths M (2020b). Suicide of Bangladeshi medical students: Risk factor trends based on Bangladeshi press reports. Asian Journal of Psychiatry, 48, e101905. Shah M, Ahmed S and Arafat SM (2017). Demography and Risk Factors of Suicide in Bangladesh: A Six- Month Paper Content Analysis. Psychiatry Journal, 2017, 1–5. Funding source: None Funding source: Tsirigotis K, Gruszczynski W and Tsirigotis M (2011). Gender differentiation in methods of suicide attempts. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research, 17, PH65-70. Conflict of interest: The authors have no conflicts of interest to disclose. Conflict of interest: Yozwiak J, Lentzsch-Parcells C and Zapolski T (2012).Suicide and suicidal ideation among college students.International Journal on Disability and Human Development, 11. REFERENCES Arafat SM (2019). Females are dying more than males by suicide in Bangladesh. Asian Journal of Psychiatry, 40, 124–125. Arafat SMY and Al Mamun MA (2019). Repeated suicides in the University of Dhaka (November 2018): Strategies to identify risky individuals. Asian Journal of Psychiatry, 39, 84–85. Arafat SMY and Al Mamun MA (2019). Repeated suicides in the University of Dhaka (November 2018): Strategies to identify risky individuals. Asian Journal of Psychiatry, 39, 84–85. International Journal of Natural and Social Sciences, ISSN: 2313-4461; www.ijnss.org
https://openalex.org/W4221078907	https://www.frontiersin.org/articles/10.3389/fimmu.2022.840887/pdf	English	null	Murine Chronic Pancreatitis Model Induced by Partial Ligation of the Pancreatic Duct Encapsulates the Profile of Macrophage in Human Chronic Pancreatitis	Frontiers in immunology	2,022	cc-by	9,578	ORIGINAL RESEARCH published: 01 April 2022 doi: 10.3389/fimmu.2022.840887 Keywords: pancreatitis, animal model, macrophage, cytokines, pyroptosis Edited by: Edited by: Giamila Fantuzzi, University of Illinois at Chicago, United States Immune responses are an integral part of the pathogenesis of pancreatitis. Studies applying the mouse model of pancreatitis induced by partial ligation of the pancreatic duct to explore the pancreatic immune microenvironment are still lacking. The aim of the present study is to explore the macrophage proﬁle and associated regulatory mechanisms in mouse pancreatitis, as well as the correlation with human chronic pancreatitis (CP). In the present study, the mouse model of pancreatitis was induced by partial ligation of the pancreatic duct. Mice in the acute phase were sacriﬁced at 0, 4, 8, 16, 32, 72 h after ligation, while mice in the chronic phase were sacriﬁced at 7, 14, 21, 28 days after ligation. We found that the pancreatic pathological score, expression of TNF-a and IL-6 were elevated over time and peaked at 72h in the acute phase, while in the chronic phase, the degree of pancreatic ﬁbrosis peaked at day 21 after ligation. Pancreatic M1 macrophages and pyroptotic macrophages showed a decreasing trend over time, whereas M2 macrophages gradually rose and peaked at day 21. IL-4 is involved in the development of CP and is mainly derived from pancreatic stellate cells (PSCs). The murine pancreatitis model constructed by partial ligation of the pancreatic duct, especially the CP model, can ideally simulate human CP caused by obstructive etiologies in terms of morphological alterations and immune microenvironment characteristics. Immune responses are an integral part of the pathogenesis of pancreatitis. Studies applying the mouse model of pancreatitis induced by partial ligation of the pancreatic duct to explore the pancreatic immune microenvironment are still lacking. The aim of the present study is to explore the macrophage proﬁle and associated regulatory mechanisms in mouse pancreatitis, as well as the correlation with human chronic pancreatitis (CP). In the present study, the mouse model of pancreatitis was induced by partial ligation of the pancreatic duct. Mice in the acute phase were sacriﬁced at 0, 4, 8, 16, 32, 72 h after ligation, while mice in the chronic phase were sacriﬁced at 7, 14, 21, 28 days after ligation. We found that the pancreatic pathological score, expression of TNF-a and IL-6 were elevated over time and peaked at 72h in the acute phase, while in the chronic phase, the degree of pancreatic ﬁbrosis peaked at day 21 after ligation. Citation: Peng C, Tu G, Yu L, Wu P, Zhang X, Li Z, Li Z and Yu X (2022) Murine Chronic Pancreatitis Model Induced by Partial Ligation of the Pancreatic Duct Encapsulates the Proﬁle of Macrophage in Human Chronic Pancreatitis. Front. Immunol. 13:840887. doi: 10.3389/fimmu.2022.840887 Murine Chronic Pancreatitis Model Induced by Partial Ligation of the Pancreatic Duct Encapsulates the Proﬁle of Macrophage in Human Chronic Pancreatitis Cheng Peng 1†, Guangping Tu 1†, Li Yu 2, Peng Wu 1, Xianlin Zhang 3, Zheng Li 3, Zhiqiang Li 1* and Xiao Yu 1* 1 Department of Hepatopancreatobiliary Surgery, Third Xiangya Hospital, Central South University, Changsha, China, 2 Department of Radiology, Third Xiangya Hospital, Central South University, Changsha, China, 3 Department of General Surgery, Renhe Hospital, Three Gorges University, Yichang, China Edited by: Pancreatic M1 macrophages and pyroptotic macrophages showed a decreasing trend over time, whereas M2 macrophages gradually rose and peaked at day 21. IL-4 is involved in the development of CP and is mainly derived from pancreatic stellate cells (PSCs). The murine pancreatitis model constructed by partial ligation of the pancreatic duct, especially the CP model, can ideally simulate human CP caused by obstructive etiologies in terms of morphological alterations and immune microenvironment characteristics. Reviewed by: Shatovisha Dey, Christiana Care Health System, United States Rand T. Akasheh, American University of Madaba, Jordan Correspondence: Xiao Yu yuxiaoyx4@126.com Zhiqiang Li li_zhiqiang6138@126.com †These authors have contributed equally to this work Correspondence: Xiao Yu yuxiaoyx4@126.com Zhiqiang Li li_zhiqiang6138@126.com †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Inﬂammation, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Inﬂammation, a section of the journal Frontiers in Immunology Received: 21 December 2021 Accepted: 14 March 2022 Published: 01 April 2022 Citation: Peng C, Tu G, Yu L, Wu P, Zhang X, Li Z, Li Z and Yu X (2022) Murine Chronic Pancreatitis Model Induced by Partial Ligation of the Pancreatic Duct Encapsulates the Proﬁle of Macrophage in Human Chronic Pancreatitis. Front. Immunol. 13:840887. doi: 10.3389/fimmu.2022.840887 Received: 21 December 2021 Accepted: 14 March 2022 Published: 01 April 2022 INTRODUCTION Pancreatitis mainly includes acute and chronic pancreatitis. Acute pancreatitis (AP) is a disease characterized by acinar cell death and local and systemic inﬂammation (1, 2), with a global incidence of 34 cases per 100,000 persons (3). Chronic pancreatitis (CP) is a progressive inﬂammatory disease of the pancreas characterized by irreversible morphological changes, progressive ﬁbrosis, as well as loss of exocrine and endocrine functions (4, 5). Epidemiological April 2022 \| Volume 13 \| Article 840887 Frontiers in Immunology \| www.frontiersin.org Macrophage Proﬁle in Pancreatitis Peng et al. studies have shown that the incidence of CP in the population ranges from 4.4 to 14 cases per 100,000 people (6, 7). Patients suffering from pancreatitis often have a great physical and mental burden, and research on pancreatitis remains a huge challenge (8). ligation of the pancreatic duct exhibited more severe pathological alterations with higher necrosis and hemorrhage scores, which is more consistent with the clinically severe acute pancreatitis (SAP) than the cerulein induced model. Moreover, the murine CP model constructed by this method is highly consistent with human CP caused by obstructive etiologies in terms of pathological morphology and pancreatic immune microenvironment and is therefore well suited for the study of CP. The etiology of pancreatitis mainly includes metabolic toxicity such as alcoholism and pancreaticobiliary obstructive disease such as pancreatic duct gallstones. In addition, a small part of pancreatitis is caused by autoimmune and genetic factors (1, 4, 9). Immune responses are an integral part of the pathogenesis of pancreatitis, determining its development and outcome (10). However, studies on the pancreatic immune microenvironment in pancreatitis are limited due to the paucity of pathological specimens from patients with pancreatitis. Therefore, an animal model that can better mimic clinical pancreatitis is of great value for the progress of pancreatitis research. Repeated multiple injections of cerulein in mice are the current mainstream protocol of constructing AP and CP, however, the signiﬁcant drawback is that this model cannot mimic the clinicopathological features of any type of pancreatitis (11, 12). In 2015, Sender and colleagues ﬁrst developed a mouse model of pancreatitis that more closely matched the clinicopathological features by partial ligation of the pancreatic duct (13). Duct dilation after ligation mimicking the state of increased pressure in the pancreatic duct from causes such as pancreatic duct stones, intense inﬂammatory response, and tissue regeneration (13). INTRODUCTION However, studies applying this model to explore the pancreatic immune microenvironment in pancreatitis are still lacking. Animals Mice were housed in a speciﬁc pathogen-free (SPF) facility. Breeding pairs of C57BL/6J mice (8-10 weeks) were purchased from Hunan SJA Laboratory Animal Co., Ltd. All mice used in the study were male and 6-8weeks old and were acclimatized for one week prior to the formal experiment. Mice were housed in a 12-hour alternating day and night environment and fed with standard laboratory chow and water ad libitum. Animal care and use were approved by Central South University institutional animal care and use committees (NO. 2019SYDW0125). Reagents g Cerulein(HY-A0190) was purchased from MedChemExpress (USA). Primary antibodies: IL-6 (GB11117), TNF-a (GB11188) were purchased from Servicebio, Inc(Wuhan, China). a-SMA (GB111364), CD68 (GB14043), F4/80 (GB11027) were purchased from Servicebio, Inc. IL-4 (AF5142) and Cleaved Caspase1 (AF4022) were purchased from Afﬁnity Biosciences (Jiangsu, China). CD206 (60143-1-IG) was purchased from Proteintech Group, Inc (Wuhan, China). Inos (13120) was purchased from Cell Signaling Technology(Shanghai, China), CD4 (SC-13573) was purchased from Santa Cruz Biotechnology Co., Ltd (Shanghai, China). Secondary antibodies: HRP-goat Anti- Rabbit IgG (H+L) (GB23303), Cy3 conjugated Goat Anti-Rabbit IgG (H+L)(GB21303), FITC conjugated Donkey Anti-Rabbit IgG (H+L)(GB22403), FITC conjugated Goat Anti-Mouse IgG (H+L) (GB22301) were purchased from Servicebio, Inc. Anti-mouse IgG (H+L), F(AB ‘)2 Fragment (Alexa Fluor® 594 Conjugate) (8890) and Anti-rat IgG (H+L), (Alexa Fluor® 488 Conjugate) were purchased from Cell Signaling Technology. Macrophages are the major innate immune cell subset characterized by heterogeneity and plasticity (14, 15). Activated macrophages are divided into M1 and M2 subtypes, both M1 and M2 macrophages are closely related to the inﬂammatory response, where M1 macrophages are mainly involved in the pro-inﬂammatory response by producing pro- inﬂammatory mediators such as interleukin (IL)-1b, IL-6, IL-12, and tumor necrosis factor (TNF-a). On the contrary, M2 macrophages are mainly involved in the anti-inﬂammatory response and tissue regeneration process by secreting IL-4, IL- 10, IL-13, and transforming growth factor-b (TGF-b) (16, 17). Studies have shown that macrophages are involved in the development of cerulein-induced AP (18, 19) and CP (20, 21) in mice. However, the dynamics of macrophages and the associated regulatory mechanisms remain unclear in mouse models of pancreatitis induced by partial ligation of the pancreatic duct, as well as in human CP. Frontiers in Immunology \| www.frontiersin.org Serum Amylase Measurement Blood samples were stored at 4°C overnight and then centrifuged at a speed of 3000 rpm for 10 min. Serum was collected as supernatant. Amylase levels were detected using the fully automatic biochemistry analyzer with standard techniques (Chemray 240, Redu Life Technologies, Shenzhen, China). Human Pancreatic Samples Human pancreatic tissue samples of three CP patients with obstructive etiologies (1 periampullary tumor, 1 obstructive gallstone and 1 main duct pancreatic stones) (25) and normal pancreas of three nonmalignant pancreatic patients (1 intraductal papillary mucinous neoplasm, 1 pancreatic lymphangioma and 1 pancreatic serous cystadenoma) undergoing surgery were obtained from the Department of Hepatobiliary and pancreatic Surgery, Third Xiangya Hospital, Central South University, with the approval of the Ethics Sirius Red and Masson Staining Sirius Red and Masson Staining The collagen content of lesions was assessed using Sirius Red staining kit (G1018, Servicebio, Wuhan, China) and Masson staining kit (G1006, Servicebio, Wuhan, China) according to the manufacturer’s instructions. In brief, the parafﬁn tissue sections were deparafﬁnized with xylene and rehydrated in serial dilutions of ethanol. For Sirius Red staining, the sections were stained with Sirius Red solution, then dehydrated quickly and seal with neutral gum. For Masson staining, the sections were stained sequentially with Masson A, B, C, D, E and F solution following the instruction of the manufacturer’s protocol, then differentiation using 1% glacial acetic acid, ﬁnally dehydrated quickly and seal with neutral gum. Collagen ﬁbers appeared red in Sirius Red staining and blue in Masson staining. The percentage of ﬁbrosis area in Masson staining was measured by Image J 1.52. Mice were euthanized by inhaling excessive CO2 after designated treatment. Specimens were ﬁxed with 4% paraformaldehyde and embedded in parafﬁn. Continuous sections of pancreas (3mm) were stained with hematoxylin and eosin (H&E) for histopathological analysis. Five random high-power ﬁelds were selected for each HE slice and scored individually. The histopathology of the AP injury was evaluated according to the criteria proposed by Schmidt et al (23), which was based on edema, necrosis, inﬂammation and hemorrhage. Severity of the CP was graded by a scoring system proposed by Demol et al. (24), which was based on abnormal architecture, glandular atrophy, ﬁbrosis, and pseudotubular complexes. The ﬁnal score is expressed as the average of these values. Immunohistochemistry assays were carried out on mice pancreatic tissues to detect and score TNF-a and IL-6 expression. The immunohistochemistry assay was based on the horseradish peroxidase system. Positive staining area was observed and separately evaluated by two experienced pathologists. Mean optical density (IOD) was measured by Image Pro Plus 6.0. Induction of Pancreatitis in Mice Pancreatitis was induced by partial ligation of the pancreatic duct at the junction of the pancreatic gastric, splenic and duodenal lobes, just below the pylorus of the stomach (Supplementary Figure 1), and received a single intraperitoneal injection of cerulein (50µg/kg/body weight) 48 hours after surgery. The mice were sacriﬁced 3, 7, 14, 21, 28 days after ligation. Mice sacriﬁced at day 3 were used as AP model, while those sacriﬁced at day 7, 14, 21 and 28 were served as CP model, as described previously (13, 22). In addition, we also constructed a group of AP mouse models that underwent partial ligation of the pancreatic duct without caerulein injection. This group of mice In the present study, we revealed the dynamic changes of macrophages in the pancreatitis of mice constructed by partial ligation of the pancreatic duct, including the acute and chronic phases. The progression of pancreatitis from the acute to the chronic phase is also accompanied by a dynamic shift of macrophages from the M1 to the M2 subtype. IL-4 is involved in the development of CP and its main source is pancreatic stellate cells (PSCs) rather than helper T cells (Th cells), which may be an important reason for the polarization of macrophages to the M2 subtype. The murine AP model constructed by partial April 2022 \| Volume 13 \| Article 840887 Frontiers in Immunology \| www.frontiersin.org 2 Macrophage Proﬁle in Pancreatitis Peng et al. sections were incubated with secondary antibodies for 1 hour at room temperature. Nuclear staining was performed with DAPI, and the spontaneous ﬂuorescence quenching agent was added. For primary antibodies of the same species origin, we used a Tyramide signal ampliﬁcation kit (G1235-100T, Servicebio, Wuhan, China) for ﬂuorescent double-label staining. The ﬂuorescence images were captured by a ﬂuorescent microscope. The number of F4/80+iNOS+, F4/80+CD206+, F4/80+Cleaved Caspase1+, CD68+iNOS+, CD68+CD206+, CD68+Cleaved Caspase1+, a-SMA+IL-4+ or CD4+IL-4+ cells as well as the mean ﬂuorescence intensity (MFI) of a-SMA or IL-4 were calculated by Image J 1.52. were sacriﬁced 0, 4, 8, 16 and 32 hours after ligation. Three mice were used in each time point. A schematic diagram for constructing the pancreatitis model is shown in Figure 1. Pancreatic tissue from sacriﬁced animals was immediately ﬁxed with 4% paraformaldehyde for histological examination. AP Induced by Partial Ligation of Pancreatic Duct Presented as Necrotizing Pancreatitis The serum amylase level in AP mice induced by partial ligation of the pancreatic duct started to increase after 4h, peaked at 16h, and then gradually decreased (Figure 2B). We explored the pathological changes of the pancreas within 72h after partial ligation of the pancreatic duct, and the results showed that the pancreatic lesions in the AP model constructed by this method were severe, with massive necrosis of pancreatic acinar cells, interstitial edema, parenchymal loss, hemorrhage, and inﬂammatory cell inﬁltration starting gradually at 4h (Figure 2A). Pancreatic edema score, inﬂammation score, hemorrhage score, necrosis score as well as total score were elevated over time and peaked at 72h (Figures 2C–G). Moreover, the expression of TNF-a and IL-6, inﬂammatory mediators of acute inﬂammation, tended to increase over time and peaked at 72h, which was consistent with the trend of the pathological score (Figures 2A, H, I). RESULTS AP Induced by Partial Ligation of Pancreatic Duct Presented as Necrotizing Pancreatitis Immunoﬂuorescence The parafﬁn sections were dewaxed to water, and the tissue sections were placed in a repair box for antigen repair. Bovine serum albumin (BSA) was dropped for blocking. Sections were incubated overnight with primary antibodies at 4°C. Then, FIGURE 1 \| Schematic diagram for constructing a mouse model of pancreatitis induced by partial ligation of the pancreatic duct. FIGURE 1 \| Schematic diagram for constructing a mouse model of pancreatitis induced by partial ligation of the pancreatic duct April 2022 \| Volume 13 \| Article 840887 Macrophage Proﬁle in Pancreatitis Peng et al. abnormal architecture score and glandular atrophy score were elevated over time and reached the maximum at 28d (Figures 3C, D), while pseudobulbar complexes and ﬁbrosis score were elevated over time and peaked at 21 days (Figures 3E, F). Eventually, the total pathological score of 21d was the highest (Figure 3G), suggesting that 21d was the time point when CP injury was the severest in our study. Masson and Sirius red staining showed that ﬁbrosis appeared from 7d, peaked on 21d, and still existed at 28d. However, due to lipomatosis (26), the ﬁbrotic area decreased compared with that at 21d (Figures 3A, H). We collected some pancreatic tissues from clinical CP patients with a background of obstructive etiologies and human nonmalignant pancreatic patients for the HE, Masson and Sirius red staining, which exhibited obvious ﬁbrosis and parenchymal loss (Figure 3B), and the percentage of ﬁbrosis area in human CP specimen was closer to that at 21d in mice (Figures 3H, I). a-SMA is a common index of ﬁbrosis in pancreatitis (27), immunoﬂuorescence staining showed that the expression of a-SMA was signiﬁcantly upregulated in CP mouse at 14d, 21d, and 28d post ligation and peaked at 21d, which was consistent with the trend of Masson and Sirius red staining (Figures 4A, C). In addition, a-SMA expression was signiﬁcantly upregulated in human CP specimens compared to normal tissue (Figures 4B, D). Therefore, we concluded that the lesions on 21d of the mouse CP model are similar to those of clinical CP and can well mimic clinical CP. Committee of the Third Xiangya Hospital of Central South University (NO. 21175) and the consent of patients. No morphological abnormalities were found in normal pancreatic samples from nonmalignant pancreatic patients, and no visible tumors were found in samples from patients with CP. Detailed clinical characteristics of each patient are provided in Table 1. Statistical Analysis All data are expressed as the mean ± standard error of the mean of at least three animals or experiments. Data analysis was performed using GraphPad Prism 8.1 software. Comparisons between two experimental groups were performed by a 2-tailed Student’s t-test, and multiple comparisons were performed using the one-way ANOVA statistical test followed by Dunnett test. A statistically signiﬁcant difference was considered when P < 0.05. Macrophage Polarization and Pyroptosis Are Involved in the Development of Pancreatitis A substantial of pancreatic inﬁltrating macrophages (F4/80+cells) were observed in both acute and chronic phases in mice with pancreatitis induced by partial pancreatic duct ligation. We found that macrophages were mainly distributed in the pancreatic interstitium at the early stage, whereas they began to migrate into the parenchyma as the disease course progressed. Only a few pancreatic resident macrophages were present in the pancreas of mice in the control group (Supplementary Figure 2), consistent with the previous report (28). The same pattern of macrophage (CD68+cells) inﬁltration and distribution was present in human pancreas specimens (Supplementary Figure 3). Macrophages are heterogeneous and their phenotype and functions are regulated by the surrounding microenvironment (29). Our results showed that in the AP model (3d), there was a large inﬁltration of macrophages in the pancreas and they mainly exhibited the M1 subtype with pro- inﬂammatory properties (Figures 5A, B, E). Notably, the April 2022 \| Volume 13 \| Article 840887 CP Induced by Partial Ligation of Pancreatic Duct Can Simulate Clinical CP After partial ligation of the pancreatic duct, the pancreatic sections of mice showed glandular atrophy, acinar cell damage, interstitial ﬁbrosis, lipomatosis, as well as replacement of exocrine tubules by pseudobulbar complexes over time (Figure 3A). We evaluated CP injury based on the Demols criteria. The results showed that the TABLE 1 \| Clinical characteristics of human pancreatic biopsies. No. Age (y) Sex Duration Etiology Pancreatic insufﬁciency Dilation of main pancreatic duct Group 1 67 Male 6 months Intraductal papillary mucinous neoplasm Absent Absent Nornal tissue 2 60 Female 2 years Pancreatic lymphangioma Absent Absent Nornal tissue 3 63 Female 6 months Pancreatic serous cystadenoma Absent Absent Nornal tissue 4 42 Male 2 years Periampullary tumor diabetes mellitus 7mm Chronic pancreatitis 5 49 Male 6 years Obstructive gallstone dyspepsia 6mm Chronic pancreatitis 6 54 Male 3 years Main duct pancreatic stones diabetes mellitus 5mm Chronic pancreatitis Frontiers in Immunology \| www.frontiersin.org April 2022 \| Volume 13 \| Article 840887 4 April 2022 \| Volume 13 \| Article 840887 Macrophage Proﬁle in Pancreatitis Peng et al. A E F G I H C B D E C FIGURE 2 \| The pancreas of mice presented signiﬁcant lesions after ligation of pancreatic duct. Serum amylase level started to increase after 4h, peaked at 16h, and then gradually decreased. Pancreatic edema score, inﬂammation score, hemorrhage score, necrosis score as well as total score, and the expression of TNF-a and IL-6 were elevated over time and peaked at 72h. (A) HE staining and immunohistochemical staining of TNF-a and IL-6 of pancreas in mice. (B) Serum amylase. (C) Pancreatic edema score. (D) Pancreatic hemorrhage score. (E) Pancreatic hemorrhage scores. (F) Pancreatic necrosis scores. (G) Total pathological scores. (H) Mean IOD of pancreatic TNF-a. (I) Mean IOD of pancreatic IL-6. Values are shown as means ± SE. n = 3 per time point. P < 0.05, P < 0.01, P < 0.001, *P < 0.0001 versus 0h. Scale bar=150mm. IOD, integrated optical density. A C B C D E D E EE EE CC D CC F G G I H I I H I G H FIGURE 2 \| The pancreas of mice presented signiﬁcant lesions after ligation of pancreatic duct. Serum amylase level started to increase after 4h, peaked at 16h, and then gradually decreased. CP Induced by Partial Ligation of Pancreatic edema score, inﬂammation score, hemorrhage score, necrosis score as well as total score, and the expression of TNF-a and IL-6 were elevated over time and peaked at 72h. (A) HE staining and immunohistochemical staining of TNF-a and IL-6 of pancreas in mice. (B) Serum amylase. (C) Pancreatic edema score. (D) Pancreatic hemorrhage score. (E) Pancreatic hemorrhage scores. (F) Pancreatic necrosis scores. (G) Total pathological scores. (H) Mean IOD of pancreatic TNF-a. (I) Mean IOD of pancreatic IL-6. Values are shown as means ± SE. n = 3 per time point. P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 versus 0h. Scale bar=150mm. IOD, integrated optical density. which seemed to be similar to the trend in M1 macrophages in mouse CP models, as well as in human CP (Figures 6A–D). which seemed to be similar to the trend in M1 macrophages in mouse CP models, as well as in human CP (Figures 6A–D). subtype of macrophages started to change when AP shifted to CP. In the early phase of CP (7d), macrophages were still predominantly M1 subtype (F4/80+iNOS+ cells), but a certain amount of M2 macrophages (F4/80+CD206+ cells) had appeared. From 14d onwards, it was the M2 subtype of macrophages that was predominant, and the proportion of M2 macrophages peaked at 21d (Figures 5A, B, E). For human CP specimens, the pancreatic inﬁltrating macrophages were also predominantly M2 subtype (Figures 5C, D, F). Thus, the mouse CP model can also ideally mimic the macrophage proﬁle of human CP. Frontiers in Immunology \| www.frontiersin.org IL-4 Is Involved in the Development of CP and Is Mainly Derived From PSCs B A B A FIGURE 3 \| The pancreatic sections of CP mice model induced by ligation of the pancreatic duct showed progressively aggravated glandular atrophy, acinar cell damage, ﬁbrosis, lipomatosis, as well as the formation of pseudobulbar complexes. The pancreatic specimens from CP patients also exhibited obvious ﬁbrosis and parenchymal loss. (A) HE, Masson and Sirius Red staining of pancreas in mice at different time points. (B) HE, Masson and Sirius Red staining of human pancreatic specimens. (C) Pancreatic abnormal architecture scores in mice. (D) Pancreatic glandular atrophy scores in mice. (E) Pancreatic pseudobulbar complexes scores in mice. (F) Pancreatic ﬁbrosis scores in mice. (G) Total pathological scores in mice. (H) Pancreatic ﬁbrosis area in mice. (I) Pancreatic ﬁbrosis area in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.05, P < 0.01, P < 0.001, *P < 0.0001 versus 0d. ##p < 0.01 versus H-Con. Scale bar=150mm. H-CP, human chronic pancreatitis; H-Con, human control. A B D C FIGURE 4 \| The expression of pancreatic a-SMA was signiﬁcantly upregulated in CP mouse at 14d, 21d, and 28d post ligation and peaked at 21d, it was also upregulated in human CP specimens compared to normal tissue. (A) Immunoﬂuorescence staining of pancreatic a-SMA in mice at different time points. (B). Immunoﬂuorescence staining of pancreatic a-SMA in human specimens. (C) MFI of pancreatic a-SMA at different time points in mice. (D) MFI of pancreatic a-SMA in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.001, *P < 0.0001 versus 0d. #P < 0.05 versus H-Con. Scale bar=20mm. H-CP, human chronic pancreatitis; H-Con, human control; MFI, mean ﬂuorescence intensity. A A D C FIGURE 4 \| The expression of pancreatic a-SMA was signiﬁcantly upregulated in CP mouse at 14d, 21d, and 28d post ligation and peaked at 21d, it was also upregulated in human CP specimens compared to normal tissue. (A) Immunoﬂuorescence staining of pancreatic a-SMA in mice at different time points. (B). Immunoﬂuorescence staining of pancreatic a-SMA in human specimens. (C) MFI of pancreatic a-SMA at different time points in mice. (D) MFI of pancreatic a-SMA in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.001, **P < 0.0001 versus 0d. #P < 0.05 versus H-Con. Scale bar=20mm. IL-4 Is Involved in the Development of CP and Is Mainly Derived From PSCs IL-4, one of the members of the Th2 cytokine family, is important for macrophage polarization toward the M2 subtype (30), it has also been reported to be associated with pancreatic ﬁbrosis (31). We explored IL-4 dynamics in mice CP models and human CP specimens. The results showed that the expression of IL-4 increased in mice CP models as the disease progressed and peaked at 21d (Figures 7A, E), which was consistent with the trend ofﬁbrosis. There was also a signiﬁcant expression of IL-4 in human CP specimens (Figures 7B, F). We further explored the source of pancreatic IL-4 in CP. PSCs are involved in the development of pancreatic ﬁbrosis (32). In addition, it is reported that helper T cells (Th cells) are associated with the M2 polarization of macrophages (33). However, we only Macrophage pyroptosis is a strong promoter of inﬂammation, characterized by assembly of inﬂammasomes, activation of caspase-1 to cleaved caspase1, cleavage, and release of downstream IL-1b and IL-18 (29). Our results showed that macrophage pyroptosis (F4/80+Cleaved-Caspase1+ cells) was associated with the development of pancreatitis, however, it occurred more in AP and started to decrease over time in CP, April 2022 \| Volume 13 \| Article 840887 Frontiers in Immunology \| www.frontiersin.org 5 Peng et al. Macrophage Proﬁle in Pancreatitis A B D E F G I H C FIGURE 3 \| The pancreatic sections of CP mice model induced by ligation of the pancreatic duct showed progressively aggravated glandular atrophy, acinar cell damage, ﬁbrosis, lipomatosis, as well as the formation of pseudobulbar complexes. The pancreatic specimens from CP patients also exhibited obvious ﬁbrosis and parenchymal loss. (A) HE, Masson and Sirius Red staining of pancreas in mice at different time points. (B) HE, Masson and Sirius Red staining of human pancreatic specimens. (C) Pancreatic abnormal architecture scores in mice. (D) Pancreatic glandular atrophy scores in mice. (E) Pancreatic pseudobulbar complexes scores in mice. (F) Pancreatic ﬁbrosis scores in mice. (G) Total pathological scores in mice. (H) Pancreatic ﬁbrosis area in mice. (I) Pancreatic ﬁbrosis area in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.05, P < 0.01, P < 0.001, *P < 0.0001 versus 0d. ##p < 0.01 versus H-Con. Scale bar=150mm. H-CP, human chronic pancreatitis; H-Con, human control. IL-4 Is Involved in the Development of CP and Is Mainly Derived From PSCs H-CP, human chronic pancreatitis; H-Con, human control; MFI, mean ﬂuorescence intensity. April 2022 \| Volume 13 \| Article 840887 6 Frontiers in Immunology \| www.frontiersin.org Peng et al. Macrophage Proﬁle in Pancreatitis A B D C E F FIGURE 5 \| The subtype of macrophages changes when AP shifted to CP. In AP (3d) and the early phase of CP (7d), macrophages were predominantly M1 subtype (F4/80+iNOS+ cells). From 14d onwards, it was the M2 subtype (F4/80+CD206+ cells) of macrophages that was predominant, which was peaked at 21d. For human CP specimens, the pancreatic inﬁltrating macrophages were also predominantly M2 subtype (CD68+CD206+ cells) rather than M1 (CD68+iNOS+ cells) subtype. (A, B) Immunoﬂuorescence staining of pancreatic inﬁltrating M1 and M2 macrophages in mice model of pancreatitis at different time points. (C, D) Immunoﬂuorescence staining of pancreatic inﬁltrating M1 and M2 macrophages in human specimens. (E) Pancreatic M1 and M2 macrophage counts per HPF in mice. (F) Pancreatic M1 and M2 macrophage counts per HPF in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.01, *P < 0.001, P < 0.0001 versus 0d. #p < 0.05, ##p < 0.01 versus H-Con. Scale bar=20mm. H-CP, human chronic pancreatitis; H-Con, human control; HPF, high-powered ﬁeld of view. A C C B D E F FIGURE 5 \| The subtype of macrophages changes when AP shifted to CP. In AP (3d) and the early phase of CP (7d), macrophages were predominantly M1 subtype (F4/80+iNOS+ cells). From 14d onwards, it was the M2 subtype (F4/80+CD206+ cells) of macrophages that was predominant, which was peaked at 21d. For human CP specimens, the pancreatic inﬁltrating macrophages were also predominantly M2 subtype (CD68+CD206+ cells) rather than M1 (CD68+iNOS+ cells) subtype. (A, B) Immunoﬂuorescence staining of pancreatic inﬁltrating M1 and M2 macrophages in mice model of pancreatitis at different time points. (C, D) Immunoﬂuorescence staining of pancreatic inﬁltrating M1 and M2 macrophages in human specimens. (E) Pancreatic M1 and M2 macrophage counts per HPF in mice. (F) Pancreatic M1 and M2 macrophage counts per HPF in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.01, *P < 0.001, *P < 0.0001 versus 0d. #p < 0.05, ##p < 0.01 versus H-Con. Scale bar=20mm. H-CP, human chronic pancreatitis; H-Con, human control; HPF, high-powered ﬁeld of view. animal experiments. IL-4 Is Involved in the Development of CP and Is Mainly Derived From PSCs Drugs-induced AP models are mainstream research methods, the most commonly used drugs are cerulein, followed by L-arginine, which has the advantages of simplicity and high rate of success (35–37). Nevertheless, the correlation between these two models and human AP remains to be discussed. More importantly, the cerulein-induced AP model is mostly mild edematous pancreatitis with only a small amount of necrosis (38, 39), which is not suitable for simulating clinical severe acute pancreatitis (SAP). AP models constructed by speciﬁc diet or surgery in mice can often better simulate the etiology of human AP and has higher clinical transformation value, such as alcoholic AP model constructed by oral administration of ethanol and palmitoleic acid (40), alcoholic AP with a background of hypophosphatemia constructed by low- phosphate diet (LPD) feeding followed by orogastric administration of ethanol (41), hypertriglyceridemic AP observed a small number of CD4+ cells in the pancreas of mice CP models and human CP, with little co-localization of CD4 and IL-4 (Figures 7C, D). Interestingly, the presence of a large number of a-SMA+IL-4+cells, which represents PSCs secreting IL-4, were found in the pancreas of mice CP models and human CP (Figures 7A, B, G, H). Therefore, we considered that PSCs are the main source of IL-4 both in murine CP and human CP, which is closely associated with pancreatic ﬁbrosis. DISCUSSION Pancreatitis is an inﬂammatory disease, which begins with the premature activation of digestive enzymes in pancreatic acinar cells and leads to cell damage (7, 34). Most of the knowledge about the pathophysiology of this disease comes mainly from Pancreatitis is an inﬂammatory disease, which begins with the premature activation of digestive enzymes in pancreatic acinar cells and leads to cell damage (7, 34). Most of the knowledge about the pathophysiology of this disease comes mainly from April 2022 \| Volume 13 \| Article 840887 Frontiers in Immunology \| www.frontiersin.org 7 Macrophage Proﬁle in Pancreatitis Peng et al. A B D C FIGURE 6 \| Pyroptotic macrophages (F4/80+Cleaved-Caspase1+cells) were observed more in AP, which decreased over time in CP. (A) Immunoﬂuorescence staining of pancreatic pyroptotic macrophages in mice model of pancreatitis at different time points. (B) Immunoﬂuorescence staining of pancreatic pyroptotic macrophages in human specimens. (C) Pancreatic pyroptotic macrophages counts per HPF in mice. (D) Pancreatic pyroptotic macrophages counts per HPF in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.05, *P < 0.001, *P < 0.0001 versus 0d. ##p < 0.01 versus H-Con. Scale bar=20mm. H-CP, human chronic pancreatitis; H-Con, human control; HPF, high-powered ﬁeld of view. A B D C B C D D FIGURE 6 \| Pyroptotic macrophages (F4/80+Cleaved-Caspase1+cells) were observed more in AP, which decreased over time in CP. (A) Immunoﬂuorescence staining of pancreatic pyroptotic macrophages in mice model of pancreatitis at different time points. (B) Immunoﬂuorescence staining of pancreatic pyroptotic macrophages in human specimens. (C) Pancreatic pyroptotic macrophages counts per HPF in mice. (D) Pancreatic pyroptotic macrophages counts per HPF in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.05, *P < 0.001, P < 0.0001 versus 0d. ##p < 0.01 versus H-Con. Scale bar=20mm. H-CP, human chronic pancreatitis; H-Con, human control; HPF, high-powered ﬁeld of view. CP, as an irreversible ﬁbrotic disease, is still challenging to study in-depth. Similar to the AP model in mice, repeated injection of cerulein is also the mainstream means to construct mouse CP with a high reliability and reproducibility. However, it has the disadvantage of low correlation with human CP (45). As a skill-dependent modality, pancreatic duct ligation is superior in simulating human CP caused by obstructive etiologies (46). DISCUSSION We explored the morphological changes of the pancreas in 7, 14, 21, and 28 days after pancreatic duct ligation. The results showed that signiﬁcant lesions appeared in the pancreas from 7d. With the passage of time, there were varying degrees of glandular atrophy, ductal dilatation, interstitial ﬁbrosis, tissue proliferation, inﬂammatory cell inﬁltration, and ﬁbrosis marker a-SMA expression. We considered that 21d was the time point when CP injury was the severest in our study based on Demols criteria. Interestingly, the pancreatic parenchyma was replaced by a large amount of adipose tissue at 28d after surgery, namely lipomatosis (26). In order to verify the correlation between the mouse CP model constructed by pancreatic duct ligation and human CP, induced by oral administration of high-fat diet (HFD) with or without intraperitoneal injection of cerulein (42, 43), biliary AP model constructed by retrograde injection of sodium taurocholate into the pancreaticobiliary duct (44), and the partial ligation of pancreatic duct proposed in recent years (13). However, the application of the surgical AP model in mice is still not common. In our study, we found that the serum amylase level of mice AP models elevated 4 hours after pancreatic duct ligation, suggesting pancreatic damage. The pancreatic injury gradually aggravated over time, as evidenced by elevated HE pathological scores and upregulated expression of IL-6 and TNF-a, and eventually reached the peak at 72h. In addition to the signiﬁcant edema and inﬂammatory cell inﬁltration of the pancreas, there is also a large amount of acinar cell necrosis and hemorrhage, which is a signiﬁcant difference from cerulein-induced AP (38, 39), and is more consistent with clinical SAP. Therefore, our model is suitable for the scientiﬁc research of SAP with a background in pancreatic ductal hypertension. induced by oral administration of high-fat diet (HFD) with or without intraperitoneal injection of cerulein (42, 43), biliary AP model constructed by retrograde injection of sodium taurocholate into the pancreaticobiliary duct (44), and the partial ligation of pancreatic duct proposed in recent years (13). However, the application of the surgical AP model in mice is still not common. In our study, we found that the serum amylase level of mice AP models elevated 4 hours after pancreatic duct ligation, suggesting pancreatic damage. The pancreatic injury gradually aggravated over time, as evidenced by elevated HE pathological scores and upregulated expression of IL-6 and TNF-a, and eventually reached the peak at 72h. DISCUSSION In addition to the signiﬁcant edema and inﬂammatory cell inﬁltration of the pancreas, there is also a large amount of acinar cell necrosis and hemorrhage, which is a signiﬁcant difference from cerulein-induced AP (38, 39), and is more consistent with clinical SAP. Therefore, our model is suitable for the scientiﬁc research of SAP with a background in pancreatic ductal hypertension. April 2022 \| Volume 13 \| Article 840887 Frontiers in Immunology \| www.frontiersin.org 8 Peng et al. Macrophage Proﬁle in Pancreatitis A B D E F G H C FIGURE 7 \| The expression of IL-4 increased in human CP specimens, as well as in mice CP models as the disease progressed and peaked at 21d. A large number of a-SMA+IL-4+cells, which represents PSCs secreting IL-4, were found in the pancreas of mice CP models and human CP. However, almost no CD4+IL-4+cells in the pancreas of mice CP models and human CP were observed. (A, C) Immunoﬂuorescence staining of pancreatic IL-4 co-localized with a-SMA or CD4 in mice model of pancreatitis at different time points. (B, D) Immunoﬂuorescence staining of pancreatic IL-4 co-localized with a-SMA or CD4 in human specimens. (E) MFI of pancreatic IL- 4 at different time points in mice. (F) MFI of pancreatic IL-4 at different time points in human specimens. (G) a-SMA+IL-4+cells counts per HPF in mice. (H) a-SMA+IL-4 +cells counts per HPF in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.01, *P < 0.001, P < 0.0001 versus 0d. #P < 0.05 versus H- Con. Scale bar=20mm. H-CP, human chronic pancreatitis; H-Con, human control; MFI, mean ﬂuorescence intensity; HPF, high-powered ﬁeld of view. B A B D H D E F G H H G FIGURE 7 \| The expression of IL-4 increased in human CP specimens, as well as in mice CP models as the disease progressed and peaked at 21d. A large number of a-SMA+IL-4+cells, which represents PSCs secreting IL-4, were found in the pancreas of mice CP models and human CP. However, almost no CD4+IL-4+cells in the pancreas of mice CP models and human CP were observed. (A, C) Immunoﬂuorescence staining of pancreatic IL-4 co-localized with a-SMA or CD4 in mice model of pancreatitis at different time points. (B, D) Immunoﬂuorescence staining of pancreatic IL-4 co-localized with a-SMA or CD4 in human specimens. DISCUSSION (E) MFI of pancreatic IL- 4 at different time points in mice. (F) MFI of pancreatic IL-4 at different time points in human specimens. (G) a-SMA+IL-4+cells counts per HPF in mice. (H) a-SMA+IL-4 +cells counts per HPF in human specimens. Values are shown as means ± SE. n = 3 per group. P < 0.01, *P < 0.001, **P < 0.0001 versus 0d. #P < 0.05 versus H- Con. Scale bar=20mm. H-CP, human chronic pancreatitis; H-Con, human control; MFI, mean ﬂuorescence intensity; HPF, high-powered ﬁeld of view. we collected three clinical samples of patients with CP caused by obstructive etiologies and three normal pancreatic samples for research. The results showed that the status of mouse pancreas at 21d was similar to that of human CP with a background of obstructive etiologies in terms of pancreatic morphological changes and degree of ﬁbrosis. Therefore, we recommend 21d as the best time point to study CP in this model. pancreatic ducts. In recent years, investigators have focused on macrophage pyroptosis, a caspase-1-dependent programmed cell death that culminates in cell lysis and release of mature IL-1b and IL-18 thereby aggravating the inﬂammatory response (47). Macrophage pyroptosis has been reported to play a role in the development of hepatitis (48), Alzheimer’s disease (49), AP (22, 50), etc. In our study, we found that there were a large number of M1 macrophages in the pancreas of AP mice constructed by partial ligation of the pancreatic duct, accompanied by a large number of F4/80+cleaved caspase1+ cells, which indicate macrophage pyroptosis. In CP mice constructed by partial ligation of the pancreatic duct, M1 and M2 macrophages in the pancreas went in opposite directions. At the early stage of CP (7 days after ligation), M1 macrophages were still far more numerous than the M2 subtype. The numbers of M1 and M2 macrophages were roughly the same at 14 days after ligation. Whereas by 21 days post ligation, the number of M2 It is now well established that immune cells are major contributors to the pathogenesis of pancreatitis, and among these, macrophages make up a key innate immune population (10). Studies have shown that in mice with cerulein-induced AP and CP, the inﬁltrating macrophages in the pancreas of the former are predominantly of the M1 subtype, whereas those of the latter are M2 (18–21). Frontiers in Immunology \| www.frontiersin.org ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee of the Third Xiangya Hospital of Central South University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by Central South University institutional animal care and use committees. DISCUSSION However, there is still a lack of evidence regarding macrophage status, their regulatory mechanisms, and relevance to human CP in murine pancreatitis models constructed by partial ligation of April 2022 \| Volume 13 \| Article 840887 9 Peng et al. Macrophage Proﬁle in Pancreatitis macrophages had peaked, a time point when the macrophage polarization state is highly similar to human CP. Macrophage pyroptosis, as a pro-inﬂammatory cell death manner, occurred more in AP and the early stage of CP in our model, then started to decrease over time, which seemed to be similar to the trend of M1 macrophages in mice. For human CP specimens, fewer pyroptotic macrophages were observed. These results suggest that a mouse CP model constructed by partial ligation of the pancreatic duct for 21 days could ideally mimic the pattern of macrophage changes in human CP and be suitable for related studies. different models and the advantages of different models in order to select a speciﬁc model for a speciﬁc purpose. FUNDING The current study was funded by the National Natural Science Foundation of China (No. 81873589). Natural Science Foundation of Hunan Province (No. 2020JJ4858). Science and technology planning project of Changsha (No. kq2004143). 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 authors. It is of great clinical value to explore the mechanism of pancreatic ﬁbrosis in CP. The role of PSCs has been studied with great interest (51). PSCs can be activated by chemokines released as a result of pancreatic inﬂammation and drive pancreatic parenchymal ﬁbrosis (8). Up-regulated expression of a-SMA is the hallmark of PSCs activation (52, 53). In addition to producing collagen and extracellular matrix, PSCs were reported to mediate pancreatic ﬁbrosis by modulating M2 macrophages in cerulein-induced CP in mice, and IL-4, as a member of the Th2 cytokine family, played a key role in this regulatory process (21). In our study, we found that in human CP, pancreatic expression of a-SMA and IL-4 was signiﬁcantly upregulated. In the CP mice model constructed by partial ligation of the pancreatic duct, the expression of pancreatic a- SMA and IL-4 began to be upregulated 7 days after ligation and reached a peak by 21 days, consistent with the trend of M2 macrophages. More importantly, there was substantial immunoﬂuorescence colocalization of a-SMA and IL-4, suggesting that PSCs are a major source of IL-4 in human CP and in murine CP constructed by partial ligation of the pancreatic duct. Next, we further explored whether Th2 cells were another source of IL-4 since Th2 cells were reported to be elevated in the peripheral blood of patients with CP (54) and could regulate M2 macrophage polarization in atherosclerosis (55). However, our results showed a low number of T cells inﬁltrating the pancreas in human CP and in CP mice model constructed by partial ligation of the pancreatic duct, as well as an almost absent immunoﬂuorescent colocalization of CD4 and IL-4, suggesting that Th2 cells are not another source of IL-4. Therefore, we speculate that in CP caused by obstructive etiologies, Th2 cells hardly inﬁltrated into the pancreas, so they did not directly regulate the polarization of macrophages. AUTHOR CONTRIBUTIONS CP and GT designed the experiment, analyzed data, and wrote the manuscript. CP and GT performed research. LY and PW assisted in completing the experiment, XZ and ZheL reviewed and modiﬁed the manuscript. ZhiL and XY provided overall guide and supervision. All authors contributed to the article and approved the submitted version. Frontiers in Immunology \| www.frontiersin.org REFERENCES 21. Xue J, Sharma V, Hsieh MH, Chawla A, Murali R, Pandol SJ, et al. Alternatively Activated Macrophages Promote Pancreatic Fibrosis in Chronic Pancreatitis. Nat Commun (2015) 6:7158. doi: 10.1038/ncomms8158 1. Banks PA, Bollen TL, Dervenis C, Gooszen HG, Johnson CD, Sarr MG, et al. Classiﬁcation of Acute Pancreatitis–2012: Revision of the Atlanta Classiﬁcation and Deﬁnitions by International Consensus. Gut (2013) 62 (1):102–11. doi: 10.1136/gutjnl-2012-302779 22. Sendler M, van den Brandt C, Glaubitz J, Wilden A, Golchert J, Weiss FU, et al. 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SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2022. 840887/full#supplementary-material In conclusion, our study explored the pancreatic pathomorphology and immune microenvironment characteristics in AP and CP in a mouse model constructed by partial ligation of the pancreatic duct, as well as the relation between this model and human CP. Overall, the murine pancreatitis model constructed by partial ligation of the pancreatic duct, especially the CP model, is highly similar to human CP caused by obstructive etiologies in terms of morphological alterations and immune microenvironment characteristics and is recommended for further studies. However, it should be recognized that no model can fully share features of the human disease, it is still necessary to understand more deeply the pathophysiological mechanisms of Supplementary Figure 1 \| Intraoperative photograph of partial ligation of the pancreatic duct in mice. Supplementary Figure 2 \| Immunoﬂuorescence staining of pancreatic macrophages (F4/80+cells) in mice at different time points with magniﬁcation of the areas indicated by the white square. Scale bar=100mm. Supplementary Figure 2 \| Immunoﬂuorescence staining of pancreatic macrophages (F4/80+cells) in mice at different time points with magniﬁcation of the areas indicated by the white square. Scale bar=100mm. Supplementary Figure 3 \| Immunoﬂuorescence staining of pancreatic macrophages (CD68+cells) in human specimens with magniﬁcation of the areas indicated by the white square. Scale bar=100mm. H-CP, human chronic pancreatitis; H-Con, human control. April 2022 \| Volume 13 \| Article 840887 10 Macrophage Proﬁle in Pancreatitis Peng et al. Signaling and M2 Polarization. Toxicol Appl Pharmacol (2020) 403:115162. doi: 10.1016/j.taap.2020.115162 Signaling and M2 Polarization. Toxicol Appl Pharmacol (2020) 403:115162. doi: 10.1016/j.taap.2020.115162 REFERENCES Macrophages and Cadherins in Fibrosis and Systemic Sclerosis. Curr Opin Rheumatol (2019) 31(6):582–8. doi: 10.1097/ bor.0000000000000657 34. Lankisch PG, Apte M, Banks PA. Acute Pancreatitis. Lancet (2015) 386 (9988):85–96. doi: 10.1016/s0140-6736(14)60649-8 35. Delaney CP, McGeeney KF, Dervan P, Fitzpatrick JM. Pancreatic Atrophy: A New Model Using Serial Intra-Peritoneal Injections of L-Arginine. Scand J Gastroenterol (1993) 28(12):1086–90. doi: 10.3109/00365529309098314 16. Yunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 Polarization. Eur J Pharmacol (2020) 877:173090. doi: 10.1016/j.ejphar.2020.173090 36. 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https://openalex.org/W3010335009	https://u-picardie.hal.science/hal-03612508/document	English	null	Plasma Treatment Reduced the Discoloration of an Acrylic Coating on Hot-Oil Modified Wood Exposed to Natural Weathering	Coatings	2,020	cc-by	10,028	To cite this version: Arash Jamali, Philip D. Evans. Plasma Treatment Reduced the Discoloration of an Acrylic Coating on Hot-Oil Modified Wood Exposed to Natural Weathering. Coatings, 2020, 10 (3), 10.3390/coat- ings10030248. hal-03612508 Plasma Treatment Reduced the Discoloration of an Acrylic Coating on Hot-Oil Modified Wood Exposed to Natural Weathering Arash Jamali, Philip D. Evans Received: 28 January 2020; Accepted: 3 March 2020; Published: 8 March 2020 Abstract: We test the hypothesis that plasma-treatment will remove oil from the surface of hot-oil modiﬁed blue-stained pine wood, and improve the adhesion and outdoor performance of a white acrylic coating on the modiﬁed wood. Modiﬁed wood was treated with water-vapour plasma, and microstructural changes at wood surfaces were examined. Plasma treatment removed oil from the surface of modiﬁed wood and etched bordered pits. The contact angle of water droplets on modiﬁed wood was 91.8◦, but plasma-treatment for only 33 s reduced contact angle to less than that of the unmodiﬁed control (48.6◦). The adhesion of the acrylic paint to modiﬁed wood was unaﬀected by plasma-treatment, but the adhesion rating of coated samples tested wet was slightly lower (3.1) than that of the coating on samples tested dry (3.5). The lightness value (CIE-L) of the acrylic coating on hot-oil modiﬁed wood samples exposed outdoors for 18 months was signiﬁcantly lower (darker, 65.5) than that of the coating on similarly modiﬁed and exposed samples pre-treated with plasma (75.8). We conclude that plasma-treatment shows promise as a way of removing oil from the surface of hot-oil modiﬁed wood and reducing the discolouration of an acrylic coating on modiﬁed wood exposed to natural weathering. Keywords: wood; plasma; pine; thermal modiﬁcation; oil; blue stain; weathering; discolouration; adhesion; bordered pits Arash Jamali 1,2 and Philip D. Evans 1,3,* Arash Jamali 1,2 and Philip D. Evans 1,3,* Arash Jamali 1,2 and Philip D. Evans 1,3,* 1 Centre for Advanced Wood Processing, Faculty of Forestry, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; arash.jamali@u-picardie.fr 2 Plateforme de Microscopie Electronique, Université de Picardie Jules Verne, 80039 Amiens, France 3 Department of Applied Mathematics, Research School of Physics, The Australian National University, Canberra, ACT 0200, Australia * Correspondence: phil evans@ubc ca Arash Jamali 1,2 and Philip D. Evans 1,3,* 1 Centre for Advanced Wood Processing, Faculty of Forestry, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; arash.jamali@u-picardie.fr 2 Plateforme de Microscopie Electronique, Université de Picardie Jules Verne, 80039 Amiens, France 3 Department of Applied Mathematics, Research School of Physics, The Australian National University, Canberra, ACT 0200, Australia * Correspondence: phil evans@ubc ca Received: 28 January 2020; Accepted: 3 March 2020; Published: 8 March 2020 coatings coatings www.mdpi.com/journal/coatings HAL Id: hal-03612508 https://u-picardie.hal.science/hal-03612508v1 Submitted on 24 Aug 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. Coatings 2020, 10, 248; doi:10.3390/coatings10030248 1. Introduction The performance of coatings on hydrophobic materials such as plastics can be improved by treating the surface of the material with plasma, a reactive mixture of charged particles [1,2]. For example, plasma treatments are widely used by industry as a pre-treatment to increase wettability and adhesion of coatings to plastic automotive components [2,3]. Plasma treatment of wood prior to coating is unnecessary because wood is not normally hydrophobic, but there are exceptions. Wood that has been heat-treated at temperatures of 180–220 ◦C to reduce its susceptibility to fungal decay and moisture-induced deformation is hydrophobic [4–6]. Accordingly, there has been interest in using plasma treatments to reverse the eﬀects of heat treatment (thermal modiﬁcation) on the surface hydrophobicity of wood [7–11]. Plasma treatment of thermally modiﬁed wood has positive eﬀects on both wettability [7–11], and coating adhesion [11]. These ﬁndings were obtained using wood that was thermally modiﬁed in air/steam or nitrogen [7–11]. However, wood can also be thermally modiﬁed using oil as the heating medium [12]. To our knowledge, there have been no studies of the eﬀects of plasma treatment on wood that has been thermally modiﬁed in hot-oil. A previous study used plasma Coatings 2020, 10, 248; doi:10.3390/coatings10030248 2 of 13 Coatings 2020, 10, 248 to treat wood surfaces that had been brush-coated with paraﬃn oil, but the aim of this research was to cross-link the oil and create a hydrophobic barrier, rather than modify the wood surface [13]. Oil modiﬁcation treatments involve heating wood in vegetable oil (linseed, soy, sunﬂower, etc.) at temperatures of 180–220 ◦C for various periods of time [5]. The hygroscopic components of wood are degraded during this heating period, and the wood absorbs oil, resulting in a hydrophobic material, which, like wood that has been thermally modiﬁed in air or nitrogen, is less susceptible to moisture-induced deformation and fungal decay [12]. Oil at the surface of hot-oil modiﬁed wood can reduce its wettability [14], and also the adhesion of coatings to wood [15]. An additional concern is that oil could migrate through coatings and encourage mould growth. In accord with this suggestion, low molecular weight wood extractives are able to migrate through coatings [16]. Furthermore, hot-oil modiﬁed wood is susceptible to colonization by mould fungi [17]. Oils also encourage pickup and retention of dirt at the surface of materials [18]. 1. Introduction However, oils can be removed from surfaces by plasma, and there are several papers that have successfully used plasma to remove oily lubricants from metals and polymers [19,20]. Plasma cleaning systems have replaced solvent cleaning systems in some applications (decontamination of oxidized metal surfaces, cleaning of coins and degreasing of oily metal sheets) because they do not generate waste that requires disposal [21]. Solvent wiping is used to remove oil from oily woods such as teak (Tectona grandis L.f.), and to improve the performance of coatings on teak wood [22]. The novelty of this work lies in its potential to assist with the development of a solvent-free method of improving the performance of coatings on oily wood substrates. We hypothesize that plasma treatment will remove residual oil from the surface of hot-oil modiﬁed wood and this will have beneﬁcial eﬀects on the wettability, adhesion and performance of an acrylic coating on wood. We test this hypothesis here. We chose blue-stained, mountain pine beetle-aﬀected lodgepole pine wood (Pinus contorta Douglas) as our substrate because large volumes of this wood are available in Canada due to the unprecedented climate-hastened infestation of boreal forests by mountain pine beetle (Dendroctonus ponderosae Hopkins), and the salvage-harvesting of diseased pine trees in aﬀected forests [23]. Furthermore, there is signiﬁcant industry interest in hot-oil treatments as a way of making blue-stained wood more suitable for outdoor applications including cladding, which requires coating [24]. For the ﬁrst experiment, ﬁve eight foot long (2438 mm) 2′′ × 4′′ blue-stained lodgepole pine studs were purchased from Home Depot in Richmond, British Columbia. These parent studs were each 2.1. Experimental Design and Preparation of Wood Samples Two separate experiments were carried out that examined the eﬀects of plasma treatments on: (1) wettability and adhesion of an acrylic primer to hot-oil modiﬁed blue-stained wood (Table 1); (2) exterior performance of an acrylic coating system on hot-oil modiﬁed wood (Table 2). Table 1. Treatments, sample sizes, and replication for an experiment that examined the eﬀects of plasma treatment on the wettability and adhesion of a white acrylic primer to hot-oil modiﬁed blue stained lodgepole pine wood. Table 1. Treatments, sample sizes, and replication for an experiment that examined the eﬀects of plasma treatment on the wettability and adhesion of a white acrylic primer to hot-oil modiﬁed blue stained lodgepole pine wood. Table 1. Treatments, sample sizes, and replication for an experiment that examined the eﬀects of plasma treatment on the wettability and adhesion of a white acrylic primer to hot-oil modiﬁed blue stained lodgepole pine wood. Plasma Treatment Time, Seconds Wettability Dry and Wet Adhesion Tests Sample Size, mm3 Replication Sample Size, mm3 Replication * 0 3 × 15 × 38 5 3 × 15 × 19 5 + 5 33 3 × 15 × 38 5 3 × 15 × 19 5 + 5 333 3 × 15 × 38 5 3 × 15 × 19 5 + 5 667 3 × 15 × 38 5 3 × 15 × 19 5 + 5 1333 3 × 15 × 38 5 3 × 15 × 19 5 + 5 * Equal numbers (5) of replicates were used for adhesion tests performed on dry and wet samples. For the ﬁrst experiment, ﬁve eight foot long (2438 mm) 2′′ × 4′′ blue-stained lodgepole pine studs were purchased from Home Depot in Richmond, British Columbia. These parent studs were each 3 of 13 Coatings 2020, 10, 248 cross-cut to produce a board that was 38 (radial) × 89 (tangential) × 240 (longitudinal) mm3 in size, free of defects, and with growth rings oriented tangentially to its wide faces. These boards provided replication at the higher level. Boards were conditioned at 20 ± 1 ◦C and 65 ± 5% r.h. for one week and modiﬁed with hot-oil (see below). Modiﬁed boards were sawn to produce 3 mm thick veneer samples, which were allocated at random to the diﬀerent plasma treatments (Table 1). The wettability of modiﬁed and plasma treated samples was measured (see below). 2.2. Hot-Oil Modiﬁcation and Plasma Treatment of Wood Samples Wood was thermally modiﬁed by placing pre-weighed and conditioned samples in a circulating oil-bath (Model HTB, Thermoline Scientiﬁc Equipment Pty Ltd., Wetherill Park, Sydney, Australia) containing soya-bean oil at 220 ◦C. Modiﬁed wood was removed from the oil bath after 2 h and blotted on paper towels to remove excess oil. Hot-oil modiﬁed blue-stained wood and unmodiﬁed wood samples (described above) were plasma-treated in a reactor designed to clean silicon wafers to produce high-energy surfaces [27,28]. One wood sample was placed in the chamber of the plasma reactor at a time, and a vacuum of 0.15 ± 0.01 Torr was drawn. A valve was opened to allow water vapour from a glass reservoir into the chamber and the vacuum was redrawn. Radio frequency energy at 125 kHz was transmitted to the treatment chamber for diﬀerent periods of time (33, 333, 667, and 1333 s). Samples subjected to vacuum acted as a control. After treatment, the chamber was vented to atmosphere. Samples were removed from the chamber, taking care to avoid touching and contaminating their upper surfaces. 2.1. Experimental Design and Preparation of Wood Samples Samples were brush-coated with a water-borne primer (CIL Dulux acrylic latex), sawn in half and allocated to either dry or wet adhesion tests. The resulting experimental design accounted for random variation between boards (blocks) and between and within samples. For the second experiment, eight conditioned blue-stained lodgepole pine studs, as above (eight experimental blocks) were sawn into four defect-free samples measuring 38 (radial) × 89 (tangential) × 55 (longitudinal) mm3, and assigned to the diﬀerent treatments (Table 2). Veneer samples, 3 × 17 × 55 mm3 in size were sawn from modiﬁed and/or plasma-treated samples and brush-coated with an acrylic latex ﬁnishing system (CIL Dulux acrylic latex primer and top coat, one coat each). The paint used as a top coat was ‘mildew resistant’ and contained a fungicide, but information on the type and quantity of fungicide in the paint is not known. We chose an acrylic paint system because it is representative of those commonly used on wood cladding (siding). Furthermore, previous research has shown that acrylic paint is susceptible to mould even when the formulation contains fungicide [25,26]. Samples were randomly assigned to “weathering” or the non-weathered controls. Separate wood samples were prepared for scanning electron microscopy and confocal proﬁlometry (see below). Table 2. Treatments, sample sizes and replication for an experiment that examined the eﬀects of hot-oil modiﬁcation and plasma treatment on the water absorption and colour (CIE L value) of blue-stained lodgepole pine samples coated with a white acrylic paint system and exposed to natural weathering for 18 months (unexposed samples acted as a control). Treatment Sample Size, mm3 Replication Unweathered and Weathered Samples * Water Absorption Test Colour Test Untreated 3 × 17 × 55 8 + 8 8 + 8 Hot oil 3 × 17 × 55 8 + 8 8 + 8 Hot oil/plasma 3 × 17 × 55 8 + 8 8 + 8 Plasma 3 × 17 × 55 8 + 8 8 + 8 * Equal numbers (8) of replicates were used for tests performed on weathered and unweathered samples. * Equal numbers (8) of replicates were used for tests performed on weathered and unweathered samples. 2.2. Hot-Oil Modiﬁcation and Plasma Treatment of Wood Samples t-Oil Modiﬁcation and Plasma Treatment of Wood Samples 2.2. Hot-Oil Modiﬁcation and Plasma Treatment of Wood Samples 2.3. Contact Angle and Adhesion Measurements The wettability of the hot-oil modiﬁed and plasma treated wood was assessed by measuring the contact angle of 5 µL droplets of distilled water on three spots on wood surfaces. A 5 µL droplet 4 of 13 Coatings 2020, 10, 248 of distilled water was placed on the wood surface using a syringe, after adjusting the needle of the syringe so it produced a sessile drop [28]. The static contact angle of the droplet on the sample was measured using an optical tensiometer (KSV CAM 101, KSV Instruments Ltd., Helsinki, Finland). Images of the droplets captured after 160 milliseconds were used to calculate the contact angles that water droplets made with wood surfaces. Contact angles were calculated using instrument software by baseline adjustment and curve ﬁtting of the captured drop proﬁle to the theoretical shape predicted by the Young–Laplace equation (KSV Instruments Ltd., Helsinki, Finland). Left and right contact angles were averaged to obtain a mean contact angle for each measurement. The contact angle was measured on three diﬀerent locations for each specimen. Droplets were wiped from the surface of samples, and samples were air dried for 30 min. They were then brush-coated with one coat of an acrylic primer (CIL Dulux primer), in accord with manufacturer’s instructions. The ﬁnished samples were sawn into two and kept in a conditioning room for one week. The samples used for the wet adhesion test were sealed on their uncoated faces and edges with two-part epoxy resin (G2, Industrial Formulators of Canada Ltd., Burnaby, BC, Canada) to reduce water uptake by uncoated areas. After curing for 48 h, samples for wet adhesion tests were weighed, ﬂoated on water on the coated (painted) side for 72 h, and reweighed. The dry and wet adhesion of the coatings on hot-oil modiﬁed and plasma treated wood and controls was assessed using a cross-hatch tape test [29]. Samples were held ﬁrmly in a jig and then six cross-cuts, 1 mm apart, were manually inscribed on coated surfaces using a scalpel, guided by a steel ruler. Detached ﬂakes or ribbons of coating were removed from coated surfaces with a soft brush. A strip of pressure-sensitive tape (3M pressure-sensitive adhesive packaging tape, 3M Co., Maplewood, MN, USA) was placed over the cross-hatched area and then pulled oﬀas described in ASTM D 3359 [30]. 2.3. Contact Angle and Adhesion Measurements The cross-hatched area was scanned using a desktop scanner (Microtek Scan Maker i800, Microtek International Inc., Hsinchu, Taiwan), and digital (TIFF) images were obtained. These images were used to assess the adhesion of coatings on each sample ranking them from 0 (no adhesion, >65% of paint removed from cross-hatched area) to 5 (complete adhesion, 0% of paint removed from cross-hatched area) [30]. Analysis of variance was used to assess the eﬀects of plasma treatment on contact angle, and treatment and testing condition (dry v. wet) on paint adhesion. Statistical computation was performed using Genstat (v. 20). Contact angle results are presented in a graph and error bars on the graph (± standard error of diﬀerence) can be used to estimate whether diﬀerences between individual means are statistically signiﬁcant at the 5% level (p < 0.05) [31]. 2.4. Scanning Electron Microscopy and Confocal Proﬁlometry Scanning electron microscopy was used to examine the eﬀects of oil modiﬁcation and plasma treatment on the micro-structure of blue-stained lodgepole pine wood. Samples measuring 15 × 15 × 30 mm3 were cut from blue-stained lumber studs, as above. These small blocks were soaked in distilled water for three days and individual blocks were clamped in a small vice beneath the stage of a low power binocular microscope (Nikon 69480, Nikon Vision Co., Ltd., Shinagawa-ku, Tokyo, Japan) with their radial face uppermost. A sharp single-edged razor blade (Type S35, Feather Safety Razor Co., Osaka, Japan) was used to slice by hand, thin (20–30 µm) sections from the radial longitudinal face of each specimen until a clean, undamaged, surface was obtained [32]. Samples were dried over silica gel at 20 ± 1 ◦C for 24 h. Three types of samples were prepared using procedures described above: (1). Unmodiﬁed blue-stained lodgepole pine wood; (2) Hot-oil modiﬁed blue-stained lodgepole pine wood; (3) Hot-oil modiﬁed blue-stained lodgepole pine wood treated with plasma for diﬀerent times, varying from 333 s to 1333 s (described above). Samples were dried over silica gel at 20 ± 1 ◦C for 24 h and reduced in size to ~5 × 5 × 8 mm3 using single-edged razor blades (Gem surgical carbon steel blades, American Safety Razor Co. Verona, VA, USA). They were then glued to separate aluminium stubs using Nylon nail polish as an adhesive. The stubs were sputter coated with an 8 nm layer of gold and they were then examined using a Hitachi S-2600 variable pressure scanning electron microscope (Hitachi Ltd., Chiyoda, Tokyo, Japan) at accelerating voltages of 5–6 kV. Secondary electron images of 5 of 13 Coatings 2020, 10, 248 samples were obtained and saved as TIFF ﬁles. Non-contact surface proﬁlometry was used to probe the surface structure of hot-oil modiﬁed and plasma treated blue-stained lodgepole pine wood [27]. Samples measuring 3 × 15 × 30 mm3 were cut from the radial surfaces of hot-oil modiﬁed blue-stained lodgepole pine wood. An area measuring 1.5 × 1.5 mm2 was marked on the radial surface of each sample, and an AltiSurf 500® proﬁlometer (Altimet, 298 Allée du Larry, 74200, Marin, France) was used to image the wood in this area. These samples were then treated with plasma, as above, and the marked surfaces were re-imaged. 2.5. Exposure of Samples to Natural Weathering and Evaluation of Coating Performance The edges of coated samples were sealed using two-part epoxy resin (G2, Industrial Formulators of Canada Ltd., Burnaby, BC, Canada). The samples were then placed on glass backing plates (750 × 120 mm) and secured against the glass plates by clamping the edges of the samples between two glass strips using alligator clips. Samples were exposed to the weather on a rack inclined at 45◦ to the vertical and facing south in Vancouver, BC, Canada for a period of 18 months. A matching set of samples was kept in a conditioning room for the duration of the weathering trial. After the weathering trial, samples were removed from the weathering rack, and kept in a conditioning room for one week. The CIE L value (lightness on a scale of 100 (white) to 0 (black)) of the white acrylic paint was measured in the middle of each sample using a spectrophotometer (CM-2600d, Minolta Co. Ltd., Osaka, Japan) [33]. The water absorption of coated samples (weathered and controls) was measured as follows. Samples were weighed using an analytical balance (AAA 300L, B.C. Scale Co. Ltd., Vancouver, BC, Canada) and a 100 µL droplet of distilled water was placed on each sample using a single-channel pipette. After 60 s, the water droplet was wiped oﬀthe surface of the sample with soft paper tissue and the sample was re-weighed. The weight of water absorbed by the surface was calculated as described by Kiguchi and coworkers and expressed as the ratio of weight of water absorbed by weathered samples to that of unweathered controls [34]. Analysis of variance was used to assess the eﬀects of hot-oil modiﬁcation and/or plasma treatment and exposure to natural weathering on CIE L value and water absorption. Lightness results are presented in a graph and an error bar (Fisher’s least signiﬁcant diﬀerence, LSD) on the graph can be used to estimate whether diﬀerences between individual means are statistically signiﬁcant at the 5% level (p < 0.05) [31]. 2.4. Scanning Electron Microscopy and Confocal Proﬁlometry The software Papermap was used to produce topographical images of oil-modiﬁed and plasma-treated wood surfaces. 3.1. Contact Angle and Adhesion Measurements The bold X symbol on the y-axis and the horizontal dotted line indicate the contact angle of a water droplet on unmodiﬁed blue-stained lodgepole pine wood. wood. 3.2. Appearance and Microstructure of Hot-Oil and Plasma Treated Wood wood. 3.2. Appearance and Microstructure of Hot-Oil and Plasma Treated Wood 3.2. Appearance and Microstructure of Hot-Oil and Plasma Treated Wood Hot-oil modified wood was a brown colour, in contrast to the blue colour of unmodified blue- stained lodgepole pine wood. The modified wood was dry and appeared to be free of residual oil, but scanning electron microscopy revealed the presence of oil at the surface of hot-oil modified wood (Figure 2). Figure 2a,b shows the surface of unmodified wood. The pine cells (longitudinal tracheids) run obliquely from left to right (Figure 2a,b). A fungal hypha is present in a tracheid lumen in Figure 2a, and circular bordered pits (connections between tracheids) are prominent in both Figure 2a,b (arrowed). After hot-oil modification, oil was present at the surface of modified wood samples (Figure 2c). The oil did not completely cover the surface of the wood, but occluded some tracheids (arrowed in Figure 2c), while others were free of oil (Figure 2c). Plasma treatment of unmodified wood removed (etched) the raised border and the internal membrane (margo and torus) of bordered pits creating large circular voids at the surface of the wood (arrowed in Figure 2d) as we have Hot-oil modiﬁed wood was a brown colour, in contrast to the blue colour of unmodiﬁed blue-stained lodgepole pine wood. The modiﬁed wood was dry and appeared to be free of residual oil, but scanning electron microscopy revealed the presence of oil at the surface of hot-oil modiﬁed wood (Figure 2). Figure 2a,b shows the surface of unmodiﬁed wood. The pine cells (longitudinal tracheids) run obliquely from left to right (Figure 2a,b). A fungal hypha is present in a tracheid lumen in Figure 2a, and circular bordered pits (connections between tracheids) are prominent in both Figure 2a,b (arrowed). After hot-oil modiﬁcation, oil was present at the surface of modiﬁed wood samples (Figure 2c). The oil did not completely cover the surface of the wood, but occluded some tracheids (arrowed in Figure 2c), while others were free of oil (Figure 2c). 3.1. Contact Angle and Adhesion Measurements Hot-oil modiﬁed blue-stained lodgepole pine wood was hydrophobic as water droplets formed contact angles with modiﬁed wood surfaces that were greater than 90◦(0 s in Figure 1). Plasma treatment for only 33 s made the surface of hot-oil modiﬁed wood slightly more wettable than unmodiﬁed wood. Increasing the treatment time to 333 s further increased wettability, but thereafter there was no signiﬁcant (p > 0.05) eﬀect of treatment time on wettability (Figure 1). We measured the adhesion of an acrylic primer to hot-oil modiﬁed and plasma treated samples using a cross-cut adhesion test. There was no signiﬁcant (p > 0.05) eﬀect of plasma treatment on adhesion, and no signiﬁcant (p > 0.05) interaction of plasma treatment and testing condition (dry vs. wet) on adhesion. However, there was a small, but statistically signiﬁcant (p = 0.026) eﬀect, of testing condition on adhesion when results were averaged across diﬀerent treatments. The average adhesion rating for samples in the dry condition was 3.5 (good to very good adhesion) versus 3.1 (good adhesion) for samples tested in the wet condition (least signiﬁcant diﬀerence was 0.349). Photographs of a representative set of cross-hatched, adhesion-tested samples can be found in the Supplementary Materials associated with this paper (Figure S1). 6 of 13 6 of 13 Coatings 2020, 10, 248 Coati 2020 10 FOR Figure 1. Effect of plasma treatment on the contact angle of water droplets on the surface of hot-oil modified blue-stained lodgepole pine wood. The bold X symbol on the y-axis and the horizontal dotted line indicate the contact angle of a water droplet on unmodified blue-stained lodgepole pine wood Figure 1. Eﬀect of plasma treatment on the contact angle of water droplets on the surface of hot-oil modiﬁed blue-stained lodgepole pine wood. The bold X symbol on the y-axis and the horizontal dotted line indicate the contact angle of a water droplet on unmodiﬁed blue-stained lodgepole pine wood. Figure 1. Effect of plasma treatment on the contact angle of water droplets on the surface of hot-oil modified blue-stained lodgepole pine wood. The bold X symbol on the y-axis and the horizontal dotted line indicate the contact angle of a water droplet on unmodified blue-stained lodgepole pine Figure 1. Eﬀect of plasma treatment on the contact angle of water droplets on the surface of hot-oil modiﬁed blue-stained lodgepole pine wood. 3.1. Contact Angle and Adhesion Measurements Plasma treatment of unmodiﬁed wood removed (etched) the raised border and the internal membrane (margo and torus) of bordered pits creating large circular voids at the surface of the wood (arrowed in Figure 2d), as we have observed previously with other wood species [27,28]. pits creating large circular voids at the surface of the wood (arrowed in Figure 2d), as we have observed previously with other wood species [27,28]. Plasma treatment of hot-oil modified wood removed deposits of oil from the surface of the wood (Figure 2e), and etched wood cell walls (Figure 2f,g), but there were differences in the etching of unmodified wood and the etching of hot-oil modified wood. In particular, the bordered pit membranes in hot-oil modified wood were resistant to plasma etching (arrowed in Figure 2h), whereas plasma completely removed pit membranes at the surface of unmodified wood (compare Plasma treatment of hot-oil modiﬁed wood removed deposits of oil from the surface of the wood (Figure 2e), and etched wood cell walls (Figure 2f,g), but there were diﬀerences in the etching of unmodiﬁed wood and the etching of hot-oil modiﬁed wood. In particular, the bordered pit membranes in hot-oil modiﬁed wood were resistant to plasma etching (arrowed in Figure 2h), whereas plasma completely removed pit membranes at the surface of unmodiﬁed wood (compare Figure 2f,g with Figure 2d). 7 of 13 Coatings 2020, 10, 248 Figure 2. SEM photomicrographs of blue-stained lodgepole pine wood before and after hot-oil modification and plasma treatment: (a) and (b) Unmodified controls; (c) Wood modified with hot-oil; (d) Unmodified wood treated with plasma for 667 s; (e) Hot-oil modified wood treated with plasma for 333 s. Note the ballooning of pit membranes through the aperture of the bordered pits; (f–h) Hot- oil modified wood treated with plasma for 1333 s. The ‘protective’ effect of hot-oil modification on plasma etching of bordered pit membranes w igure 2. SEM photomicrographs of blue-stained lodgepole pine wood before and after hot-oi modiﬁcation and plasma treatment: (a) and (b) Unmodiﬁed controls; (c) Wood modiﬁed with hot-oi d) Unmodiﬁed wood treated with plasma for 667 s; (e) Hot-oil modiﬁed wood treated with plasma fo 33 s. Note the ballooning of pit membranes through the aperture of the bordered pits; (f–h) Hot-oi modiﬁed wood treated with plasma for 1333 s. Figure 2. 3.1. Contact Angle and Adhesion Measurements SEM photomicrographs of blue-stained lodgepole pine wood before and after hot-oil modification and plasma treatment: (a) and (b) Unmodified controls; (c) Wood modified with hot-oil; (d) Unmodified wood treated with plasma for 667 s; (e) Hot-oil modified wood treated with plasma for 333 s. Note the ballooning of pit membranes through the aperture of the bordered pits; (f–h) Hot- oil modified wood treated with plasma for 1333 s. Figure 2. SEM photomicrographs of blue-stained lodgepole pine wood before and after hot-oil modiﬁcation and plasma treatment: (a) and (b) Unmodiﬁed controls; (c) Wood modiﬁed with hot-oil; (d) Unmodiﬁed wood treated with plasma for 667 s; (e) Hot-oil modiﬁed wood treated with plasma for 333 s. Note the ballooning of pit membranes through the aperture of the bordered pits; (f–h) Hot-oil modiﬁed wood treated with plasma for 1333 s. The protective effect of hot oil modification on plasma etching of bordered pit membranes was confirmed by confocal profilometry of hot-oil modified wood samples before and after plasma treatment. Figure 3 shows topographic images of the surface of hot-oil modified wood before (Figure 3a) and after plasma treatment (Figure 3b,c). Figure 3a shows the raised borders around two pit apertures at the surface of hot-oil modified wood. After exposure to plasma the border is etched away, as well as some of the adjacent cell wall material (Figure 3b,c). However, the central part of the pit, the pit membrane is still intact (Figure 3b,c). The ‘protective’ eﬀect of hot-oil modiﬁcation on plasma etching of bordered pit membranes was conﬁrmed by confocal proﬁlometry of hot-oil modiﬁed wood samples before and after plasma treatment. Figure 3 shows topographic images of the surface of hot-oil modiﬁed wood before (Figure 3a) and after plasma treatment (Figure 3b,c). Figure 3a shows the raised borders around two pit apertures at the surface of hot-oil modiﬁed wood. After exposure to plasma the border is etched away, as well as some of the adjacent cell wall material (Figure 3b,c). However, the central part of the pit, the pit membrane is still intact (Figure 3b,c). Coatings 2020, 10, 248 Coatings 2020 10 x FO 8 of 13 8 of 13 8 of 13 8 of 13 (a) (b) (c) Figure 3. 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering The appearance of a representative set of coated wood samples that had been hot-oil modified and/or treated with plasma, and then exposed to the weather in Vancouver for 18 months can be seen in Figure 4b,d,f,h. Unweathered controls stored in a conditioning room for the duration of the weathering trial are shown on the left-hand side of Figure 4a,c,e,g. The most obvious change in appearance of the samples during exposure to the weather is the more pronounced discolouration of the acrylic coating on the hot-oil modified wood sample (Figure 4d). Some discolouration of the ends of most weathered samples occurred where they were covered with glass strips used to fix them to The appearance of a representative set of coated wood samples that had been hot-oil modiﬁed and/or treated with plasma, and then exposed to the weather in Vancouver for 18 months can be seen in Figure 4b,d,f,h. Unweathered controls stored in a conditioning room for the duration of the weathering trial are shown on the left-hand side of Figure 4a,c,e,g. The most obvious change in appearance of the samples during exposure to the weather is the more pronounced discolouration of the acrylic coating on the hot-oil modiﬁed wood sample (Figure 4d). Some discolouration of the ends of most weathered samples occurred where they were covered with glass strips used to ﬁx them to weathering racks. of most weathered samples occurred where they were covered with glass strips used to fix them to weathering racks. The white colour of coated samples was measured using a spectrophotometer, and the CIE parameter L (white to black on a scale of 100 (white) to 0 (black)) was used to assess changes in the colour of the white paint. The colour of coated wood samples that had been hot-oil modified and/or treated with plasma, and stored in a conditioned room for 18 months, or exposed to the weather for a similar period of time can be seen in Figure 5. There were no significant (p > 0.05) effects of hot-oil treatment and/or plasma modification on the colour of the white acrylic paint on unweathered samples. The hot-oil and oil and plasma treated samples were slightly darker than the unmodified samples (control and plasma treated), in accord with their appearance in Figure 4, but the differences in colour are not statistically significant (p > 0.05) (Figure 5). 3.1. Contact Angle and Adhesion Measurements Confocal profilometry images of the wood cell wall in hot-oil modified wood samples before and after plasma treatment: (a) Two bordered pits in the cell wall of a wood sample modified with hot-oil; (b) and (c) Bordered pit in the cell wall of a wood sample modified with hot-oil and then treated with plasma for 1333 s. Note that the cell wall around the bordered has been etched but the central part of the pit is intact. Performance of an Acrylic Coating on Hot Oil and Plasma Treated Wood after Natural Weathering Figure 3. Confocal proﬁlometry images of the wood cell wall in hot-oil modiﬁed wood samples before and after plasma treatment: (a) Two bordered pits in the cell wall of a wood sample modiﬁed with hot-oil; (b) and (c) Bordered pit in the cell wall of a wood sample modiﬁed with hot-oil and then treated with plasma for 1333 s. Note that the cell wall around the bordered has been etched but the central part of the pit is intact. . Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering (b) (a) (b) (c) (a) (b) (c) (c) Figure 3. Confocal profilometry images of the wood cell wall in hot-oil modified wood samples before and after plasma treatment: (a) Two bordered pits in the cell wall of a wood sample modified with hot-oil; (b) and (c) Bordered pit in the cell wall of a wood sample modified with hot-oil and then treated with plasma for 1333 s. Note that the cell wall around the bordered has been etched but the central part of the pit is intact. Figure 3. Confocal proﬁlometry images of the wood cell wall in hot-oil modiﬁed wood samples before and after plasma treatment: (a) Two bordered pits in the cell wall of a wood sample modiﬁed with hot-oil; (b) and (c) Bordered pit in the cell wall of a wood sample modiﬁed with hot-oil and then treated with plasma for 1333 s. Note that the cell wall around the bordered has been etched but the central part of the pit is intact. 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weatherin 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering Unweathered controls on left-hand side (samples a,c,e,g) were stored in a conditioning room for 18 months. Scale bars = 11 mm. Figure 4. Effects of hot-oil treatment and/or plasma modification on the appearance of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering (samples b,d,f,h). Unweathered controls on left-hand side (samples a,c,e,g) were stored in a conditioning room for 18 months. Scale bars = 11 mm. Figure 4. Eﬀects of hot-oil treatment and/or plasma modiﬁcation on the appearance of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering (samples b,d,f,h). Unweathered controls on left-hand side (samples a,c,e,g) were stored in a conditioning room for 18 months. Scale bars = 11 mm. Figure 4. Effects of hot-oil treatment and/or plasma modification on the appearance of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering (samples b,d,f,h). Unweathered controls on left-hand side (samples a,c,e,g) were stored in a conditioning room for 18 months. Scale bars = 11 mm. There was no significant (p > 0.05) effect of plasma treatment on water uptake of samples and no significant (p > 0.05) interaction of plasma treatment and exterior exposure (unweathered vs. weathered) on water uptake. There was no signiﬁcant (p > 0.05) eﬀect of plasma treatment on water uptake of samples and no signiﬁcant (p > 0.05) interaction of plasma treatment and exterior exposure (unweathered vs. weathered) on water uptake. There was no significant (p > 0.05) effect of plasma treatment on water uptake of samples and no significant (p > 0.05) interaction of plasma treatment and exterior exposure (unweathered vs. weathered) on water uptake. There was no significant (p > 0.05) effect of plasma treatment on water uptake of samples and no significant (p > 0.05) interaction of plasma treatment and exterior exposure (unweathered vs. weathered) on water uptake. There was no signiﬁcant (p > 0.05) eﬀect of plasma treatment on water uptake of samples and no signiﬁcant (p > 0.05) interaction of plasma treatment and exterior exposure (unweathered vs. weathered) on water uptake. There was no significant (p > 0.05) effect of plasma treatment on water uptake of samples and no significant (p > 0.05) interaction of plasma treatment and exterior exposure (unweathered vs. weathered) on water uptake. Figure 5. 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering The paint on all samples became significantly (p < 0.05) darker after weathering, but the darkening was most pronounced on hot-oil modified samples. Plasma treatment clearly reduced the darkening of the hot-oil modified samples, although hot-oil modified, plasma treated and weathered samples were significantly (p < 0.05) darker The white colour of coated samples was measured using a spectrophotometer, and the CIE parameter L (white to black on a scale of 100 (white) to 0 (black)) was used to assess changes in the colour of the white paint. The colour of coated wood samples that had been hot-oil modiﬁed and/or treated with plasma, and stored in a conditioned room for 18 months, or exposed to the weather for a similar period of time can be seen in Figure 5. There were no signiﬁcant (p > 0.05) eﬀects of hot-oil treatment and/or plasma modiﬁcation on the colour of the white acrylic paint on unweathered samples. The hot-oil and oil and plasma treated samples were slightly darker than the unmodiﬁed samples (control and plasma treated), in accord with their appearance in Figure 4, but the diﬀerences in colour are not statistically signiﬁcant (p > 0.05) (Figure 5). The paint on all samples became signiﬁcantly (p < 0.05) darker after weathering, but the darkening was most pronounced on hot-oil modiﬁed samples. Plasma treatment clearly reduced the darkening of the hot-oil modiﬁed samples, although hot-oil modiﬁed, plasma treated and weathered samples were signiﬁcantly (p < 0.05) darker than the controls that were not modiﬁed with hot-oil (Figure 5). Coatings 2020, 10, 248 9 of 13 Figure 4. Effects of hot-oil treatment and/or plasma modification on the appearance of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering (samples b,d,f,h). Unweathered controls on left-hand side (samples a,c,e,g) were stored in a conditioning room for 18 months. Scale bars = 11 mm. Figure 4. Eﬀects of hot-oil treatment and/or plasma modiﬁcation on the appearance of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering (samples b,d,f,h). Unweathered controls on left-hand side (samples a,c,e,g) were stored in a conditioning room for 18 months. Scale bars = 11 mm. Figure 4. Effects of hot-oil treatment and/or plasma modification on the appearance of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering (samples b,d,f,h). 4. Discussion In the introduction to this paper, we hypothesized that plasma treatment would remove residual oil from the surface of hot-oil modiﬁed blue-stained lodgepole pine wood, and this would have beneﬁcial eﬀects on the adhesion and performance of an acrylic coating on wood. Our results partially support this hypothesis. Oil was present at the surface of hot-oil modiﬁed wood and signiﬁcantly increased the hydrophobicity of the wood (contact angle of greater than 90 degrees). However, after plasma modiﬁcation the contact angle of hot-oil modiﬁed wood was lower than that of wood in its native state suggesting that plasma removed oil from wood surfaces. Scanning electron microscopy conﬁrmed that oil was absent from hot-oil modiﬁed wood surfaces that were treated with plasma, and it also revealed extensive etching of wood cell walls. Hence, we conclude that plasma is able to remove oil from the surface of hot-oil modiﬁed wood, in accord with previous studies that have shown that plasma can remove oil from the surface of metals and polymers [19,20]. Plasma treatments have been tested to determine if they can improve the adhesion of coatings to diﬀerent wood species [35], heat-treated wood [11] and wood composites [36]. Oil at the surface of wood is reported to reduce coating adhesion [15], but we found that removal of oil from the surface of hot-oil modiﬁed blue-stained lodgepole pine wood by plasma did not alter the adhesion of an acrylic primer to the modiﬁed wood. This runs counter to our hypothesis, but accords with the ﬁndings of Podgorski and coworkers who found that a plasma treatment did not improve the adhesion of coatings to heat-treated wood, even though their plasma treatment made the surface more wettable [7]. A number of other studies have shown that plasma pre-treatments have no, or even negative, eﬀects on the adhesion of coatings to unmodiﬁed wood [37,38]. The adhesion of the acrylic coating to replicates obtained from diﬀerent wood samples was highly variable, and this would have reduced the ability to detect statistically signiﬁcant (p < 0.05) plasma-treatment eﬀects on coating adhesion. In general, our ﬁndings accord with those of Acda and coworkers who concluded that “eﬀects of plasma treatment on the adhesion of coatings to wood vary with wood substrate and process parameters” [35]. 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering 3.3. Performance of an Acrylic Coating on Hot-Oil and Plasma Treated Wood after Natural Weathering Effects of hot-oil treatment and/or plasma modification on the white colour (CIE L value) of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering. Discussion Figure 5. Effects of hot-oil treatment and/or plasma modification on the white colour (CIE L value) of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering. Discussion Figure 5. Eﬀects of hot-oil treatment and/or plasma modiﬁcation on the white colour (CIE L value) of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering. Figure 5. Effects of hot-oil treatment and/or plasma modification on the white colour (CIE L value) of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering. Figure 5. Effects of hot-oil treatment and/or plasma modification on the white colour (CIE L value) of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering. Figure 5. Eﬀects of hot-oil treatment and/or plasma modiﬁcation on the white colour (CIE L value) of an acrylic paint on blue-stained lodgepole pine samples exposed to 18 months of natural weathering. Coatings 2020, 10, 248 10 of 13 10 of 13 4. Discussion Nevertheless, removal of surface oil from hot-oil modiﬁed wood samples by plasma improved the performance of the acrylic paint by reducing the discolouration of the paint on samples exposed outdoors to the weather. The discolouration of paint ﬁlms is an important criterion used to assess the performance of paint ﬁlms exposed outdoors [39]. Several approaches are used to reduce such discolouration including modiﬁcations to coatings to reduce pickup by dirt, and the use of biocides to reduce colonization of paint by micro-organisms [39]. Therefore, our ﬁnding that plasma treatment signiﬁcantly reduced the discolouration of an acrylic paint on hot-oil modiﬁed wood is noteworthy, but further research is needed to determine whether the same eﬀect occurs with other coatings, some of which are less permeable than acrylic paints, for example water-borne alkyds and mixed alkyd-acrylic systems [40]. Further research is also needed to determine the identity of the agents responsible for the discolouration of the acrylic paint on hot-oil modiﬁed wood, which could include dirt [39], mould [17,25,26] and algae [41]. Plasma treatment did not completely eliminate discolouration of the acrylic paint on hot-oil modiﬁed wood. One possible explanation for this observation is that plasma only removed oil from the surface of the wood, and subsequent migration of oil from the interior of samples to the surface of wood during weathering encouraged some discolouration of hot-oil modiﬁed plasma-treated wood samples. Further research would be needed to conﬁrm this hypothesis, which is supported by previous studies that have shown that oils migrate to the surface of preservative treated wood exposed to natural weathering [42,43]. One unexpected ﬁnding arising from our work was the eﬀect that oil modiﬁcation had on the etching of bordered pits. These pits act as conduits allowing the passage of liquids (including oils) and gases between cells (tracheids) in softwood species. They contain a cellulosic membrane (the margo) and a centrally thickened torus. Pit membranes in untreated wood are rapidly etched by plasma [27,28], and they are also highly susceptible to microbial degradation [44]. However, remnants of pit membranes were commonly observed at the surface of hot-oil modiﬁed and plasma treated wood, whereas they were absent from the surface of unmodiﬁed plasma treated controls. 4. Discussion This ﬁnding 11 of 13 Coatings 2020, 10, 248 suggests that hot-oil modiﬁcation reduced the susceptibility of pit membranes to plasma etching, possibly in accord with previous research that has shown that oils increase the oxidative stability of cellulosic substrates [45]. One feature of the hot-oil thermal modiﬁcation process is that oil is absorbed and retained by the modiﬁed wood [46]. This oil contributes to the desirable water repellency of hot-oil modiﬁed wood [5], but as we have shown here, it appears to have an undesirable eﬀect on the exterior performance of an acrylic coating. We are unable to compare our results with those of others because there has been no related work on the use of plasma treatments to improve the performance of coatings on hot-oil modiﬁed wood. Plasma treatments are used to remove oils from the surfaces of metals and polymers, as mentioned above [19–21], and are preferred to solvent cleaning systems because they do not generate waste that requires disposal. Solvent wiping is used to remove oil from oily woods such as teak, and to improve the performance of coatings on the wood, as mentioned in the introduction [22]. Therefore, it is possible that plasma cleaning systems could emerge to replace solvent wiping systems and improve coating performance on woods that contain oil, either naturally or as a result of hot-oil modiﬁcation. However, the plasma treatment system used here would not be suitable for commercial applications because it required high vacuum, relatively long treatment times, and it is unable to treat large samples. Furthermore, our ﬁndings need to be replicated with additional wood species and oils that are used to manufacture hot-oil modiﬁed wood, and complimented by further studies that examine chemical and physical changes resulting from plasma treatment of hot-oil modiﬁed wood (FTIR and polar and dispersive components of surface energy changes). Nevertheless, we conclude that plasma treatment shows promise as a way of improving the performance of exterior acrylic coatings on hot-oil modiﬁed wood, but commercial applications would require the development of treatment technology that can more rapidly remove oil from the surface of modiﬁed wood. 5. Conclusions A water-vapor plasma pre-treatment reduced the discolouration of a water-borne acrylic latex coating on hot-oil modiﬁed, blue-stained lodgepole pine wood exposed to natural weathering for 18 months. We identiﬁed that the ability of plasma to remove oil from the surface of the modiﬁed wood was responsible for the reduced discolouration of the coating on hot-oil modiﬁed wood. We conclude that plasma treatment shows promise as a way of improving the outdoor performance of white acrylic paint on hot-oil modiﬁed wood. Further research on plasma modiﬁcation to improve the performance of coatings on hot-oil modiﬁed wood will focus on trialing the treatment on additional wood species and using diﬀerent oils that are used to manufacture hot-oil modiﬁed wood, and developing treatment technology that can more rapidly remove oil from the surface of modiﬁed wood. Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6412/10/3/248/s1, Figure S1: Images of hot-modiﬁed and plasma-treated samples after cross-cut adhesion testing. Author Contributions: A.J. and P.D.E. conceived and designed the experiments; A.J. performed all experimental work. P.D.E. analyzed all data and wrote the ﬁrst draft of the paper. Both authors discussed and commented on the results and contributed to the ﬁnal submitted manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by ForValueNet–NSERC strategic network on forest management for value-added products, grant numbers NSERC 340830-06 and 03701-CG08822; NSERC Collaborative Research and Development Grant (CRDPJ 485007-15); Forestry Innovation Investment Market Development Program 08-52; Rix Family Foundation Award to A.J. Acknowledgments: We thank Ian Cullis, Mohammad Jahangir Chowdhury and Lukie H. 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https://openalex.org/W3094298740	https://periodicos.ufpb.br/ojs/index.php/arf/article/download/55825/31749	Spanish; Castilian	null	Jano bifronte: propiedad e impropiedad del Dasein frente a la transformación ontológica del Ge-stell	Aufklärung	2,020	cc-by	7,455	* Doctor en Filosofía por la Universidad de Chile. Académico e investigador del área de Informática & Telecomunicaciones en el Instituto INACAP Chile, sede Temuco, Chile. Email: leopoldo.tilleria@inacapmail.cl Leopoldo Tillería Aqueveque * Instituto INACAP, Chile RESUMEN: El artículo discute la noción de Ge stell planteada por Heidegger en su filosofía tardía de la técnica. Tal idea se entiende en una doble posibilidad: como dispositivo de control tecnológico al modo de una imposición, es decir, como la esencia de la técnica moderna, o como destinación otorgante de la salvación, esto es, como desocultamiento de la verdad del ser. Como Jano bifronte, el Dasein se encuentra permanentemente en la encrucijada de elegir mantenerse en el modo de la impropiedad y, por lo mismo, perder su autonomía ante la omnipotencia tecnológica del Gestell, o bien, decidirse por una existencia desde la propiedad y proyectar su ser a partir de la posibilidad de la intercambiabilidad ontológica del Gestell, resguardando de este modo su autonomía de la dictadura metafísica del dispositivo. ABSTRACT: The article discusses the notion of Gestell raised by Heidegger in his late philosophy of technique. Such an idea is understood in a double possibility: as a device of technological control in the manner of an imposition, that is, as the essence of modern technique, or as a destination that grants salvation, that is, as uncovering the truth of being. Like Twofaced Janus, Dasein is permanently at the crossroads of choosing to remain in the mode of impropriety and, therefore, lose his autonomy before the technological omnipotence of Gestell, or else decide on an existence from ownership and projecting its being from the possibility of the ontological interchangeability of the Gestell, thus protecting its autonomy from the device's metaphysical dictatorship. KEYWORDS: Dasein; Gestell; Heidegger; ontology; technique KEYWORDS: Dasein; Gestell; Heidegger; ontology; technique PALABRAS CLAVE: Dasein; Gestell; Heidegger; ontología; técnica AUFKLÄRUNG,JoãoPessoa,v.7,n.2.,Mai.Ago.,2020,p.6778 DOI:https://doi.org/10.18012/arf.v7i2.55825 Recebido:22/06/2020\|Aceito:25/09/2020 Licença:CreativeCommons4.0International(CCBY4.0) AUFKLÄRUNG,JoãoPessoa,v.7,n.2.,Mai.Ago.,2020,p.6778 DOI:https://doi.org/10.18012/arf.v7i2.55825 Recebido:22/06/2020\|Aceito:25/09/2020 Licença:CreativeCommons4.0International(CCBY4.0) AUFKLÄRUNG,JoãoPessoa,v.7,n.2.,Mai.Ago.,2020,p.6778 DOI:https://doi.org/10.18012/arf.v7i2.55825 Recebido:22/06/2020\|Aceito:25/09/2020 Licença:CreativeCommons4.0International(CCBY4.0) JANO BIFRONTE: PROPIEDAD E IMPROPIEDAD DEL DASEIN FRENTE A LA TRANSFORMACIÓN ONTOLÓGICA DEL GE-STELL TWOFACED JANUS: PROPRIETY AND IMPROPRIETY OF DASEIN FACING ONTOLOGICAL TRANSFORMATION OF THE GESTELL Leopoldo Tillería Aqueveque * Instituto INACAP, Chile Leopoldo Tillería Aqueveque * Instituto INACAP, Chile 1.INTRODUCCIÓN U U na revisión somera de la literatura especializada permite ver que la figura de Heidegger es traída una y otra vez a colación, ya sea desde la filosofía del lenguaje, ya desde la pura ontología y su inagotable pregunta por el ser, o ya desde la crítica prácticomoral, probablemente uno de los principales flancos del filósofo de Friburgo. Ni qué hablar de los cuestionamientos políticos, que han levantado, como se sabe, una lapidaria crítica filosófica a raíz de la militancia nacionalsocialista del pensador alemán. A través de este trabajo se pretende hacer una observación ontológica a una de las ideas más enigmáticas que Heidegger dejó en su última filosofía, la del Ge stell, precisamente cuando sus observaciones del potencial técnicotecnológico –o LeopoldoTilleríaAqueveque atómico, si se prefiere mostraban al hombre contemporáneo en la más apremiante de sus posibilidades. Este escrito se plantea entre dos regiones del pensamiento heideggeriano: la ontología fundamental y su preocupación por la técnica (Technik), meditación que definitivamente es una sola si aceptamos el argumento de que la técnica es el último gran tema de la filosofía heideggeriana. Por lo demás, en la filosofía de la técnica de Heidegger –desde luego mucho más explícita en sus conferencias de la década del 50 que en sus escritos más tempranos parece hallarse incuestionablemente la pregunta por el ser. 68 g Heidegger denomina Gestell a la esencia de la técnica moderna, pero advirtiendo, en su conocida y paradójica frase, que no es nada técnico. En lo que sigue, intentaré, dicho coloquialmente, desmitificar al Gestell heideggeriano como esa especie de monstruo de siete cabezas que, montado en su carro de guerra tecnológico, termina por controlar hasta la última mente humana existente en el planeta. Apoyándome en la tesis autonomista de Peck y en la de la mutabilidad ontológica de Malabou, sugeriré que el Gestell, interpretado como el mundo tecnológico (technische Welt) que nos rodea e incumbe y, por tanto, nos define en nuestro particular modo de ser, no constituye por ello ningún determinismo ontológico, sino que, muy por el contrario, se revela como una suerte de límite en el acceso a la posibilidad de encontrarnos con nuestro ser más propio. 1.INTRODUCCIÓN El artículo se organiza en dos partes: en la primera, se plantea y discute la noción de Gestell, sobre todo en el contexto de la ambigüedad con que el mismo Heidegger la presenta en su filosofía tardía, específicamente en La pregunta por la técnica (1953). En la segunda, tomaré partido por la tesis que considera al Gestell como algo parecido a un límite ontotecnológico desde donde el Dasein tiene la posibilidad de definir una relación más auténtica con su propio ser. sino en relación a su significado original: El término GeStell es compuesto por el sufijo “Ge”, que es un sufijo colectivo porque indica un conjunto, una multiplicidad reunida bajo una mirada abarcadora, y la raíz “stell” que remite al verbo “stellen”, que significa “poner”. GeStell entonces reúne en sí todas las modalidades esenciales que concurren a formar el mundo de la técnica: “Herstellen, Vorstellen, Bestellen”: el producir, representar, ordenar (GIUBILATO, 2016, p. 60). El término GeStell es compuesto por el sufijo “Ge”, que es un sufijo colectivo porque indica un conjunto, una multiplicidad reunida bajo una mirada abarcadora, p q j , p j , y la raíz “stell” que remite al verbo “stellen”, que significa “poner”. GeStell entonces reúne en sí todas las modalidades esenciales que concurren a formar el mundo de la técnica: “Herstellen, Vorstellen, Bestellen”: el producir, representar, ordenar (GIUBILATO, 2016, p. 60). 69 De esta manera, el GeStell reúne en sí todas las modalidades esenciales que determinan el mundo de la técnica: Herstellen, Vorstellen y Bestellen, esto es, el producir, el representar y el ordenar. Lo que se desoculta mediante el Gestell, se hace al modo de una productividad técnica dominada por su condición de dispositivo. De otra forma: como el mundo tecnológico que se nos impone. Sin embargo, la ambigüedad del término es patente cuando Heidegger menciona en la misma conferencia que esta idea del Gestell debe ser puesta en liza por medio de este poner lo presente como ποίησις. Observa Hernández: Dice Heidegger: «La palabra “poner” (stellen), en la denominación “imposición” (Gestell), no mienta sólo la provocación; debe conservar a la vez la resonancia de otro “poner” del que proviene, a saber, de aquel producir y exponer (Her und Darstellen) que, en el sentido de la ποίησις, deja que lo presente aparezca en el estadodenooculto» (HERNÁNDEZ, 2006). El Gestell, o sea, el ser de la era de la técnica, no puede en ningún caso (si creemos que la filosofía de Heidegger constituye una unidad, aunque matizada, respecto de la pregunta ontológica fundamental) dejar de disponerse en un sentido temporal. Ese es el legado irrenunciable de Ser y Tiempo (en adelante ST) en la filosofía contemporánea (zeitgenössische Philosophie). De este modo, como dispositivo, el Ge stell no puede sino acontecer desde la existencia fáctica de los seres humanos, existencia que eminente e irreversiblemente se ve impactada por el despliegue calculante de la técnica y de la tecnología (Technologie). 2.DAS GE-STELL En La pregunta por la técnica el filósofo plantea: “Nosotros llamamos ahora aquella interpelación provocante, que reúne al hombre en ella a establecer el desocultar como constante, lo dispuesto [das Gestell]” (HEIDEGGER, 1984, p. 87). El Gestell entonces es aquella suerte de llamada que provoca a la conciencia del hombre a desocultar el mundo del pensar calculante. El núcleo de la definición está en el binomio provocar/desocultar, cuestión que hablaría efectivamente de una interpelación: La esencia de la técnica moderna es designada por Heidegger con la palabra Ge stell: imposición. También se ha vertido esta palabra como dispuesto, posición total, dispositivo, instalación, estructura de emplazamiento, disposición. Con este término se apunta hacia una instancia que escapa al mero arbitrio humano, aunque en su emergencia han cooperado los hombres, ni su aparición ni su despliegue están sin más bajo directrices humanas” (ACEVEDO, 2012). No obstante referirse esencialmente a la técnica, el Gestell, como modo del desocultar, y como se ha repetido tantas veces, no es algo técnico: “Dispuesto significa el modo del desocultar que impera en la esencia de la técnica moderna y que él mismo no es nada técnico” (HEIDEGGER, 1984, p. 88). De hecho, su semántica es ontológica. A decir verdad, el Gestell es el dispositivo tecnológico de la época posmoderna, dicho de otro modo, el ser tecnológico. Sin embargo, el binomio provocación/desocultación requiere de una suerte de garantía que lo conecte con el modo de este desocultar. Tal garantía está dada por la palabra “poner” (stellen), en las varias acepciones en que Heidegger la utiliza en esta conferencia. Es decir, no solo en el sentido del provocar Janobifronte:propiedadeimpropiedaddelDaseinfrentealatransformaciónontológica... sino en relación a su significado original: Como efectividad técnica, tal dispositivo constituiría literalmente un mecanismo de conectividad: “La técnica convierte a la naturaleza y a la realidad en un engranaje (Gestell) en el que todo se relaciona con todo según la lógica de la compatibilidad y de la producción, en el que todo se hace presente a partir de la conexión causaefecto” (LOZANO, 2004, p. 207). Ahora, a diferencia del ser que fue tematizado en ST como proyección del Dasein, precisamente formando parte de una densidad temporal que la historia de la metafísica había pasado siempre por alto, el Gestell Es lo radicalmente primero, lo que constituye al serahí y a la vez lo interpela, lo que lo hace ser. De hecho, en diversas ocasiones Heidegger sustituye la palabra alemana actual para referirse al Ser ‘Sein’, por su grafía más antigua ‘Seyn’, o incluso tacha la palabra ‘Sein’ en aspa, intentando acentuar el hecho de que aquello por lo que se pregunta es el Ser mismo, no el Ser como mera verdad del ente tematizado por la tradición metafísica (LOZANO, 2004, p. 205). En el curso del semestre de verano de 1941 de Friburgo, titulado Conceptos fundamentales, Heidegger ya anticipa esta comprensión radical del ser como Gestell con un par de definiciones suficientemente abarcadoras: “El ser es lo más vacío y al mismo tiempo lo exuberante. El ser es lo más común de todo y al mismo tiempo la unicidad. El ser es lo más comprensible y al mismo tiempo la ocultación. El ser es lo más desgastado y al mismo tiempo el origen” (HEIDEGGER, 1997, p. 99). Y escribe algunas páginas después: “El ser es más coactivo y al mismo tiempo la liberación” LeopoldoTilleríaAqueveque (HEIDEGGER, 1997, p. 107). Dicha coacción reaparece en la noción de Gestell en el sentido de una dominación en cuanto a nuestra respectividad de lo técnico: no podemos sino estarenelmundo de la técnica. De ahí que para Linares el mundo tecnológico contemporáneo sea el ámbito de la técnica, en cuanto fuerza planetaria dominada por el Gestell como una especie de imperativo a priori (LINARES, 2003, p. 17). Desde luego, esta visión sustancialista de Heidegger de la técnica ha sido blanco de severas críticas desde el campo de la filosofía de la tecnología. sino en relación a su significado original: Así pues, Riu considera que el filósofo de Messkirch abandona el campo óntico de la técnica y de la tecnología y se sitúa en el de la mistificación de la realidad técnica, o para decirlo con sus propias palabras, en el plano de la constelación semántica a partir de la presentación del verbo stellen: “Lo más sorprendente, sin embargo, es la ausencia de toda referencia al sistema económico capitalista, sin cuya mediación es imposible en absoluto entender el alcance y significado real de la tecnología” (citado en ARAUJO, 2013, p. 178). Sin embargo, nos parece sumamente relevante la postura de Malabou, para quien el Gestell –dicho, en todo caso, por una discípula de Derrida “es la revelación del punto donde el ser, la esencia y los seres son susceptibles de perder (o ceder) su posición. El ser, la esencia y los seres ‘pierden esas cualidades con las que la metafísica los ha dotado’” (MALABOU, 2011, p. 169). Esta condición, pudiéramos decir, deconstructiva del Ge stell, es fundamental para avanzar en las posibles repercusiones de esta tesis respecto de la filosofía del ser del último Heidegger. (HEIDEGGER, 1997, p. 107). Dicha coacción reaparece en la noción de Gestell en el sentido de una dominación en cuanto a nuestra respectividad de lo técnico: no podemos sino estarenelmundo de la técnica. De ahí que para Linares el mundo tecnológico contemporáneo sea el ámbito de la técnica, en cuanto fuerza planetaria dominada por el Gestell como una especie de imperativo a priori (LINARES, 2003, p. 17). Desde luego, esta visión sustancialista de Heidegger de la técnica ha sido blanco de severas críticas desde el campo de la filosofía de la tecnología. Así pues, Riu considera que el filósofo de Messkirch abandona el campo óntico de la técnica y de la tecnología y se sitúa en el de la mistificación de la realidad técnica, o para decirlo con sus propias palabras, en el plano de la constelación semántica a partir de la presentación del verbo stellen: “Lo más sorprendente, sin embargo, es la ausencia de toda referencia al sistema económico capitalista, sin cuya mediación es imposible en absoluto entender el alcance y significado real de la tecnología” (citado en ARAUJO, 2013, p. 178). sino en relación a su significado original: Sin embargo, nos parece sumamente relevante la postura de Malabou, para quien el Gestell –dicho, en todo caso, por una discípula de Derrida “es la revelación del punto donde el ser, la esencia y los seres son susceptibles de perder (o ceder) su posición. El ser, la esencia y los seres ‘pierden esas cualidades con las que la metafísica los ha dotado’” (MALABOU, 2011, p. 169). Esta condición, pudiéramos decir, deconstructiva del Ge stell, es fundamental para avanzar en las posibles repercusiones de esta tesis respecto de la filosofía del ser del último Heidegger. 70 3.EL GE-STELL COMO DISPOSITIVO DE CONTROL TECNOLÓGICO En la misma línea parece inscribirse Angela Luzia Miranda, para quien y citando a Towarnicki y Palnier, el Ge stell deformaría el desocultamiento de la verdad: llevarnos a la ilusión de suponer que nos hemos convertido, como dice el propio Heidegger, en “señores de la tierra” (PECK, 2015, p. 1011). En la misma línea parece inscribirse Angela Luzia Miranda, para quien y citando a Towarnicki y Palnier, el Ge stell deformaría el desocultamiento de la verdad: 71 La retirada del ser pone en riesgo el propio sentido de la verdad, según Heidegger, porque el rasgo fundamental de la técnica, a saber, este hacer salir lo oculto, no aparece ya como tal. En ese sentido, ‘la estructura de emplazamiento [Gestell] deforma el resplandecer y el prevalecer de la verdad’” (MIRANDA, 2017, p. 204). Ciertamente, debemos considerar en esta tesis de la “amenaza” un asunto esencial a la concepción del Gestell y que es el referido al “lugar” desde donde Heidegger filosofa, es decir, el Heidegger histórico. Tal como observa Méndez, “podemos considerar el pensamiento heideggeriano como una reacción al impacto de la industrialización en las comunidades rurales centroeuropeas” (MÉNDEZ, 2007, p. 24), lo cual significa que en la Alemania de Heidegger no habría predominado una recepción fundamentalmente ilustrada de la tecnología, “sino una visión romántica que, admirando la creatividad que se pone en juego, se mueve en el ámbito de lo sublime sombrío” (MÉNDEZ, 2007, p. 24). De aquí que para Heidegger la verdadera contradicción de la razón técnica estaría dada por una visión naturalista, asociada al modo de producción o a la técnica artesanal representada por el campesinado alemán, versus una visión industrializada de la técnica y de la naturaleza, propia de la modernidad. Escribe Heidegger en La pregunta por la técnica: AUFKLÄRUNG,JoãoPessoa,v.7,n.2,Mai.,Ago.,2020,p.6778 El desocultar que domina a la técnica moderna tiene el carácter del poner en el sentido de la provocación. Esta acontece de tal manera que se descubren las energías ocultas en la naturaleza; lo descubierto es transformado; lo transformado acumulado; lo acumulado, a su vez, repartido y lo repartido, se renueva cambiado. Descubrir, transformar, acumular, repartir, cambiar, son modos del desocultar (HEIDEGGER, 1984, p. 83). Esta prerrogativa del Gestell de poner el sentido de la provocación, o dicho más coloquialmente, de impulsarnos bajo un estricto régimen de control a la utilización, valga la redundancia, instrumental de la técnica, constituye precisamente la amenaza absoluta de la técnica moderna. 3.EL GE-STELL COMO DISPOSITIVO DE CONTROL TECNOLÓGICO En La pregunta por la técnica, Heidegger dirá que el Gestell no solo es una provocación. En la segunda parte del texto se muestra un poco menos ambiguo y señala con claridad que también se trata, y esencialmente, de una amenaza: “Emplazado entre estas posibilidades, el hombre, desde el destino, está en peligro. El destino del desocultamiento es en cuanto tal y en todos sus modos, y por eso necesariamente, peligro” (HEIDEGGER, 1984, p. 95). Ahora ¿en qué términos?, es una cuestión que ha sido muy discutida y que seguramente no está del todo zanjada. Feenberg parece ser parte de los comentaristas de Heidegger que ven en el Gestell una amenaza, por así decir, del peor tipo, que pone en embargo la totalidad de la autonomía del hombre respecto de la tecnología. Para el filósofo canadiense, se trataría de resistir a los sistemas tecnológicos que imponen el gerenciamiento técnico sobre los seres humanos: El punto de vista estratégico privilegia las consideraciones relativas al control y la eficiencia, y busca saliencias (affordances), precisamente lo que Heidegger le critica a la tecnología. Mi queja más básica con respecto a Heidegger es que él mismo adopta irreflexivamente el punto de vista estratégico sobre la tecnología para condenarla. La ve exclusivamente como un sistema de control y pasa por alto su papel en las vidas de quienes están subordinados a ella (FEENBERG, 2005, p. 117). Esta idea del Gestell como dispositivo de control tecnológico, a su vez, ha dado pie para ver en el segundo Heidegger lo que se ha llamado la tesis de la tecnología autónoma. En efecto, y Peck lo expone con toda lucidez en su texto Das Gestell and Human Autonomy: On Andrew Feenberg's Interpretation of Martin Heidegger (2015), la principal recusación respecto de este súper dispositivo proviene del argumento que considera que la autonomía de la tecnología elimina, o si se quiere, destruye la autonomía humana. Esta versión, por llamarla de algún modo, catastrófica del Gestell, sugiere que este dispositivo no solo podría ocultar la esencia humana, sino que, además, Janobifronte:propiedadeimpropiedaddelDaseinfrentealatransformaciónontológica... llevarnos a la ilusión de suponer que nos hemos convertido, como dice el propio Heidegger, en “señores de la tierra” (PECK, 2015, p. 1011). 4.CONVERTIBILIDAD ONTOLÓGICA Y PROPIEDAD Heidegger cerrará La pregunta por la técnica desde el más absoluto misticismo: “Cuanto más nos acerquemos al peligro, tanto más claramente comienza a destellar el camino hacia lo salvador, tanto más preguntadores llegamos a ser. Pues el preguntar es la devoción del pensar” (HEIDEGGER, 1984, p. 107). El pasaje, además de poner en liza el concepto de claro (Lichtung) como nota esencial respecto de la noción de salvación, confirma, más que la ambigüedad, la convertibilidad del Gestell. Como indicábamos, Malabou parece avizorar en la propia estructura del Dasein la posibilidad de comprender al Gestell literalmente como transformador: La esencia, das Wesen, es de hecho el centro neurálgico del pensamiento heideggeriano del Gestell. Todo lo que acabo de recordar sobre el tema del sujeto mediano del Gestell, la cabeza de Jano entre dos maneras de cambiar, ahora puede ser devuelto y reducido a su origen: el metabolismo de lo esencial (MALABOU, 2011, p. 162). La esencia, das Wesen, es de hecho el centro neurálgico del pensamiento heideggeriano del Gestell. Todo lo que acabo de recordar sobre el tema del sujeto mediano del Gestell, la cabeza de Jano entre dos maneras de cambiar, ahora puede ser devuelto y reducido a su origen: el metabolismo de lo esencial (MALABOU, 2011, p. 162). La noción del Gestell como destino (Geschick) remite, pues, a la posibilidad de que el Dasein se encuentre con su fundamento (Grund), paradójicamente, en el propio centro del dispositivo: “Más bien, precisamente la esencia de la técnica tiene que albergar en sí el crecimiento de lo salvador” (HEIDEGGER, 1984, p. 98). Es el mismo Gestell el que le permite al Dasein de una manera u otra “intuir la más elevada dignidad de su esencia e ingresar a ella” (HEIDEGGER, 1984, p. 102). Lo que se aclara entonces (más allá de considerar que Heidegger roza acá el camino de la teología) es que el propio Dasein tiene la posibilidad de retornar a su esencia y de reencontrarse con esta idea casi sublime del destino del ser. De lo que se trata, dice Heidegger, es de que el Dasein recupere su morada. 3.EL GE-STELL COMO DISPOSITIVO DE CONTROL TECNOLÓGICO Como afirma Luna, yendo todavía más lejos, el Gestell es un sistema transversal en el sentido de la hipervinculación que establece con las cosas: Ahora bien, el dispositivo no es, como quiere Heidegger, una cierta disposición de los entes y ni siquiera del ser humano. El dispositivo lo que hace es situar a la máquina en disposición de hacer cosas. El dispositivo es un sistema que está preparado para generar determinadas respuestas ante determinados inputs. Esa relación entre entradas y salidas halla su fundamento en la disposición (LUNA, 2013, p. 60). En síntesis, el Gestell es esencialmente el peligro de la técnica, desplegado en su ensamblaje de los entes respecto del uso que de ellos hace el hombre, incluyendo esta relación depredadora con la naturaleza. El hombre es parte del despliegue del Ge stell, está peligrosamente atrapado en él. Dice Forment: “En la época técnica actual, el hombre se ve obligado a tomar una serie de resoluciones, pero por su visión unilateral está impreparado para ello” (FORMENT, 1990, p. 50). Esta tematización del Gestell como el peligro de la técnica moderna, ha requerido que Heidegger haya previsto esta posibilidad a partir de la propia fenomenología desarrollada en ST. Según observa LeopoldoTilleríaAqueveque Boutot, Heidegger aborda fenomenológicamente la técnica poniéndola en perspectiva en el marco de la historia del ser: “Más precisamente, intenta preparar, actualizando el peligro inherente a la esencia de la técnica, el apresamiento de lo que desde siempre pide ser pensado, es decir, el ser mismo” (BOUTOT, 1991, p. 86). De modo que mediante la misma fenomenología Heidegger intentará romper la ambigüedad y confusión con que hasta ahora ha presentado el significado del Gestell. Es en el propio centro del Gestell donde nuestro filósofo verá el contrapeso del peligro que representa como imposición. Tal contrapeso está dado por la posibilidad de pensar el Gestell como lo salvador (Das Rettende): “Así pues, lo esente de la técnica alberga en sí lo que nosotros menos presumiríamos, el posible surgimiento de lo salvador” (HEIDEGGER, 1984, p. 103). En sintonía con el planteamiento inicial de Heidegger, esta posibilidad de ver en el Gestell la idea de salvación no tendrá que ver con la técnica, sino primordialmente con el modo que tenemos de comprenderla, vale decir, con su ontología. Este modo de ser de la técnica, bloqueada la idea de Gestell como peligro, es la ποἰησις. 72 4.CONVERTIBILIDAD ONTOLÓGICA Y PROPIEDAD Ahora, todo este argumento revelador del Gestell, comprendido ahora como destinación otorgante de la salvación, está en nuestras manos, en nuestra decisión de resguardar esta posibilidad y de que empecemos a prestar atención a la esencia de la técnica: “Todo estriba en que meditemos el surgimiento y lo custodiemos conmemoradoramente” (HEIDEGGER, 1984, p. 103). La pregunta es cómo el Dasein emprende tamaña tarea. La mirada debe dirigirse hacia la analítica existenciaria de ST, sobre todo porque nuestra conjetura inicial considera que la filosofía de la técnica de Heidegger es una sola unidad y que su fase preliminar se halla justamente en la fenomenología del Dasein. Es este, por tanto, quien debe jugarse la Janobifronte:propiedadeimpropiedaddelDaseinfrentealatransformaciónontológica... posibilidad de transformación de la esencia del Gestell. En todo caso, es evidente que Heidegger no ha logrado mediante su analítica existenciaria especificar los pasos concretos para el acceso ontológico del Dasein. Tal como observa Giannina Burlando, enfatizando la crítica de Stein a la posición ontológica unidimensional que Heidegger adopta respecto del símismo del Dasein, “no tenemos noticias de que el Ser se revele a sí mismo a otros Daseins o a una comunidad intersubjetiva de Daseins, el Ser mismo del cual el Dasein es el custodio” (BURLANDO, 2015, p. 379). De esta forma, y como subraya la filósofa chilena, la fenomenología temprana de Heidegger no le permite aclarar objetiva y suficientemente el sentido del ser, principalmente porque “la comprensión [del ser del Dasein] no es conceptual, sino una experiencia preconceptual de la realidad” (BURLANDO, 2015, p. 381). 73 ( p ) Para que sea posible que el Dasein recobre su lugar en la morada del ser, el destino de esta recuperación debe ser necesariamente un movimiento ontológico. Así pues: “En este modo de expresarse [«el habitar del Dasein en la luz del Ser»], Heidegger parece implicar que está dando primacía al Ser, y subordinando el papel del hombre al del Ser. Esto se hace notar cuando Heidegger analiza la estructura del Dasein como eksistencia” (BURLANDO, 2015, p. 375). Ahora, tal modo de comprensión del Gestell debe ser, a su vez, distinto a como se ha presentado en la primera parte de La pregunta por la técnica, vale decir, como “la forma en la cual el ser se desoculta al hombre contemporáneo” (CADAHIA, 2012, p. 172). Esta segunda versión, por tanto, deberá entenderse como posibilidad de una ontología del cambio. 4.CONVERTIBILIDAD ONTOLÓGICA Y PROPIEDAD En otras palabras: su condición peyorativa como dispositivo ontológico, debe convertirse en una disposición positiva, en una destinación de la posibilidad de una nueva ontología. Tal es, grosso modo, la tesis de la identidad metabólica del ser de Catherine Malabou. Dice la filósofa francesa: AUFKLÄRUNG,JoãoPessoa,v.7,n.2,Mai.,Ago.,2020,p.6778 Como un mutante ontológico, el Gestell revela la mutabilidad original de la ontología. El Gestell es la revelación del punto donde el ser, la esencia y los seres son susceptibles de perder (o ceder) su posición. El ser, la esencia y los seres “pierden esas cualidades con las que la metafísica los ha dotado” (MALABOU, 2011, p. 169). Como un mutante ontológico, el Gestell revela la mutabilidad original de la ontología. El Gestell es la revelación del punto donde el ser, la esencia y los seres son susceptibles de perder (o ceder) su posición. El ser, la esencia y los seres “pierden esas cualidades con las que la metafísica los ha dotado” (MALABOU, 2011, p. 169). El que el Gestell pueda ser comprendido, por un lado, como una suerte de estructura ontoteológica de la tecnología, y por otro, “como una formación extrema del ser” (MALABOU, 2011, p. 25), hace completamente plausible esta tesis de la mutación ontológica. En todo caso la postura de Malabou, por muy arriesgada que parezca, responde casi literalmente a las expresiones de Heidegger en su comentario a la sentencia de Anaximandro de 1941: El tránsito es cada vez la irrupción de la presencia en la que esencian ante todo el provenir y el escapar. Así, el tránsito contiene en sí aquel Mismo de donde proviene el provenir y a donde va la desaparición, esenciando; más aún: el tránsito es la pura proveniencia de eso Mismo. Este Mismo es el ser mismo (HEIDEGGER, 1997, p. 168). Dicha convertibilidad ontológica del Gestell, parece tener su correlato en la propia estructura trascendental del Dasein a partir de sus dos modos de realización: la propiedad (Eigentlichkeit) y la impropiedad (Uneigentlichkeit). Esta proyección del Dasein en torno a la tarea de repensar el Gestell como claridad ontológica, implica necesariamente incluir esta posibilidad en su propio poder ser, es decir, como efectividad (Faktizität) de la existencia. 4.CONVERTIBILIDAD ONTOLÓGICA Y PROPIEDAD En el parágrafo 64 de ST Heidegger dice lo siguiente: “La orientación fenoménica por el sentido del ser del podersersímismo propio permite establecer el derecho ontológico que se le puede asignar a la LeopoldoTilleríaAqueveque sustancialidad, simplicidad y personalidad como caracteres de la mismidad” (HEIDEGGER, 2002, p. 340). ¿A dónde apunta con esta aclaración? Según creemos, este pasaje desarrolla la relación entre cuidado (Sorge) y mismidad (Selbigkeit) en un capítulo que intenta explicar justamente el sentido ontológico del cuidado a partir de la temporeidad (Zeitlichkeit) del poderestarentero propio. En primer lugar, y sin entrar en detalles engorrosos, digamos que en la analítica existenciaria de ST se considera la impropiedad como el modo de ser cotidiano del Dasein en su estarenelmundo. La cotidianidad (Alltäglichkeit) representa el modo de ser inmediato y regular del estaren mediode del Dasein entre las cosas del mundo. Esto no debiera sorprendernos, pues como se planteó al inicio, el Dasein no es nunca el mero ente, sino el ente que es ahí siempre, arrojado entremedio del mundo. Ahora bien, el cuidado es aquella preocupación que el propio Dasein tiene respecto de sí, en términos de la existencialidad y de la facticidad, o sea, estando siempre enmediode. Sin embargo, el cuidado ya presupone en el Dasein el modo caído de la cotidianidad. Es decir, “que el Dasein ya se ha cuidado, en su ser, desde la cotidianidad” (GARRIDO, 2019, p. 171). De lo que se trata entonces es de si el Dasein, determinado por este modo de preocupación que es el cuidado, por un lado, se deja arrastrar “impropiamente” por la medianía de lo habitual, vale decir, por una mismidad indiferenciada y mediocre, o, por otro lado, se resuelve “propiamente” a pensar, comprender y actuar de un modo diferente a su comportamiento cotidiano guiado por el unomismo (Mansselbst). Como subraya GarridoPeriñán: “El sentimiento de que ‘algo no cuadra’, mueve al Dasein a la no conformación con lo regulado, afectivacomprensivamente, por la estructura denominada el uno” (GARRIDO, 2019, p. 172). 74 5.PROPIEDAD, RESOLUCIÓN De esta manera, la propiedad pareciera ser un estado excepcional en la cotidianidad del Dasein: el modo en que el Dasein comprende que el Gestell amenaza radicalmente su autonomía. Se hace necesario, decíamos, que el Dasein se resuelva a conducirse propiamente como modo de su estarenelmundo. Tal resolución (Entschlossenheit) “no se da lejos del mundo ni aparte de la cotidianidad del uno, sino que se da propiamente desde la solicitud, la ocupación, y la posibilidad de que el Dasein sea conciencia de otros que no lo son” (GÓMEZ, 2016, p. 57). Pues bien, la relación fundamental entre caída y propiedad, o para decirlo bien, con la impropiedad, la especifica Heidegger en el mismo parágrafo 38 de ST: El Dasein está inmediata y regularmente en medio del “mundo” del que se ocupa. Este absorberse en… tiene ordinariamente el carácter de un estar perdido en lo público del uno (…) el Dasein ha desertado siempre de sí mismo en cuanto poder sersímismo propio, y ha caído en el “mundo” (HEIDEGGER, 2002, p. 198). Mas, dejando de estar caído, el Dasein recupera su modo de ser propiamente sí mismo. Sabemos que esto ocurre cuando el Dasein atiende la llamada de la conciencia (Ruf des Gewissens). Tal llamada es el cuidado para dejar de estar caído. Sin embargo, ocurre aquí algo de la mayor trascendencia. Se trata efectivamente de un llamado al Dasein (hecho desde él mismo, por lo demás) a hacerse cargo de su propiedad, y esto, por medio de la resolución. Lo dicho lleva a Gómez a afirmar que: “Es con la resolución que el Dasein sale del uno, que logra tener un dominio de sí mismo y conocerse como lo que es propiamente” (GÓMEZ, 2016, p. 57). Esta podría ser la pieza faltante en el engranaje de la argumentación, que permitiese conectar la posibilidad Janobifronte:propiedadeimpropiedaddelDaseinfrentealatransformaciónontológica... salvífica del Gestell con la estructura existenciaria del Dasein. De esta forma, el Dasein parece encaminarse a custodiar la realidad de esta transformada diferencia ontológica, solo en el modo de ser de la propiedad. Heidegger puntualiza en el parágrafo 60 de ST: “Al mismo tiempo, los proyectos fácticos inmediatos quedan dirigidos por el estarperdido en el uno en medio de las ocupaciones. Este estarperdido puede ser interpelado por el propio Dasein; la interpelación puede ser comprendida en el modo de la resolución” (HEIDEGGER, 2002, p. 315). 5.PROPIEDAD, RESOLUCIÓN Desde este punto de vista, es el conjunto de la analítica del Dasein la que, como quien dice, blinda el modo de ser propio contra una lectura más superficial, que pudiera ver en la impropiedad, por ejemplo, el modo de resolución históricoreligiosa que Heidegger no se habría atrevido a reconocer. En tal sentido, Hoberg afirmará que “sólo ella [la existencia resuelta] aporta la Durchsichtigkeit, la perfecta transparencia consigo mismo [Sein und Zeit 299, 307], mientras que la existencia de la vida ordinaria se establece y se mueve en la no verdad [Sein und Seit, p. 222, 256, 257; Wgr 106]” (citado en LYTHGOE, 2002, p. 264). Convendría aclarar, para no enfrentar objeciones metafísicas de ningún tipo, que la estructura formal misma de la propiedad, tal como lo expone Lythgoe, supone la imposibilidad de mantenerse en la propiedad. Esta observación ratificaría la tesis del acceso ontológico de Burlando: 75 La propiedad establece una vía de acceso al sentido del ser del Dasein, en la que lo fundamental no es el contenido de la situación abierta, su qué, sino la relación con esta certeza, su cómo. Dado que el proceso de acceso es más importante que la ‘verdad alcanzada’, el Dasein “…no debe obstinarse en la situación, sino que debe comprender que, por su propio sentido aperiente, el acto resolutorio tiene que mantenerse libre y abierto para la correspondiente posibilidad fáctica. La certeza del acto resolutorio significa: mantenerse libre para su posible y acaso fácticamente necesaria revocación” (LYTHGOE, 2002, p. 266). Esta autonomía que el Dasein salvaguarda de la impropiedad, de la cotidianidad, o si se quiere, de la inauntenticidad (Uneigentlichkeit), constituiría algo así como el impulso de una resolución orientada a contrarrestar las determinaciones ontológicas y prácticoestéticas del Gestell. Es lo mismo que parece pensar Peck: “Alternativamente, auténticas concepciones del símismo presumiblemente aumentarían la autonomía al revelar formas prácticas en las que podemos aumentar nuestra autonomía auténticamente” (PECK, 2015, p. 21). Por cierto, toda resolución del Dasein se realiza con una finalidad práctica determinada, o como dice Moreno, frente a un para qué concreto: “Ya se trate de San Pablo, Hitler o Calvino, o se trate de un ser simple y anónimo, la resolución acontece frente a algo, siempre ante una posibilidad de ser y otras varias de no ser” (MORENO, 2002, p. 226). El Dasein, siendo propiamente sí mismo, parece destinado a contrarrestar y superar la amenaza del Gestell. 5.PROPIEDAD, RESOLUCIÓN La propiedad del Dasein es, pues, una cuestión ontológica, que lo sitúa no solo ante el desafío, sino ante la exigencia de su más auténtica posibilidad de poder ser: Precisamente, en virtud de la homologación que parece existir entre verdad D i l d i l f ó d l d d á i i i l Precisamente, en virtud de la homologación que parece existir entre verdad Dasein, el modo propio, o sea el fenómeno de la verdad más originario, es la “verdad de la existencia” [Wahrheit der Existenz], entendida como acción proyectivoejecutiva que, “desde el modo de la propiedad” [im Modus der Eigentlichkeit], coloca al Dasein ante su más genuino poder ser (SuZ,p. 221) (GARRIDO, 2020, p. 221). En definitiva, resolución y propiedad son concomitantes a esta posibilidad de la mutabilidad ontológica del Gestell. Al respecto, recojo por su lucidez las palabras de Dastur: “Lo que atestigua la resolución es que la ‘autenticidad’ del Dasein no es una LeopoldoTilleríaAqueveque noción vacía ni una idea completamente inventada (SZ, p. 301). El Dasein resuelto alcanza efectivamente la transparencia propia de sí mismo (SZ, p. 299)” (DASTUR, 2006, p. 71). Después de todo, y como lo ha sugerido Chiodi, la Kehre del pensamiento heideggeriano consiste en el hecho de que la exigencia metafísica predomina sobre la exigencia existencialista (VATTIMO, 2006, p. 139). noción vacía ni una idea completamente inventada (SZ, p. 301). El Dasein resuelto alcanza efectivamente la transparencia propia de sí mismo (SZ, p. 299)” (DASTUR, 2006, p. 71). Después de todo, y como lo ha sugerido Chiodi, la Kehre del pensamiento heideggeriano consiste en el hecho de que la exigencia metafísica predomina sobre la exigencia existencialista (VATTIMO, 2006, p. 139). 76 AUFKLÄRUNG,JoãoPessoa,v.7,n.2,Mai.,Ago.,2020,p.6778 0.3 Como Jano bifronte –parafraseando a Malabou, el Dasein heideggeriano se encuentra permanentemente en la encrucijada de elegir mantenerse en el modo de la impropiedad y, por lo mismo, perder su autonomía ante la omnipotencia tecnológica del Gestell, o decidirse por una existencia desde la propiedad y proyectar su ser a partir de la posibilidad de la intercambiabilidad ontológica del Gestell, resguardando así su autonomía de la dictadura metafísica del dispositivo. 0.4 El camino del Dasein en torno a tomar posición ante la mutabilidad ontológica del Gestell, no puede darse por predefinido y ni siquiera por conocido. Por lo demás, el Dasein mismo es pura posibilidad, aun en su trascendencia como fundamento. Más bien deberíamos guardar silencio ante este encaminarse ontológico, pues, como reflexiona Barone: “Lo nulo, la nada –que es la verdad del ser debería por consiguiente ser entendido, percibido, y al mismo tiempo continuar no siendo identificado; debería aparecer en todos los fenómenos y al mismo tiempo permanecer, por así decir, invisible” (BARONE, 1999, p. 39). 0.4 El camino del Dasein en torno a tomar posición ante la mutabilidad ontológica del Gestell, no puede darse por predefinido y ni siquiera por conocido. Por lo demás, el Dasein mismo es pura posibilidad, aun en su trascendencia como fundamento. Más bien deberíamos guardar silencio ante este encaminarse ontológico, pues, como reflexiona Barone: “Lo nulo, la nada –que es la verdad del ser debería por consiguiente ser entendido, percibido, y al mismo tiempo continuar no siendo identificado; debería aparecer en todos los fenómenos y al mismo tiempo permanecer, por así decir, invisible” (BARONE, 1999, p. 39). 6.CONCLUSIÓN 0.1 Ante la irresolución como posibilidad permanente del Dasein, este se adelanta por medio de la adopción del modo propio de la resolución. Mediante esta forma, en el más propio poderser, el Dasein es capaz de hacer frente a la amenaza del Gestell de convertirse en el marco ontológico dominante en la era tecnológica. Por medio de este resolverse, el Dasein queda en la disposición de su poderser más propio. Lo que abre tal resolución es la posibilidad del Dasein de enfrentar las determinaciones 0.1 Ante la irresolución como posibilidad permanente del Dasein, este se adelanta por medio de la adopción del modo propio de la resolución. Mediante esta forma, en el más propio poderser, el Dasein es capaz de hacer frente a la amenaza del Gestell de convertirse en el marco ontológico dominante en la era tecnológica. Por medio de este resolverse, el Dasein queda en la disposición de su poderser más propio. Lo que abre tal resolución es la posibilidad del Dasein de enfrentar las determinaciones del Gestell y tal como indica Peck experimentar la ruptura momentánea de su reinado q p Gestell y, tal como indica Peck, experimentar la ruptura momentánea de su reinad 0.2 No es posible interpretar al Dasein simplemente y sin más como el mero ser humano que deambula por entre las cosas del mundo, sino como su radical posibilidad de no llegar a estar nunca en posesión de su ser. Es decir, como pura posibilidad proyectivoejecutiva siempre ya fácticamente dispuesta (GARRIDO, 2020). Paradójicamente, es esta misma finitud de su posibilidad la que hace posible que el Dasein se resuelva a contragolpear al Gestell y se plantee la tarea de desocultar su fondo ontológico. ARAUJO, C. Un problema posmoderno en la tecnología: limitar los excesos de la autonomía. Scientia Helmantica, Salamanca, n. 1, p. 167192, 2013. Disponible en: <http://revistascientiahelmantica.usal.es/docs/Vol.01/09.Unproblemaposmodernoen latecnolog%C3%ADa.pdf>. Acceso en: 4 de enero de 2020. ACEVEDO J. [Jorge Quezada]. (2012, Junio 28). Conferencia de Jorge Acevedo sobre la esencia de la técnica moderna en Heidegger [Archivo de video]. Disponible en: <https://www.youtube.com/watch?v=rIfZoCy2Djs&t=49s>. Acceso en: 12 de febrero de 2020. 7.REFERENCIAS ACEVEDO J. [Jorge Quezada]. (2012, Junio 28). Conferencia de Jorge Acevedo sobre la esencia de la técnica moderna en Heidegger [Archivo de video]. Disponible en: <https://www.youtube.com/watch?v=rIfZoCy2Djs&t=49s>. Acceso en: 12 de febrero de 2020. ARAUJO, C. Un problema posmoderno en la tecnología: limitar los excesos de la autonomía. Scientia Helmantica, Salamanca, n. 1, p. 167192, 2013. Disponible en: <http://revistascientiahelmantica.usal.es/docs/Vol.01/09.Unproblemaposmodernoen latecnolog%C3%ADa.pdf>. Acceso en: 4 de enero de 2020. BARONE, P. (1999). Presente y utopía. Notas sobre Heidegger y Celan. En VATTIMO, G. (comp.). Filosofía y poesía: dos aproximaciones a la verdad. Barcelona: Gedisa, 1999, p. 3764. Janobifronte:propiedadeimpropiedaddelDaseinfrentealatransformaciónontológica... BOUTOT, A. Heidegger. México D.F.: Cruz O, 1991. 88 p. BURLANDO, G. Un análisis interno del dasein ontológico de Heidegger: críticas externas de Edith Stein. Mirabilia, Brasil, n. 20, p. 364382, 2015. Disponible en: <https://dialnet.unirioja.es/servlet/articulo?codigo=5180531>. Acceso en: 17 de enero de 2020. 77 CADAHIA, L. El dispositivo de la crisis como nuevo Orden Mundial. En: CADAHIA, L. y VELASCO, G. (comps.). Normalidad de la crisis/crisis de la normalidad. MadridB. Aires: Katz, 2012, p. 171188. , , p DASTUR, F. Heidegger y la cuestión del tiempo. B. Aires: Ed. del Signo, 2006. 132 p FEENBERG, A. Teoría crítica de la tecnología. Revista Iberoamericana de Ciencia, Tecnología y Sociedad, B. Aires, v. 2, n. 5, p. 109123, 2005. Disponible en: <http://www.sfu.ca/~andrewf/theoriacritica.htm>. Acceso en: 11 de febrero de 2020. p FORMENT, E. La civilización tecnológica como problema en Heidegger. Espíritu, Barcelona, a. 39, n. 101102, p. 4158, 1990. Disponible en: <http://www.revistaespiritu.org/wp content/uploads/2018/02/ESP101102Arti%CC%81culo.FormentGiralt.pdf>. Acceso en: 19 de enero de 2020. GARRIDO, J. Vinculabilidad entre cuidado y mismidad en los §§. 3942 de Ser y Tiempo: Heidegger y la mismidad del Dasein. Alpha, Osorno, n. 49, p. 159175, 2019. 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https://openalex.org/W2943688048	https://www.frontiersin.org/articles/10.3389/fncel.2019.00175/pdf	English	null	Lipids at the Crossroad of α-Synuclein Function and Dysfunction: Biological and Pathological Implications	Frontiers in cellular neuroscience	2,019	cc-by	17,796	Abbreviations: AA, arachidonic acid; Chol, cholesterol; DHA, docosahexaenoic acid; FFA, free fatty acids; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LB, Lewy bodies; LD, lipid droplet; PA, phosphatidic acid; PC, phosphatidylcholine; PD, Parkinson’s disease; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipid; PLA2, phospholipases A2; PLC, phospholipase C; PLD, phospholipase D; PS, phosphatidylserine; PUFA, polyunsaturated fatty acids; SN, substantia nigra pars compacta; TAGs, triacylglycerols. REVIEW published: 01 May 2019 doi: 10.3389/fncel.2019.00175 Natalia P. Alza1,2, Pablo A. Iglesias González1†, Melisa A. Conde1,3†, Romina M. Uranga1,3 and Gabriela A. Salvador1,3* 1 Instituto de Investigaciones Bioquímicas de Bahía Blanca, Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas, Universidad Nacional del Sur, Bahía Blanca, Argentina, 2 Departamento de Química, Universidad Nacional del Sur, Bahía Blanca, Argentina, 3 Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Argentina Since its discovery, the study of the biological role of α-synuclein and its pathological implications has been the subject of increasing interest. The propensity to adopt different conformational states governing its aggregation and ﬁbrillation makes this small 14-kDa cytosolic protein one of the main etiologic factors associated with degenerative disorders known as synucleinopathies. The structure, function, and toxicity of α-synuclein and the possibility of different therapeutic approaches to target the protein have been extensively investigated and reviewed. One intriguing characteristic of α-synuclein is the different ways in which it interacts with lipids. Though in-depth studies have been carried out in this ﬁeld, the information they have produced is puzzling and the precise role of lipids in α-synuclein biology and pathology and vice versa is still largely unknown. Here we provide an overview and discussion of the main ﬁndings relating to α-synuclein/lipid interaction and its involvement in the modulation of lipid metabolism and signaling. Keywords: α–synuclein, lipids, lipid metabolism, lipid signal transduction, membrane lipids INTRODUCTION α-Synuclein is a cytosolic protein of 140 amino acids which was discovered in 1988 together with β- and γ-synucleins by Maroteaux et al. (1988). The name α-synuclein derives from the fact that it was originally described as being located in presynaptic endings and also as being associated with nuclear envelopes. In the central nervous system, α-synuclein is abundantly expressed in neurons of diﬀerent brain areas such as the neocortex, hippocampus, SN, thalamus, and cerebellum (Iwai et al., 1995; Asi et al., 2014). α-Synuclein is also present in the peripheral nervous system, muscle, liver, heart, lungs, kidney, hematopoietic cells of the bone marrow, and circulating blood cells Received: 21 December 2018 Accepted: 11 April 2019 Published: 01 May 2019 Edited by: Lavinia Alberi, SICHH, Switzerland Reviewed by: Luigi Bubacco, University of Padova, Italy Natalia Ninkina, Cardiff University, United Kingdom Jean-Christophe Rochet, Purdue University, United States Edited by: Lavinia Alberi, SICHH, Switzerland Reviewed by: Luigi Bubacco, University of Padova, Italy Natalia Ninkina, Cardiff University, United Kingdom Jean-Christophe Rochet, Purdue University, United States Correspondence: Gabriela A. Salvador salvador@inibibb-conicet.gob.ar; salvador@criba.edu.ar Correspondence: Gabriela A. Salvador salvador@inibibb-conicet.gob.ar; salvador@criba.edu.ar *Correspondence: Gabriela A. Salvador salvador@inibibb-conicet.gob.ar; salvador@criba.edu.ar †These authors have contributed equally to this work †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience Citation: Alza NP, Iglesias González PA, Conde MA, Uranga RM and Salvador GA (2019) Lipids at the Crossroad of α-Synuclein Function and Dysfunction: Biological and Pathological Implications. Front. Cell. Neurosci. 13:175. doi: 10.3389/fncel.2019.00175 May 2019 \| Volume 13 \| Article 175 1 Frontiers in Cellular Neuroscience \| www.frontiersin.org α Synuclein and Lipids: Biological Intersections Alza et al. (Gardai et al., 2013; Burré et al., 2018; Mohamed Badawy et al., 2018). β-Synuclein is also located at presynaptic terminals in the central nervous system (Jakes et al., 1994; Wilhelm et al., 2014) while γ-synuclein is primarily expressed in the peripheral nervous system, and the ocular and adipose tissues (Buchman et al., 1998; Surguchov et al., 2001). It has been reported that γ-synuclein is overexpressed in diﬀerent human tumors (Bruening et al., 2000; Guo et al., 2007; Hibi et al., 2009). forms are considered the most toxic species, disrupting cellular homeostasis and triggering neuronal death. Moreover, α-synuclein can exert a deleterious eﬀect by spreading from cell to cell and thus contributing to progressive neurodegeneration (Desplats et al., 2009). Identiﬁcation of the factors promoting the toxic conversion of mutated or wild-type forms of α-synuclein is a topic of intense interest. Deeper insight into the physiological function and pathological features of α-synuclein would not only contribute toward a better understanding of the pathogenesis but also help to develop biomarkers for early disease detection and progression and to design speciﬁc disease-modifying therapies for synucleinopathies. Though numerous studies have been carried out on the biology of α-synuclein, its physiological function remains a matter of debate. The search for a fuller understanding of this function is driven by the fact that it has been linked with several devastating diseases known as synucleinopathies, including neurodegenerative disorders such as: PD, Lewy body dementia, neurodegeneration with brain iron accumulation, Krabbe disease, dementia with LB, diﬀuse Lewy body disease, Lewy body variant of Alzheimer’s disease, among others (Burré, 2015; Burré et al., 2018). y p The α-synuclein protein is composed of three well-described regions that confer the biological and functional characteristics that implicate it in PD pathology. The N-terminal domain comprises residues 1–60 and bestows lipid binding properties on the protein, in particular, the ability to interact with membranes and lipid micelles. Citation: This distinctive feature of α-synuclein is explained by the presence of amphipathic repeats of 11 amino acids, predominantly with the highly conserved KTKEGV motif, similar to that present in apolipoprotein domains (Bussell and Eliezer, 2003). This structural characteristic is shared with β- and γ-synucleins (Clayton and George, 1998). The missense mutations A53T, A30P, H50Q, G51D, A53E, and E46K reside in the N-terminal domain of α-synuclein (Polymeropoulos et al., 1997; Krüger et al., 1998; Zarranz et al., 2004; Flagmeier et al., 2016). The central hydrophobic region (residues 61–95) called NAC (non-amyloid β component) is essential for the conformational change of α-synuclein from random coil to β-sheets, leading to aggregation, and ﬁbrillation (El-Agnaf et al., 1998; Rodriguez et al., 2015). Giasson et al. (2001) demonstrated through diﬀerent assays that amino acids 71–82 represent the speciﬁc sequence of the NAC core which is necessarily involved in α-synuclein ﬁbrillation. Biophysical studies using methods like nuclear magnetic resonance, electron paramagnetic resonance, circular dichroism and transmission electron microscopy,with recombinant proteins of α-synuclein and synthetic fragments of NAC were the ﬁrst and are still used to demonstrate the importance of this region in the ﬁbrillation pathway (El- Agnaf et al., 1998; Giasson et al., 2001; Der-Sarkissiant et al., 2003; Tashiro et al., 2008; Waxman et al., 2009; Shaltiel- Karyo et al., 2010). The elimination of the NAC region or its targeting with antibodies showed to inhibit aggregation and toxicity in cell culture (Lynch et al., 2008; Luk et al., 2009). In vivo models were also used to evaluate the pathological eﬀects of this region. For instance, the deletion of amino acids 71–82 in Drosophila prevents α-synuclein aggregation and neurotoxicity, thus testifying to the importance of this region in the pathogenicity of the protein (Periquet et al., 2007). Diﬀerences in the amino acid sequence of the NAC region have been attributed to be responsible for the non-ﬁbrillation feature of β-synuclein (Uversky and Fink, 2002). The third region of α-synuclein, enriched in glutamate, aspartate, and proline residues, is the acidic C-terminal domain (residues 96–140). It constitutes the main site of post-translational modiﬁcations, such as phosphorylation and nitration, although other modiﬁcations (ubiquitination, glycation, and methionine Parkinson’s disease is the second most prevalent neurodegenerative age-associated disorder after Alzheimer’s disease and is mainly characterized by movement impairments such as resting tremor, bradykinesia, and rigidity. Frontiers in Cellular Neuroscience \| www.frontiersin.org FUNCTIONS OF α-SYNUCLEIN The physiological cellular functions of α-synuclein are still a matter of intense debate, despite continuous eﬀorts over the last 30 years to clarify the issue. Since it is highly concentrated in presynaptic terminals, where it is associated with synaptic vesicles (Maroteaux et al., 1988), the involvement of α-synuclein in neurotransmission, and synaptic plasticity has been extensively investigated (Burré, 2015; Zaltieri et al., 2015). Although there is disagreement as to whether it promotes or inhibits neurotransmitter release, it is well-established that α-synuclein mediates this process by regulating the availability of synaptic vesicles in diﬀerent pools, facilitating their clustering, recycling, and docking to the cell membrane (Chandra et al., 2005; Burré et al., 2010; Zaltieri et al., 2015; Miraglia et al., 2018). The involvement of α-synuclein in synaptic vesicle endocytosis has been demonstrated (Vargas et al., 2014). In addition, α-synuclein can act as a chaperone molecule thus contributing to the assembly of the SNARE complex (Bonini and Giasson, 2005; Diao et al., 2013; Burré et al., 2014). The modulation of dopamine levels by α-synuclein has been demonstrated as a consequence of the inhibition of the neurotransmitter synthesis through the regulation of tyrosine hydroxylase activity (Abeliovich et al., 2000; Peng, 2005) and the inhibition of the vesicular monoamine transporter-2 (Guo et al., 2008). It also has a regulatory eﬀect on the targeting and activity of the dopamine transporter DAT (Swant et al., 2011; Oaks et al., 2013; Butler et al., 2015). From a conformational point of view, under physiological conditions α-synuclein is considered to be an intrinsically unstructured protein. The soluble α-synuclein in the cytosol is natively unfolded (Burré et al., 2013). Conformational ﬂexibility is largely attributed to the α-synuclein structure because it can adopt a range of conformations depending on its interactions with membranes or proteins (Lashuel et al., 2013). When α-synuclein/PL interaction occurs, the N-terminal domain takes on an α-helical conﬁguration that occurs physiologically in a dynamic equilibrium with the soluble state (Eliezer et al., 2001). An α-helical homo-tetramer of the protein has recently been identiﬁed in neurons and other cells (Bartels et al., 2011; Wang et al., 2011; Fanning et al., 2018), occurring in equilibrium with α-synuclein monomers. As the tetramer is resistant to pathological aggregation, the promotion of its formation could be protective against α-synuclein oligomerization (Bartels et al., 2011; Dettmer et al., 2013, 2015a,b, 2017). Citation: Increased levels of the phosphorylated protein at serine 87 have been found in human brains from LB dementia patients (Paleologou et al., 2010) and at serine 129 in LB from PD patients (Fujiwara et al., 2002). Although this latter has been widely investigated, its role on the pathogenesis of PD has not been determined yet. Some authors propose that it is implicated in α-synuclein pathological aggregation (Fujiwara et al., 2002) whereas others suggest that it has a protective role on the aggregation and toxicity (Oueslati et al., 2010; Chen et al., 2019). The protein also suﬀers C-terminal truncation linked to an increase of α-synuclein aggregation in vitro (Murray et al., 2003); the loss of SN neurons in transgenic mice overexpressing this form has been reported (Wakamatsu et al., 2008). Both the N- and C-terminal domains have also been involved in the interaction with proteins and metal ions (Miotto et al., 2014; Carboni and Lingor, 2015; Billings et al., 2016). oxidation) also occur in the N-terminal domain (Chen et al., 2019). These modiﬁcations alter α-synuclein structure and provoke changes in hydrophobicity and protein-protein and protein-lipid interactions (Burré et al., 2018). Oxidation, nitration and phosphorylation are thought to contribute to diﬀerent extents to α-synuclein aggregation and ﬁbrillation (Barrett and Timothy Greenamyre, 2015). Phosphorylation is the most relevant post-translational modiﬁcation related to synucleinopathies. Increased levels of the phosphorylated protein at serine 87 have been found in human brains from LB dementia patients (Paleologou et al., 2010) and at serine 129 in LB from PD patients (Fujiwara et al., 2002). Although this latter has been widely investigated, its role on the pathogenesis of PD has not been determined yet. Some authors propose that it is implicated in α-synuclein pathological aggregation (Fujiwara et al., 2002) whereas others suggest that it has a protective role on the aggregation and toxicity (Oueslati et al., 2010; Chen et al., 2019). The protein also suﬀers C-terminal truncation linked to an increase of α-synuclein aggregation in vitro (Murray et al., 2003); the loss of SN neurons in transgenic mice overexpressing this form has been reported (Wakamatsu et al., 2008). Both the N- and C-terminal domains have also been involved in the interaction with proteins and metal ions (Miotto et al., 2014; Carboni and Lingor, 2015; Billings et al., 2016). FUNCTIONS OF α-SYNUCLEIN Moreover, under pathological conditions, the induction of α-synuclein aggregation results in the formation of an initial population of oligomers enriched in insoluble cross- β-sheets (Conway et al., 2000; Pineda and Burré, 2017). These species may act as nuclei for the next steps of elongation and assembly into ﬁbrils, which are ﬁnally deposited in LB (Wood et al., 1999; Lashuel et al., 2013). Another proposed mechanism for the formation of ﬁbrillar aggregates is the lateral association of the oligomeric granules without nuclei formation (Bhak et al., 2009; Makwana and Sundd, 2016). Oxidative stress, post-translational modiﬁcations, accumulation of α-synuclein and changes in the levels of metal ions, PL, and fatty acids have been proposed as modulators of the aggregation process (Ruipérez et al., 2010; Lashuel et al., 2013; Galvagnion, 2017). α-Synuclein also has roles which are unrelated to synaptic functions. Surguchev and Surguchov have reviewed recent ﬁndings focused on its implication in the regulation of gene expression (Surguchev and Surguchov, 2017). A plethora of other biological functions have been attributed to α-synuclein such as regulation of apoptosis, modulation of glucose and calmodulin levels, and neuronal diﬀerentiation (Emamzadeh, 2016). Even though these latter functions have not been explored in depth, they provide evidence for proposing α-synuclein as a pleiotropic molecule. β- and γ-synucleins have not been linked to the pathogenesis of PD. However, several reports have shown that β-synuclein has an inhibitory eﬀect on the aggregation of α-synuclein (Brown et al., 2016) suggesting a neuroprotective role against synucleinopathies. Otherwise, it has been proposed that γ-synuclein is involved in cancer progression and metastasis (Hibi et al., 2009; Hua et al., 2009; Dunn et al., 2015). Citation: This review focuses on the intriguing characteristic of α-synuclein to pleiotropically interact with and modulate a variety of lipids and on how these interactions participate in the protein’s biological and pathological functions. Citation: The loss of dopaminergic neurons in the SN has been identiﬁed as the cause of the typical motor disablement (Dunnett and Björklund, 1999). Current therapies focus on the reestablishment of the neurotransmitter dopamine for controlling motor symptoms, but speciﬁc treatments are still not available owing mainly to a lack of knowledge of the molecular mechanisms that trigger neuronal degeneration and death (Oertel and Schulz, 2016). Since diagnosis occurs when the loss of dopaminergic neurons is massive, a better understanding of the molecular events involved in the neurodegenerative process would undoubtedly help in the discovery of speciﬁc treatments. Parkinson’s disease is a multifactorial neurodegenerative disorder whose etiopathogenesis is still largely unknown, though a common ﬁnding in patients’ brain is the abnormal accumulation, and aggregation of α-synuclein. Intracellular aggregates of α-synuclein, named LB, constitute the histological hallmark of the PD brain. LB are also the main histopathological ﬁndings in the above-mentioned synucleinopathies. The role of α-synuclein as one of the leading causes of dopaminergic cell death was described after the identiﬁcation of the ﬁrst missense mutations (A30P, E46K, and A53T) in the SNCA gene (Polymeropoulos et al., 1997; Krüger et al., 1998; Zarranz et al., 2004). The involvement of α-synuclein mutants in familial inherited PD has been conﬁrmed by genome-wide association studies (Chang et al., 2017). In addition, SNCA duplication and triplication causing elevated levels of the protein are associated with early-onset PD (Singleton et al., 2003; Chartier-Harlin et al., 2004). Although the α-synuclein protein does not display any mutations in sporadic PD and LB dementia, its involvement in neuronal damage is widely recognized. Of the several mechanisms that could be responsible for the association of α-synuclein with PD pathogenesis, there is an overall consensus that its aggregation leading to the formation of oligomers is a central event related to neuronal dysfunction (Ingelsson, 2016; Bengoa-Vergniory et al., 2017). These α-synuclein oligomeric May 2019 \| Volume 13 \| Article 175 2 α Synuclein and Lipids: Biological Intersections Alza et al. oxidation) also occur in the N-terminal domain (Chen et al., 2019). These modiﬁcations alter α-synuclein structure and provoke changes in hydrophobicity and protein-protein and protein-lipid interactions (Burré et al., 2018). Oxidation, nitration and phosphorylation are thought to contribute to diﬀerent extents to α-synuclein aggregation and ﬁbrillation (Barrett and Timothy Greenamyre, 2015). Phosphorylation is the most relevant post-translational modiﬁcation related to synucleinopathies. α-SYNUCLEIN AND MEMBRANE LIPIDS The nature of the interactions between α-synuclein and PLs has been extensively reviewed in several papers that mainly address this topic through biophysical studies (Dikiy and Eliezer, 2012; Pineda and Burré, 2017; Burré et al., 2018; O’Leary and Lee, 2018). Here we will discuss the most biologically relevant ﬁndings. One of the points included in this section is how speciﬁc PLs participate in the induction of conformational changes of α-synuclein and how this aﬀects its biological and/or pathological functions. The preferred PLs for α-synuclein membrane binding are those with an acidic head group such as PS and PI (Shvadchak et al., 2011). The charged nature of these PLs makes lysine residues candidates for α-synuclein binding sites (Jo et al., 2000; Perrin et al., 2000). The interaction between the N-terminal of α-synuclein and PS seems to be critical for C-terminal SNARE-dependent vesicle docking (Lou et al., 2017). Since SNARE-dependent vesicle docking is necessary for calcium-mediated neurotransmitter release, it is reasonable to assume that α-synuclein/PS interaction is critical to the neurotransmission process at the synapse. Indeed, as previously mentioned, one of the best characterized biological functions of α-synuclein is its participation in vesicle recycling (Scott and Roy, 2012; Wang et al., 2014). Experiments performed in giant unilamellar vesicles composed of diﬀerent proportions of dioleoyl-PC and several anionic PLs demonstrated that wild- type α-synuclein binding is dependent on the presence of negatively charged head groups. Binding to anionic PLs is also dependent of the liquid-ordered state of the vesicles, thus indicating that protein/membrane interactions are governed not only by electrostatic forces but also by lipid packaging and hydrophobic forces (O’Leary and Lee, 2018). The interaction of β- and γ-synucleins with membranes depends on the presence of PL acidic head group and is also determined by the curvature of the binding surface (Ducas and Rhoades, 2012). After adipose tissue, the brain is the organ with the highest lipid content. PL content in the brain is approximately 6% of dry weight and is represented by two main classes of molecules: glycerophospholipids and sphingolipids (Lahiri and Futerman, 2007). A glycerophospholipid molecule consists of two fatty acids, one saturated and one unsaturated, esteriﬁed in sn-1 and sn-2 of the glycerol backbone, respectively. The brain is particularly enriched in two PUFA: arachidonic acid (AA, 20:4- ω-6) and docosahexaenoic acid (DHA, 22:6-ω-3) (Chen et al., 2008). α-SYNUCLEIN AND MEMBRANE LIPIDS Although the native α-synuclein three-dimensional conformation is still under discussion, diﬀerential folding states for physiological and pathological conditions have been assigned. May 2019 \| Volume 13 \| Article 175 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 3 α Synuclein and Lipids: Biological Intersections Alza et al. equilibrium between the cytosol and the membrane fraction (i.e., plasma membrane or vesicles). The occurrence of these two physiological states has generated some controversy, but consensus has now been reached that α-synuclein mainly occurs as an intrinsically disordered monomer (Burré et al., 2013). Very recently, a helically folded tetramer of the soluble protein has been described in equilibrium with the monomer (Bartels et al., 2011; Wang et al., 2011; Fanning et al., 2018). Soluble α-synuclein exists as a random coil three dimensional structure that increases α-helical conformation after interacting with membrane PLs, suggesting that protein/PL interaction triggers structural changes that could modulate biological function (Davidson et al., 1998; Zhu et al., 2003). Furthermore, biophysical studies using tryptophan- and spin-labeled determinations demonstrated that the portion of the protein that interacts with the membrane adopts an α-helix conformation which is entirely buried within the depth of the membrane whereas the rest of the segments present lower membrane penetration and higher ﬂexibility (Bussell, 2005; Wietek et al., 2013). One of the cutting-edge questions is the role of membrane lipids in α-synuclein conformation since protein-membrane binding has been associated with both normal and pathological functions. It has been reported that the α-synuclein/membrane interaction induces diﬀerent α-helix states that could participate in protein function or drive aggregation (Bodner et al., 2010; Dikiy and Eliezer, 2012; Fares et al., 2014; Ysselstein et al., 2015; Burré et al., 2018; O’Leary et al., 2018). At the same time, the targeting of α-synuclein in its diﬀerent conformations at the membrane surface can alter lipid composition, thus promoting pathological eﬀects (van Rooijen et al., 2009; Reynolds et al., 2011; Tosatto et al., 2012; Hellstrand et al., 2013). A signiﬁcant body of experimental evidence reinforces the hypothesis that oligomers, the initial state of protein aggregation, are the most neurotoxic species because of their ability, after interaction, to disrupt biological membranes (Danzer et al., 2007; Winner et al., 2011; Fusco et al., 2017; Galvagnion, 2017). Frontiers in Cellular Neuroscience \| www.frontiersin.org α-SYNUCLEIN AND MEMBRANE LIPIDS PLs are the main constituents of cellular membranes and because of their amphipathic nature, they provide the necessary biophysical environment for ensuring the proper functioning of structural proteins, receptors, enzymes, and ion channels located at the cell surface or in intracellular membranes. The plasma and organelle membranes are asymmetric; this characteristic determines speciﬁc and diﬀerential lipid composition in the inner and the outer leaﬂets. PC, PE, sphingomyelin, and Chol are the most abundant lipids in the outer hemimembranes. PS and PI are acidic PLs and are predominantly located in the inner hemimembranes. This particular lipid composition is determinant for the formation of lipid rafts, speciﬁc membrane liquid-ordered microdomains that govern protein recruitment (van Meer et al., 2008; Marquardt et al., 2015). A fact that argues in favor of the role of α-synuclein/lipid interaction in the etiopathogenesis of synucleinopathies is that all missense mutations responsible for familial PD are localized in the 11-residue repeat domain that has membrane-binding properties; indeed, these mutations alter the lipid binding properties (Jo et al., 2002; Fares et al., 2014; Ghosh et al., 2014; Robotta et al., 2017). A30P and E46K mutants display altered binding to anionic PLs, thus suggesting that membrane interaction could be disrupted in the familial forms of PD (Stöckl et al., 2008). In native conditions, α-synuclein can be mainly found in two states: a soluble unfolded monomeric form or a membrane-associated multimeric form. The pathological state of the protein is composed of predominantly β-sheet oligomers and amyloid ﬁbrils (Burré, 2015). PLs have been reported to participate in and modulate the diﬀerent states of α-synuclein. Originally, α-synuclein was described as a monomer in Binding to PS has been shown to induce an increase in α-synuclein oligomerization, thus providing evidence of the role of protein/PL interaction in the modulation of the pathological May 2019 \| Volume 13 \| Article 175 4 α Synuclein and Lipids: Biological Intersections Alza et al. modiﬁcations; however, it has been shown that post-translational modiﬁcations such as phosphorylated or acetylated forms of the protein would interact in a diﬀerent way (Burré et al., 2018). One of the physiologically relevant post-translational modiﬁcations of α-synuclein is its acetylation in the N-terminal, which enhances binding to PC, a zwitterionic PL presented in micelles and small unilamellar vesicles with high curvature (r∼16–20 nm). Chol, another important membrane constituent, has been shown to reduce N-acetylated α-synuclein-PC binding. α-SYNUCLEIN AND MEMBRANE LIPIDS Cardiolipin-mediated binding of α-synuclein could represent a mechanism by which the cytosolic protein is targeted to mitochondria (Ghio et al., 2016). Once attached to the membrane surface, the protein is also able to extract lipids, which could explain the loss of mitochondrial integrity during dopaminergic neuronal degeneration (Pozo Devoto and Falzone, 2017). Phospholipids also participate in the spreading of α-synuclein, mainly attributed to exosomes, and are thus involved in synucleinopathy propagation. Mass spectrometry studies show the presence of PC, PE, PI, PS, and the gangliosides GM2 and GM3 in exosomes isolated from neuroblastoma cells (Marie et al., 2015). Moreover, experiments carried out on small unilamellar vesicles to ascertain the role of gangliosides in α-synuclein conformation demonstrated that GM1 and GM3, as well as exosomes, are able to accelerate the aggregation of the protein (Marie et al., 2015). Despite the mounting evidence mentioned above, the question of how PL interaction intervenes in α-synuclein physiological and pathological function remains unanswered (Figure 1). In order to shed further light on the speciﬁc role of lipid interactions in α-synuclein biology and pathology and how these interactions occur in vivo, it is necessary to carry out additional experiments using models that mimic living cell lipid composition, membrane asymmetry, and the intracellular environment. The structural changes that α-synuclein undergoes after interacting with PLs in membranes and how these changes contribute to amyloidogenesis is nevertheless still under debate. There is agreement that acidic PLs are the preferred lipids for membrane targeting and that the protein/lipid ratio could be determinant for both biological function and amyloidogenic propensity. Very recently, it has been shown that the conformational state of α-synuclein depends on the protein/lipid ratio. A high α-synuclein/lipid ratio would promote protein/protein interaction and in consequence increase the propensity to form oligomers enriched in β-sheet conformation which in turn could also ﬁbrillate and induce amyloid aggregation (Powers et al., 2017; Terakawa et al., 2018). A low α-synuclein/lipid ratio, on the other hand, would establish a condition of protein dilution, thus blocking amyloid ﬁbrillation, and indeed maintaining physiological interactions with the membrane (Terakawa et al., 2018). Previously mentioned evidence describes the interaction of α-synuclein with membranes in the absence of post-translational α-SYNUCLEIN AND MEMBRANE LIPIDS These in vitro results reported by O’Leary and coworkers suggest that N-acetylation promotes the binding of α-synuclein to highly curved membranes and that Chol interferes with this interaction (O’Leary et al., 2018). Moreover, N-acetylated α-synuclein has been shown to diminish the aggregation propensity triggered by ganglioside GM1 binding (Bartels et al., 2014). Phosphorylated α-synuclein in serine 129 has been studied in depth in the last decade, with disparate data being reported in relation to membrane interaction. However, the most accepted theory to emerge from in vitro and in vivo experiments is that the phosphorylated form has an inhibitory eﬀect on α-synuclein association with the membrane (Fiske et al., 2011; Kuwahara et al., 2012; Nübling et al., 2014; Oueslati, 2016). aggregation of the protein (Hu et al., 2016). After interacting with PLs, the impairing eﬀects of α-synuclein on biological membranes could be explained by the increase in membrane tension and lipid extraction that promote pore formation and membrane lysis (Jo et al., 2000; Zhu et al., 2003; Tosatto et al., 2012; Braun and Sachs, 2015; Pan et al., 2018). In line with this, it has been proposed that the interaction between acidic PLs and α-synuclein plays a role during nuclear extrusion in erythroblast diﬀerentiation (Araki et al., 2018). Phosphatidic acid (PA), another anionic PL, diﬀers from PS, and PI mainly in terms of the small size of its head group; this particular characteristic allows its localization very close to the hydrophobic core of the lipid bilayer. PA is also an α-synuclein partner for membrane targeting (Perrin et al., 2000). Speciﬁcally, PA esteriﬁed with saturated/monounsaturated fatty acids is the preferred molecular species for α-synuclein binding and also induces an enhancement in protein aggregation through the induction of changes in the secondary structure (Mizuno et al., 2017). It has been reported that α-synuclein overexpression is also able to modulate enzyme pathways that produce PA (Dikiy and Eliezer, 2012) (see the section “α-Synuclein and Lipid Signaling”). α-Synuclein has also been shown to be associated with mitochondrial membranes (Ghio et al., 2016). Binding of α-synuclein to mitochondrial membranes has been detected in SN dopaminergic neurons from mice and human brain (Li et al., 2007; Devi et al., 2008). The nature of protein association with mitochondria was determined by using liquid-disordered membranes enriched in cardiolipin, an essential PL very abundant in the inner mitochondrial membrane and also essential for organelle function. Frontiers in Cellular Neuroscience \| www.frontiersin.org α-SYNUCLEIN AND LIPID DROPLETS Neutral lipids are part of a large, heterogeneous family of compounds with the distinctive characteristic of being hydrophobic molecules. Another of their particular features is the lack of charged groups. The two main classes of neutral lipids in mammals are TAGs and Chol esters, both of which reside in the cytosol, contained in a simple membrane-surrounded organelle, the LD (Welte, 2015). LD membranes contain PLs, unesteriﬁed Chol, and speciﬁc anchored proteins with signaling and lipolytic functions (Gao et al., 2017; Sztalryd and Brasaemle, 2017); as highly dynamic storage organelles, they provide a rapidly mobilizable TAG pool to act as an energy source or as a fatty acid donor for lipid remodeling processes. May 2019 \| Volume 13 \| Article 175 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 5 α Synuclein and Lipids: Biological Intersections Alza et al. FIGURE 1 \| α-Synuclein/lipid interactions: from the plasma membrane to intracellular compartments and extracellular vesicles. Soluble α-synuclein exists as a random coil three-dimensional structure. Membrane targeting with acidic PLs (PS and PI) triggers α-helical folding at the N-terminal domain. Protein pathological conformation is predominantly represented by β-sheet oligomers and amyloid ﬁbrils. α-Synuclein pathological aggregation is dependent on the protein/lipid ratio. Spreading of α-synuclein, largely attributed to exosomes, is also regulated by its interaction with the PLs of the extracellular vesicles. Free fatty acids, mainly DHA, are able to induce α-synuclein oligomerization. After the interaction with lipid peroxidation products, α-synuclein is prone to the formation of toxic stable oligomers. α-Synuclein also regulates Chol efﬂux via ABCA1 transporter. DHA, docosahexaenoic acid; PA: phosphatidic acid; PI, phosphatidylinositol; PS, phosphatidylserine. FIGURE 1 \| α-Synuclein/lipid interactions: from the plasma membrane to intracellular compartments and extracellular vesicles. Soluble α-synuclein exists as a random coil three-dimensional structure. Membrane targeting with acidic PLs (PS and PI) triggers α-helical folding at the N-terminal domain. Protein pathological conformation is predominantly represented by β-sheet oligomers and amyloid ﬁbrils. α-Synuclein pathological aggregation is dependent on the protein/lipid ratio. Spreading of α-synuclein, largely attributed to exosomes, is also regulated by its interaction with the PLs of the extracellular vesicles. Free fatty acids, mainly DHA, are able to induce α-synuclein oligomerization. After the interaction with lipid peroxidation products, α-synuclein is prone to the formation of toxic stable oligomers. α-Synuclein also regulates Chol efﬂux via ABCA1 transporter. DHA, docosahexaenoic acid; PA: phosphatidic acid; PI, phosphatidylinositol; PS, phosphatidylserine. LDs (Colebc et al., 2002). α-SYNUCLEIN AND LIPID DROPLETS α-Synuclein mutants display a diﬀerent behavior: whereas A53T strongly associates with LDs, A30P remains cytosolic. These ﬁndings were corroborated by the heterologous expression of human α-synuclein in Saccharomyces cerevisiae (Sere et al., 2010). The biological signiﬁcance of the α-synuclein-induced increase in LDs observed in yeasts was addressed in a mutant strain unable to synthesize neutral lipids and more tolerant to α-synuclein overexpression (Sere et al., 2010). In line with these ﬁndings, we recently reported that A53T α-synuclein overexpression in dopaminergic neurons induces an increase in TAG production and LD accumulation (Sánchez Campos et al., 2018). Fe exposure in A53T α-synuclein- overexpressing neurons increases protein aggregation, thus also augmenting TAG and LD content (Sánchez Campos et al., 2018). The nature of the α-synuclein/LD interaction has been shown to depend on PL packing at the LD membrane, being not fully packed monolayers the preferred surface for protein binding (Thiam et al., 2013). LD surface has been described as a higher hydrophobic environment than the PL bilayer (Thiam et al., 2013; Kory et al., 2016). The need for this highly hydrophobic organelle has been reported in other biological processes such as protein viral assembly (Ogawa et al., 2009; Welte and Gould, 2017). Though LDs are present in almost all cell types, their major occurrence is in adipocytes and hepatocytes. They also serve as cellular stress indicators and are associated with pathological states including starvation, infection, cancer, and liver steatosis (Gluchowski et al., 2017; Tirinato et al., 2017). Scarce information on the precise function of LDs on the nervous system is available in the literature. However, recent works correlate the appearance of LDs with pathological situations in neurodegenerative disorders. Glial cells were reported to be LD-positive cells that accumulate neutral lipids in response to the injury of neighbor neurons in several neurodegenerative conditions (Liu et al., 2015; Cabirol-Pol et al., 2018). LDs were detected in Aplysia axons and neuronal primary cultures and neurons of Huntington’s disease models (Savage et al., 1987; Martinez-Vicente et al., 2010; Welte, 2015). In addition, iron accumulation, an event intensively described in several neurodegenerative processes and also in PD, has been shown to increase LD formation in dopaminergic neurons (Salvador, 2010; Schneider and Bhatia, 2012; Sánchez Campos et al., 2015). A large body of evidence points to α-synuclein/LD interaction. Experiments performed in vitro demonstrate that α-synuclein binds to artiﬁcial 60 nm diameter LDs (Thiam et al., 2013). Frontiers in Cellular Neuroscience \| www.frontiersin.org α-SYNUCLEIN AND FATTY ACIDS Another interesting discussion is the role of FFA in α-synuclein biology. Brain membranes exclusively contain a high concentration of PUFA, DHA, and AA being the most abundant. DHA is an essential fatty acid that needs to be incorporated from the diet, and disturbances in its metabolism have been reported to be associated with both neurodevelopment and neurodegenerative disorders (Chen et al., 2008). Some years ago, several in vitro observations proposed that α-synuclein could act as a fatty acid binding protein in the brain (Sharon et al., 2001). Soon afterward, PUFA were shown to promote the assembly of α-synuclein soluble oligomers (Sharon et al., 2003a; Karube et al., 2008). Both DHA and to a lesser extent AA are able to induce α-synuclein oligomerization as demonstrated by transmission electron microscopy, electrophoresis of native gels, and ﬂuorescence assays with thioﬂavin T (Broersen et al., 2006). Relative concentrations of α-synuclein and DHA appear to govern the free and bound states of the protein and also its conformational changes, by increasing its α-helix structure (Sharon et al., 2001; Fecchio et al., 2018). Moreover, prolonged exposure to PUFA triggers the formation of α-synuclein ﬁbrils. The establishment of oligomeric or ﬁbrillar conformations in the presence of DHA depends on the protein/FFA ratio. Whereas ﬁbril formation is favored by a 1:10 molar ratio, oligomeric species characterized by lack of seeding properties for ﬁbrillation are stabilized by a 1:50 ratio (De Franceschi et al., 2011). The general consensus among the broad range of studies on the eﬀect of fatty acids on α-synuclein biology is that PUFA elicit the formation of diverse oligomeric forms with diﬀerential structural and biological properties (Perrin et al., 2001; Fecchio et al., 2013). Oligomeric species generated in vitro by DHA exposure showed a partial α-helical structure, with the capacity to alter the membrane permeability and thus to trigger cytotoxicity (Fecchio et al., 2013). β- and γ-synucleins are also able to interact with PUFA trough their lipid binding domain (Perrin et al., 2001). The reported ﬁndings highlight the connection between oligomeric α-synuclein and fatty acid oxidation in dopaminergic toxicity, suggesting the existence of a positive loop of increased damage when α-synuclein is allowed to interact with PUFA and their oxidation products. One of the most accepted theories for the onset of neurodegenerative diseases associated with aging postulates the increase in oxidative stress as one of the main gating factors. α-SYNUCLEIN AND FATTY ACIDS Since the well-described decrease in antioxidant defenses associated with the aging process increases oxidative stress, it is arguable that lipid peroxides could be available for the interaction with α-synuclein thus triggering the generation of toxic oligomeric forms. Moreover, the extensively documented decrease in brain PUFA content during aging could also be responsible for an increase in saturated PLs at the membranes that in turn could decrease the association with α-synuclein and augment the cytosolic levels of the protein available for binding with lipid peroxides (López et al., 1995; Giusto et al., 2002; Mateos et al., 2010; Ledesma et al., 2012). Studies on oligodendroglial cells overexpressing the A53T mutant have shown that oxidative stress exposure after supplementation with DHA is able to promote the genesis of inclusion bodies whose composition includes phosphorylated α-synuclein (serine 129) and ubiquitinated and SUMOylated proteins, all reported to be aggregation inducers (Riedel et al., 2011). A number of PD animal models have been used to corroborate some of the above-discussed in vitro results. The use of transgenic mice overexpressing diﬀerent α-synuclein forms and subjected to high-DHA diet has served to support some of the earlier ﬁndings. Yakunin et al. (2012) showed increased accumulation of soluble and insoluble α-synuclein forms accompanied by astrocytosis and neuritic defects in an A53T transgenic mouse model fed a DHA-enriched diet. Experiments in vivo with transgenic mice expressing the A53T α-synuclein mutant demonstrated that a diet with high DHA content reduces lipoxidative damage (Muntané et al., 2010). A very recent report demonstrated that a diet supplemented with n-3 PUFA promotes the accumulation of a 42-kDa oligomeric form of the protein (Yakunin et al., 2012; Coulombe et al., 2018). Analysis of post-mortem human brains corroborates some of the previous in vitro and in vivo experimental ﬁndings. Protein proﬁle of synucleinopathy patients was enriched with A rise in free DHA levels in cytosolic fractions has already been found in brains of patients with PD and LB dementia, and this could be responsible for the oligomerization of α-synuclein and the underlying neuronal damage (Sharon et al., 2003b). Biophysical studies combined with transmission electron microscopy demonstrated that DHA in solution is able to form oil droplets. These oil droplets can be remodeled by α-synuclein, reducing their size and fatty acid concentration (Broersen et al., 2006; De Franceschi et al., 2009; Figure 1). α-SYNUCLEIN AND LIPID DROPLETS Moreover, it has been shown that in HeLa cells incubated with a high concentration of fatty acid, wild-type α-synuclein translocates from the cytosol to the surface of The relevance of the biological interaction between α-synuclein/LD has not been explored in vivo, but it can be proposed that LDs are early markers of neurodegenerative May 2019 \| Volume 13 \| Article 175 Frontiers in Cellular Neuroscience \| www.frontiersin.org 6 α Synuclein and Lipids: Biological Intersections Alza et al. products of DHA, mainly through its histidine residue in position 50 (De Franceschi et al., 2017). Moreover, it has been observed that lysine residues, acting as nucleophiles, are able to interact with electrophilic products of lipid peroxidation and that this protective eﬀect is weakened after lysine acetylation. Additionally, α-synuclein binding to liposomes containing PC and PA enriched with PUFA has been reported to have a protective eﬀect against nitration and oxidation (Trostchansky et al., 2006). The interaction of α-synuclein with 4-hydroxy- 2-nonenal, one of the main products of PUFA peroxidation, is able to promote the formation of toxic stable oligomers, and also prevent the passage toward the ﬁnal state of ﬁbrils (Qin et al., 2007; Puspita et al., 2017; Shamoto-Nagai et al., 2018). Furthermore, dopaminergic cells incubated with free PUFA showed decreased viability associated with the presence of oligomeric α-synuclein (Assayag et al., 2007). processes and the characteristic of these vesicles make them good candidates for the modulation of α-synuclein conformational changes and pathological aggregation. Further research is required for a deeper understanding of these phenomena and their implications in synucleinopathies (Figure 1). Frontiers in Cellular Neuroscience \| www.frontiersin.org α-SYNUCLEIN AND FATTY ACIDS In addition, the interaction with DHA droplets promotes diﬀerential susceptibility of proteolytic degradation (De Franceschi et al., 2009). α-Synuclein has been shown not only to form covalent bonds with DHA but also to act as a scavenger of the oxidation May 2019 \| Volume 13 \| Article 175 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 7 α Synuclein and Lipids: Biological Intersections Alza et al. both soluble and insoluble oligomers and a positive correlation between the accumulation of α-synuclein oligomers and cytosolic DHA content was reported (Sharon et al., 2003a,b). both soluble and insoluble oligomers and a positive correlation between the accumulation of α-synuclein oligomers and cytosolic DHA content was reported (Sharon et al., 2003a,b). Several experimental evidences have demonstrated that both the absence and the overexpression of α-synuclein are able to disturb lipid homeostasis by increasing neutral lipid storage. In astrocytes and whole brain, the absence of α-synuclein switches the distribution of fatty acids to neutral lipid reserves and also increases neutral lipid mass (Castagnet et al., 2005; Barceló- Coblijn et al., 2007). Surprisingly, α-synuclein overexpression gives rise to abnormal lipid metabolism in yeasts, characterized by an increase in LD content (Outeiro and Lindquist, 2003; Su et al., 2010). In line with this, overexpression of wild-type and mutant forms of α-synuclein triggers the accumulation of LDs in HeLa cells, and in human-induced pluripotent stem cell (Colebc et al., 2002; Fanning et al., 2018). Regarding these ﬁndings, it can be suggested that physiological levels of α-synuclein are necessary for lipid homeostasis. γ-Synuclein, another member of the synuclein family, has also been implicated in the maintenance of LD formation and in the modulation of lipid composition in adipocytes and the nervous system in mice (Guschina et al., 2011; Voshol et al., 2012). All these ﬁndings postulate DHA in soluble and esteriﬁed forms as an important candidate for modulating both the targeting and the biological function of α-synuclein. Thus, the regulation of DHA availability under pathological conditions could be an important event for regulating α-synuclein oligomerization and aggregation processes. In this sense, lipid pathways involved in DHA acylation, and deacylation could constitute a promising strategy for new therapeutic approaches in synucleinopathies (see section “α-Synuclein and Lipid Signaling”) (Figures 1, 3). α-SYNUCLEIN AND LIPID METABOLISM The clear involvement of a crosstalk between α-synuclein and lipid metabolism was investigated by Murphy’s group using protein knock-out mice (Castagnet et al., 2005; Golovko et al., 2006, 2007, 2009; Barceló-Coblijn et al., 2007; Golovko and Murphy, 2008). SNCA (−/−) mice oppositely metabolize the two main brain PUFA, AA, and DHA. The lower intake of AA reported both in neurons and astrocytes from SNCA (−/−) mice was reverted by exogenous wild-type α-synuclein through the modulation of acyl-CoA synthetase activity (Golovko et al., 2006). However, DHA turnover and acylation in PE, PI, and PS was found to be increased in SNCA (−/−) mice when compared with SNCA (+/+) mice (Golovko et al., 2007). This diﬀerential eﬀect in AA and DHA metabolism as a consequence of the silencing of α-synuclein argues in favor of compensatory mechanisms associated with PUFA content in brain membranes (Figure 2). In harmony with these results, a recent study in our laboratory showed that the overexpression of A53T α-synuclein induces an increase in TAG content and the accumulation of LDs in N27 dopaminergic neurons (Sánchez Campos et al., 2018). The rise in neuronal TAG content was associated with increased fatty acid synthase expression and Acyl-CoA synthetase activity, with no variations in TAG lipase activity or in fatty acid β-oxidation, thus demonstrating that the overexpression of A53T α-synuclein triggers a lipid metabolic switch in dopaminergic neurons. Pharmacological blockage of TAG de novo synthesis renders the neurons more susceptible to iron-induced oxidative stress (Sánchez Campos et al., 2018). α-Synuclein biology and Chol metabolism have been shown to interact in many diﬀerent ways. Such intersections regulate FIGURE 2 \| Crosstalk between α-synuclein and lipid metabolism. Knock-down and overexpression of α-synuclein have demonstrated the involvement of the protein in lipid metabolism. In α-synuclein knock-out mice, AA and DHA, the main polyunsaturated fatty acids in the brain, are oppositely metabolized: whereas AA intake is diminished, DHA turnover and acylation in PI and PS are increased. α-Synuclein overexpression triggers an increase in TAG content and LD accumulation by stimulation of acyl-CoA synthetase activity and probably the modulation of HMG-CoA reductase. AA, arachidonic acid; DHA, docosahexaenoic acid; HMG-CoA, 3-Hydroxy-3-methyl glutaryl coenzyme A; LD, lipid droplet; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglycerol. FIGURE 2 \| Crosstalk between α-synuclein and lipid metabolism. Knock-down and overexpression of α-synuclein have demonstrated the involvement of the protein in lipid metabolism. α-SYNUCLEIN AND LIPID METABOLISM In α-synuclein knock-out mice, AA and DHA, the main polyunsaturated fatty acids in the brain, are oppositely metabolized: whereas AA intake is diminished, DHA turnover and acylation in PI and PS are increased. α-Synuclein overexpression triggers an increase in TAG content and LD accumulation by stimulation of acyl-CoA synthetase activity and probably the modulation of HMG-CoA reductase. AA, arachidonic acid; DHA, docosahexaenoic acid; HMG-CoA, 3-Hydroxy-3-methyl glutaryl coenzyme A; LD, lipid droplet; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglycerol. FIGURE 2 \| Crosstalk between α-synuclein and lipid metabolism. Knock-down and overexpression of α-synuclein have demonstrated the involvement of the protein in lipid metabolism. In α-synuclein knock-out mice, AA and DHA, the main polyunsaturated fatty acids in the brain, are oppositely metabolized: whereas AA intake is diminished, DHA turnover and acylation in PI and PS are increased. α-Synuclein overexpression triggers an increase in TAG content and LD accumulation by stimulation of acyl-CoA synthetase activity and probably the modulation of HMG-CoA reductase. AA, arachidonic acid; DHA, docosahexaenoic acid; HMG-CoA, 3-Hydroxy-3-methyl glutaryl coenzyme A; LD, lipid droplet; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglycerol. May 2019 \| Volume 13 \| Article 175 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 8 α Synuclein and Lipids: Biological Intersections Alza et al. one another in a complex and not yet fully understood manner (Yeger-Lotem et al., 2009; Galvagnion, 2017). Controversial information coexists regarding plasma Chol level since it has been considered as either positive or negative risk factor for PD, or even not being linked at all to PD (Galvagnion, 2017). More concrete evidence links oxidized Chol derivatives with the onset and progression of PD (Bosco et al., 2006; Marwarha and Ghribi, 2015). In this regard, 27-hydroxycholesterol, a product of Chol oxidation, has been found to be increased in the brain, cortex and plasma, of PD patients (Lee et al., 2009; Seet et al., 2010; Cheng et al., 2011). Moreover, exogenous 27-hydroxycholesterol is able to induce the expression and accumulation of α-synuclein in human dopaminergic neurons through a transcriptional mechanism mediated by the transcription factor LXRβ (Marwarha et al., 2011). A possible mechanism that supports these ﬁndings could be the regulation of Chol eﬄux from neuronal cultures via ABCA1 transporter associated with the increased levels of oxidative stress reported in PD (Hsiao et al., 2017). the overexpression of α-synuclein (Velázquez et al., 2016; Garcia et al., 2018; Pitcairn et al., 2018). α-SYNUCLEIN AND LIPID SIGNALING Apart from their structural and biophysical role in biological membranes, lipids also serve as reservoirs of intracellular and extracellular messengers. One of the most investigated events in lipid signaling is the action of phospholipases and PL kinases and phosphatases that produce diﬀerent lipid and non-lipid messengers from membrane PLs. Regarding Chol biosynthesis, in silico analysis from the Yeger-Lotem laboratory identiﬁed the ergosterol-mevalonate pathway as a candidate for the cellular response to α-synuclein toxicity. One of the identiﬁed genes is Hrd1, a ubiquitin protein ligase related with the regulation of HMG-CoA reductase, the rate-limiting enzyme for the de novo Chol synthesis (Yeger- Lotem et al., 2009). They also found that pharmacological inhibition of HMG-CoA reductase by statins makes yeasts more vulnerable to α-synuclein toxicity (Yeger-Lotem et al., 2009). Phospholipases catalyze the hydrolysis of acyl and phosphate esters and are named according to their hydrolyzed position on the PL molecule. Among them, PLA2 and phospholipases C and D (PLC and PLD) are widely studied and many of them have been implicated in neurodegenerative processes. Our results in dopaminergic neurons and those obtained in yeasts suggest a protective role of neutral lipids (TAG and Chol) during α-synuclein overexpression either against proteotoxicity or against oxidative stress (Yeger-Lotem et al., 2009; Auluck et al., 2010; Sánchez Campos et al., 2018). However, contrasting eﬀects have been reported in primary human neurons treated with statins (Bar-On et al., 2008; Roy and Pahan, 2011). In view of the above, additional studies are needed to understand the biological signiﬁcance of these controversial experimental evidences. Concerning this matter, the clinical relevance of statins is still under debate and is currently being addressed in a trial with PD patients treated with simvastatin which ends in 2020 (Carroll and Wyse, 2017). Phospholipases A2 catalyzes the hydrolysis of the fatty acid esteriﬁed in the sn-2 of the glycerol backbone of PL moieties and releases FFA and a lysophospholipid. There are currently six diﬀerent known types of PLA2: secreted (groups I, II, III, V, IX, X, XI, XII, XIII, and XIV), cytosolic (cPLA2, group IV), calcium-independent (iPLA2, group VI), lipoprotein (groups VII and VIII), adipose (group XVI) and lysosomal (group XV), together constituting an enzyme superfamily (Dennis et al., 2011; Vasquez et al., 2018). In terms of neurodegenerative processes, and speciﬁcally PD, the most studied PLA2 isotypes are cPLA2 and iPLA2. α-SYNUCLEIN AND LIPID METABOLISM The other possibility is that α-synuclein could be involved in the regulation of gene expression associated with lipid metabolism. This latter possibility relates to the high DNA aﬃnity of amyloidogenic proteins, the nuclear localization of α-synuclein, and its participation in epigenetic modiﬁcations through histone acetylation (Ma et al., 2014; Surguchev and Surguchov, 2017). Speciﬁcally, it has been described that α-synuclein modulates gene expression related with cell survival by decreasing H3 acetylation (Pavlou et al., 2017) and with ubiquitination (Martins et al., 2011). Additional studies will be required to ascertain which of the above-mentioned mechanisms are biologically and pathologically involved and their link with lipid metabolism. Frontiers in Cellular Neuroscience \| www.frontiersin.org α-SYNUCLEIN AND LIPID SIGNALING The classical role assigned to iPLA2 enzymes is their active participation in membrane homeostasis through PL remodeling in Lands cycle where phospholipase activity operates coordinately with PL-acyltransferases. Later ﬁndings postulate other important functions for iPLA2s: while lysophosphatidyl moieties have been shown to modulate store-operated calcium entry, the released fatty acid, mostly DHA, has been described as the preferred substrate for the production of resolvins, lipid mediators involved in the resolution of inﬂammatory processes (Bazan, 2005; Serhan and Petasis, 2011). iPLA2 are mainly encoded by the PLA2G6 gene. In the nervous system, iPLA2β has been shown to be essential for remodeling membrane PLs in axons and synapses. Mutations in PLA2G6 have been associated with several neurodegenerative disorders, including adult-onset dystonia-parkinsonism, PARK14. In this regard, PLA2G6 knock-out mice have been widely used for the study of neuronal membrane-associated degeneration (Sumi-Akamaru et al., 2016). Indeed, increased expression of α-synuclein has The accumulated data allows us to propose a biological function for α-synuclein as a lipid metabolism modulator. Both at physiological levels and when overexpressed, α-synuclein is able to trigger a metabolic switch involving several aspects of lipid biology (Figure 2). The main biological response to α-synuclein overexpression is an increase in neutral lipid content that could be related to a neuroprotective strategy, whereas under physiological conditions the modulation of AA levels by the protein appears to be associated with prostaglandin generation and thus with a role in the inﬂammatory cascade (Golovko et al., 2007). The open question is how α-synuclein triggers the above-mentioned metabolic changes. One possibility is that the switch in neutral lipid metabolism in terms of LD accumulation could be a consequence of endoplasmic reticulum stress and autophagy impairment, triggered by proteotoxicity due to May 2019 \| Volume 13 \| Article 175 9 α Synuclein and Lipids: Biological Intersections Alza et al. been associated with mitochondrial injury triggered by iPLA2 dysfunction in PLA2G6 knock-out animals (Sumi-Akamaru et al., 2016). Moreover, the impairment of iPLA2-dependent calcium signaling has recently been implicated in neuronal loss through autophagic dysfunction in dopaminergic neurons derived from ﬁbroblasts from PD patients and PLA2G6 knock-out animals (Zhou et al., 2016). question is whether the dyshomeostasis between cPLA2 and iPLA2 triggered by α-synuclein overexpression could be responsible for a diﬀerential AA/DHA ratio and availability, thus modulating the balance between inﬂammation and resolution processes and promoting the neuroinﬂammatory phenotype in PD (Figure 3). α-SYNUCLEIN AND LIPID SIGNALING Among the classical signaling pathways associated with G-protein-coupled receptors is PI-speciﬁc PLC. Upon receptor activation, PLC hydrolyzes PI 4,5-bisphosphate to produce the lipid messenger diacylglycerol and the soluble inositol triphosphate. There are now six diﬀerent families of PLC (β, γ, δ, ε, ζ, and η) described in diﬀerent tissues, with pleiotropic functions associated with the membrane receptor to which they are coupled (Kadamur and Ross, 2013). It has recently been shown that α-synuclein overexpression interferes with Gq/PLC-β signaling by preventing the rise in cytosolic calcium and ERK 1/2 activation (Volta et al., 2017). α-Synuclein is also able to trigger the impairment of G-protein signaling associated with sphingosine-1-phosphate by promoting the exclusion of the receptor from lipid rafts (Mohamed Badawy et al., 2018). cPLA2 is mainly associated with the release of AA, the obligate substrate for prostaglandins generation catalyzed by cyclooxygenase-2, which is considered one of the main pathways mediating inﬂammatory response. Though neuroinﬂammation is a hallmark of PD, the speciﬁc PLA2 isoforms that are activated in dopaminergic neuronal damage have not been fully described. Besides the classical inﬂammatory process, the exposure to extracellular α-synuclein is able to activate cPLA2 and to promote synapse damage induced by phospholipase-dependent activity (Bate and Williams, 2015). Upon PLA2 activation, it is important that fatty acid release be properly coupled to PL acylation activity; impairment of the Lands cycle could be responsible for an increase in FFA availability (DHA, AA). This increase in FFA availability, could in turn, be associated with α-synuclein and promote its oligomerization. The rise in cytosolic/soluble DHA content reported by Sharon and coworkers in post-mortem brains in PD and LB dementia patients is in agreement with the activation of any speciﬁc PLA2 that could consecutively induce α-synuclein oligomerization and aggregation (Sharon et al., 2003a). The workings of this mechanism still lack clarity. Phospholipase D signaling impairment has been reported in Alzheimer’s disease and the signaling pathway is, therefore, an emerging therapeutic target for this neurodegenerative disorder (Brown et al., 2017). The role of PLD in α-synuclein- associated pathologies, however, still lacks clarity. Classical PLDs, PLD1 and PLD2, catalyze the hydrolysis of the PC head group in order to produce PA. PA has pleiotropic functions both as a lipid messenger, regulating the activity of signaling proteins, and as an important modulator of membrane curvature. CONCLUDING REMARKS enzymes participate in brain development and cognitive function (Burkhardt et al., 2014). The role of classical PLDs in the several processes of the nervous system has been well-established, but their participation in neurodegenerative processes associated with synucleinopathies is not clear. In vitro and in vivo assays have given rise to a number of divergent reports of the role of α-synuclein in PLD signaling. In vivo experiments have demonstrated that α-synuclein-induced PLD2 inhibition is able to prevent dopaminergic neurodegeneration (Gorbatyuk et al., 2010), whereas in vitro assays using recombinant α-synuclein and puriﬁed PLD1 and PLD2 have shown no inhibition of PLD activity (Rappley et al., 2009). However, protein proﬁle studies in post-mortem brains from PD patients display diminished PLD1 expression (Bae et al., 2014). In yeast, it has been demonstrated that PLD inhibition elicited by α-synuclein was related to dysregulation of lipid metabolism and traﬃcking (Outeiro and Lindquist, 2003). In line with this, our laboratory has recently described that the overexpression of human α-synuclein in neuroblastoma cells is able to induce a decrease in PLD1 mRNA and protein levels and to impair ERK1/2 signaling (Conde et al., 2018). In this cellular model of synucleinopathy, we demonstrated that the inhibition of PLD1 expression and the impairment of ERK1/2 signaling triggered by α-synuclein overexpression are associated with a decrease in neuroﬁlament light chain expression and in consequence with alteration of the neuronal cytoskeleton. The reported ﬁndings highlight the biological signiﬁcance of PLD regulation exerted by α-synuclein that could modulate PA levels in a compartment-speciﬁc manner, thus impacting on diﬀerent cellular functions: (i) vesicular traﬃcking, (ii) membrane curvature and membrane protein targeting, (iii) lysosomal activity, and (iv) PA-dependent signaling, among others (Figure 3). This review covers the diversity of ways in which α-synuclein interacts and connects with lipid biology. Much of the experimental evidences dealing with the physical interaction of the protein with diﬀerent lipid moieties derive from biophysical studies performed on artiﬁcial membranes. It is clear that the α-synuclein N-terminal domain is able to interact with the majority of PLs and with Chol with diﬀerent aﬃnities. Further research is required in order to shed more light on precisely which of these α-synuclein/lipid interactions occur in vivo; clear ﬁndings will undoubtedly contribute to a fuller understanding of both the biological and pathological implications of α-synuclein. CONCLUDING REMARKS Eﬀorts toward a more detailed characterization of α-synuclein interventions in lipid metabolism and signaling will open the way to a more in-depth assessment of the protein’s implications for therapeutic purposes. One hypothesis to be tested is whether α-synuclein pleiotropic properties are able to trigger a lipid metabolic and signaling switch, creating a propensity to interact and aggregate thus establishing a positive feedback. Unraveling this paradigm could provide not only new insights into the biological role of α-synuclein but also innovative ways of devising strategies for the treatment of synucleinopathies. α-SYNUCLEIN AND LIPID SIGNALING Whereas neuronal PLD1 has been associated with cytoskeleton architecture, PLD2 activity is linked to molecular events triggered by the activation of membrane receptors (Watanabe et al., 2011; Comoglio et al., 2014). Both PLD1 and PLD2 have been reported to participate in astroglial diﬀerentiation (Burkhardt et al., 2015). Genetic deletion of PLD1 or PLD2 in transgenic mice demonstrates that both The fact that SNCA knock-out mice present enhanced prostaglandin generation during neuronal injury argues in favor of a biological role for α-synuclein in the regulation of the inﬂammatory cascade (Golovko and Murphy, 2008). It has been demonstrated that DHA, mainly released from PLs by iPLA2 action, has a long half-life compared with other fatty acids and that its free form is the preferred substrate for neuroprotectin synthesis, and the resolution of the inﬂammatory process (Murphy, 2013). An important FIGURE 3 \| α-Synuclein and the modulation of lipid signaling. α-Synuclein is able to intervene lipid signaling by the modulation of phospholipase activities. The α-synuclein -induced impairment of cPLA2 and iPLA2 signaling triggers an imbalance in the AA/DHA ratio that ﬁnally could affect the balance of inﬂammation and resolution processes. Calcium signaling and ERK 1/2 activation are modulated by α-synuclein through PLC pathway impairment. α-Synuclein intervention in PLD signaling impacts on diverse cellular functions as vesicular trafﬁcking, membrane curvature, and membrane protein targeting, lysosomal activity and PA-dependent signaling. AA, arachidonic acid; DHA, docosahexaenoic acid; PA, phosphatidic acid; cPLA2, calcium-sensitive cytosolic phospholipase A2; iPLA2, calcium-independent phospholipase A2; PLC, phospholipase C; PLD, phospholipase D. FIGURE 3 \| α-Synuclein and the modulation of lipid signaling. α-Synuclein is able to intervene lipid signaling by the modulation of phospholipase activities. The α-synuclein -induced impairment of cPLA2 and iPLA2 signaling triggers an imbalance in the AA/DHA ratio that ﬁnally could affect the balance of inﬂammation and resolution processes. Calcium signaling and ERK 1/2 activation are modulated by α-synuclein through PLC pathway impairment. α-Synuclein intervention in PLD signaling impacts on diverse cellular functions as vesicular trafﬁcking, membrane curvature, and membrane protein targeting, lysosomal activity and PA-dependent signaling. AA, arachidonic acid; DHA, docosahexaenoic acid; PA, phosphatidic acid; cPLA2, calcium-sensitive cytosolic phospholipase A2; iPLA2, calcium-independent phospholipase A2; PLC, phospholipase C; PLD, phospholipase D. May 2019 \| Volume 13 \| Article 175 Frontiers in Cellular Neuroscience \| www.frontiersin.org 10 α Synuclein and Lipids: Biological Intersections Alza et al. FUNDING GS, NA, and RU are research members of the CONICET. MC and PIG are research fellows of the CONICET. Articles from our laboratory that have been cited in this review were granted by ANPCyT (PICT 2013-0987 and PICT 2017-0224), CONICET (PIP 11220120100251), and UNS (PGI 24/B226). GS, NA, and RU are research members of the CONICET. MC and PIG are research fellows of the CONICET. Articles from our laboratory that have been cited in this review were granted by ANPCyT (PICT 2013-0987 and PICT 2017-0224), CONICET (PIP 11220120100251), and UNS (PGI 24/B226). AUTHOR CONTRIBUTIONS GS contributed to conception and design of the study. GS, NA, and PIG organized the database and wrote the sections of the manuscript. MC designed and performed the ﬁgures. RU performed the critical reading and editing. All authors contributed to manuscript revision, read, and approved the submitted version. It is clear from the above that α-synuclein, mainly when it is overexpressed, is able to disturb lipid signaling. The plethora of lipid messengers (PA, AA, DHA, and diacylglycerol) able to be produced due to α-synuclein could have diﬀerent impacts on important cellular processes in the nervous system such as vesicular traﬃcking, autophagy, cytoskeleton architecture, and neuroinﬂammation. Dissecting the signaling pathways responsible for the production of each lipid messenger and the speciﬁc cellular compartment where they work, could contribute to the development of new strategic therapies for synucleinopathies. 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Phospholipase D2 activation by p38 MAP kinase is involved in neurite outgrowth. Biochem. Biophys. Res. Commun. 413, 288–293. doi: 10.1016/j.bbrc.2011.08.088 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Waxman, E. A., Mazzulli, J. R., and Giasson, B. I. (2009). Characterization of hydrophobic residue requirements for α-synuclein ﬁbrillization. Biochemistry 48, 9427–9436. doi: 10.1021/bi900539p Copyright © 2019 Alza, Iglesias González, Conde, Uranga and Salvador. 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. 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https://openalex.org/W4362616066	https://figshare.com/articles/journal_contribution/Supplementary_Tables_1-3_from_A_Novel_Tumor-Promoting_Role_for_Nuclear_Factor_IX_in_Glioblastoma_Is_Mediated_through_Transcriptional_Activation_of_GINS1/22527980/1/files/39990935.pdf	English	null	Supplementary Tables 1-3 from A Novel Tumor-Promoting Role for Nuclear Factor IX in Glioblastoma Is Mediated through Transcriptional Activation of GINS1	null	2,023	cc-by	198	Table S1. Primer sequences for mRNA expression analysis. Supplementary Tables Supplementary Tables Supplementary Tables Gene name Sequences (5’ to 3’) NFIX Forward CGGCTCTACAAGTCGCCTC Reverse GCAGTGGTTTGATGTCCGC CDC45 Forward TGGATGCTGTCCAAGGACCTGA Reverse CAGGACACCAACATCAGTCACG MCM2 Forward TGCCAGCATTGCTCCTTCCATC Reverse AAACTGCGACTTCGCTGTGCCA MCM3 Forward CGAGACCTAGAAAATGGCAGCC Reverse GCAGTGCAAAGCACATACCGCA MCM4 Forward CTTGCTTCAGCCTTGGCTCCAA Reverse GTCGCCACACAGCAAGATGTTG MCM5 Forward GACTTACTCGCCGAGGAGACAT Reverse TGCTGCCTTTCCCAGACGTGTA MCM6 Forward GACAACAGGAGAAGGGACCTCT Reverse GGACGCTTTACCACTGGTGTAG MCM7 Forward GCCAAGTCTCAGCTCCTGTCAT Reverse CCTCTAAGGTCAGTTCTCCACTC GINS1 Forward GCAAAGTCAGGTGGACGAAGTG Reverse CTGATCCGAAGCAAGCGGTCAT GINS2 Forward AGCCAAACTCCGAGTGTCTGCT Reverse CTTGTGTGAGGAAAGTCCCGCT GINS3 Forward GCTCCTGCATTTTGACAGTCCC Reverse TCCATCTCGTCTAGCCTGGCTA GINS4 Forward CTGGAGAGCAAGCCTGAGATTG Reverse GCAAGTAGCTGCTGAGGACGTA p53 Forward TAACAGTTCCTGCATGGGCGGC Reverse AGGACAGGCACAAACACGCACC p21 Forward TGGAGACTCTCAGGGTCGAAA Reverse GGCGTTTGGAGTGGTAGAAATC GAPDH Forward ATCAATGGAAATCCCATCACCA Reverse GACTCCACGACGTACTCAGCG Table S1. Primer sequences for mRNA expression analysis. shRNAs Sequences (5’ to 3’) GINS1 shRNA1 Sense CCGGGGAGATGAAAGCTTTGTATGACTCGATCAT ACAAAGCTTTCATCTCCTTTTTTG Antisense AATTCAAAAAGGAGATGAAAGCTTTGTATGACT CGATCATACAAAGCTTTCATCTCC GINS1 shRNA2 Sense CCGGGCGTCTTGCCAAATGCATTACCTCGAGTAA TGCATTTGGCAAGACGCTTTTTTG Antisense AATTCAAAAAGCGTCTTGCCAAATGCATTACCTC GAGTAATGCATTTGGCAAGACGC GINS1 shRNA3 Sense CCGGGCTGCACTGTAGCATACCTGTCTCGAACA GGTATGCTACAGTGCAGCTTTTTTG Antisense AATTCAAAAAGCTGCACTGTAGCATACCTGTCTC GAACAGGTATGCTACAGTGCAGC GINS1 shCont Sense CCGGGCTTCTCCGAACGTGTCACGTCTCGAGAC GTGACACGTTCGGAGAAGCTTTTTG Antisense AATTCAAAAAGCTTCTCCGAACGTGTCACGTCT CGAGACGTGACACGTTCGGAGAAGC Table S2. Sequences of shRNAs. Table S3. Antibodies for immunoblotting. Antibodies Catalog Number GINS1 #ab181112; Abcam NFIX #ab101341; Abcam p53 #2524; Cell Signaling Technology p21 #2947; Cell Signaling Technology HSP90 #4874; Cell Signaling Technology β-tubulin #2146; Cell Signaling Technology GAPDH #2118; Cell Signaling Technology Anti-mouse secondary antibody #7076S; Cell Signaling Technology Anti-rabbit secondary antibody #7074S; Cell Signaling Technology
W2096216950.txt	https://ijcsm.springeropen.com/counter/pdf/10.1007/s40069-015-0108-5	en		Physical and Mechanical Properties of Cementitious Specimens Exposed to an Electrochemically Derived Accelerated Leaching of Calcium	International journal of concrete structures and materials	2,015	cc-by	7,237	International Journal of Concrete Structures and Materials Vol.9, No.3, pp.295–306, September 2015 DOI 10.1007/s40069-015-0108-5 ISSN 1976-0485 / eISSN 2234-1315 Physical and Mechanical Properties of Cementitious Specimens Exposed to an Electrochemically Derived Accelerated Leaching of Calcium Arezou Babaahmadi1),* , Luping Tang1), Zareen Abbas2), and Per Mårtensson3) (Received October 28, 2014, Accepted August 5, 2015, Published online August 22, 2015) Abstract: Simulating natural leaching process for cementitious materials is essential to perform long-term safety assessments of repositories for nuclear waste. However, the current test methods in literature are time consuming, limited to crushed material and often produce small size samples which are not suitable for further testing. This paper presents the results from the study of the physical (gas permeability as well as chloride diffusion coefﬁcient) and mechanical properties (tensile and compressive strength and elastic modulus) of solid cementitious specimens which have been depleted in calcium by the use of a newly developed method for accelerated calcium leaching of solid specimens of ﬂexible size. The results show that up to 4 times increase in capillary water absorption, 10 times higher gas permeability and at least 3 times higher chloride diffusion rate, is expected due to complete leaching of the Portlandite. This coincides with a 70 % decrease in mechanical strength and more than 40 % decrease in elastic modulus. Keywords: nuclear waste management, service life, concrete, mechanical properties, leaching, acceleration. 1. Introduction In repositories for nuclear waste, concrete and other cementitious materials are extensively used in both the structural components such as the barrier construction and to ﬁll the voids between the waste containers inside the barrier construction. During the very long periods of time considered in an analysis of the long term safety of such a repository exchange of ions between the cementitious materials and the surrounding groundwater (Berner 1992; Reardon 1992) due to concentration differences will occur. This will result in dissolution or precipitation of minerals, and consequently in alteration of the microstructure as well as the chemical and mineralogical composition of the cementitious materials. There are a lot of concerns in normal constructions such as chloride corrosion of the reinforcement, carbonation 1) Division of Building Technology (Building Materials), Chalmers University of Technology, Gothenburg, Sweden. *Corresponding Author; E-mail: arezou.babaahmadi@chalmers.se 2) Department of Chemistry, University of Gothenburg, Gothenburg, Sweden. 3) Division of Low and Intermediate Level Nuclear Waste, Swedish Nuclear Fuel and Waste Management Company, Stockholm, Sweden. Copyright The Author(s) 2015. This article is published with open access at Springerlink.com or sulfate attack (Morga and Marano 2015; Park and Choi 2012; Pham and Prince 2014; Pritzl et al. 2014), however, here the most important process is the decalciﬁcation of the material through the dissolution of the calcium hydrates (Portlandite Ca(OH)2 and the Calcium silicate hydrates (CSH) phases, which constitute the major portion of hydrated cementitious material (Hinsenveld 1992). Safety assessments of repositories for low and intermediate level radioactive waste (LILW), require prediction of changes in the properties of the cementitious barriers over a very long period of time, up to 100,000 years. In order to improve the accuracy and reduce the uncertainties of the assessments a detailed understanding of the processes occurring in the repository and their effect on the properties of the cementitious materials is of great importance. The effect of degradation on the properties of cementitious materials has been reported in several studies in the literature (Adenot and Buil 1992; Carde et al. 1997; Carde and François 1997; Carde et al. 1996; Faucon et al. 1996, 1998; Haga et al. 2005; Maltais et al. 2004). In these studies leaching of calcium from cementitious materials have been accomplished both through immersion of the solid cementitious specimens in water (Faucon et al. 1996; Haga et al. 2005; Mainguy et al. 2000) and through immersion of the specimens in chemical agents in order to accelerate the leaching process (Adenot and Buil 1992; Carde and François 1997; Faucon et al. 1996, 1998; Haga et al. 2005; Heukamp et al. 2001; Mainguy et al. 2000; Maltais et al. 2004; Revertegat et al. 1992; Ryu et al. 2002; Saito et al. 1992; Wittmann 1997). The results in these studies indicate that a layered structure is developed in the leached samples comprising an unaltered core delineated by total 295 dissolution of Portlandite followed by different zones separated by dissolution/precipitation fronts and progressive decalciﬁcation of the CSH gel (Adenot and Buil 1992). Moreover, it is concluded that depletion in calcium changes the bulk density and the pore structure of the hydrated cement paste (Haga et al. 2005; Mainguy et al. 2000) and the changes in pore volume also alters the mechanical properties of the cementitious materials (Carde and François 1997; Heukamp et al. 2001; Saito and Deguchi 2000). However, although some important conclusions have been drawn from these studies regarding in particular the chemical properties of the Ca-depleted materials, they have been limited to the use of crushed materials or small solid samples. This has limited the possibilities to study the mechanical and physical properties, e.g. compressive strength and diffusivity, which require the use of larger samples. In addition it should be noted that there are not many studies reported in the literature with implication of concrete specimens (Choi and Yang 2013; Marinoni et al. 2008; Nguyen et al. 2007; Sellier et al. 2011) of proper size but instead paste specimens or powder samples have been used (Adenot and Buil 1992; Carde and François 1997; Carde et al. 1996; Faucon et al. 1996, 1998; Haga et al. 2005; Heukamp et al. 2001; Maltais et al. 2004; Revertegat et al. 1992; Ryu et al. 2002; Saito and Deguchi 2000; Ulm et al. 2003; Wittmann 1997). Finally, although the common feature for both natural and accelerated leaching scenarios will be a total dissolution of Portlandite and a signiﬁcant decalciﬁcation of the CSH phases, other effects of the aging processes may differ considerably between specimens aged by different acceleration methods and comparably natural leaching methods. This emphasizes the importance of reproducing accelerating tests and characterizing the aged samples to account for the comparability of the ageing function of different methods in order to demonstrate properties of degraded cementitious materials. All this implies that effective acceleration methods with comprehensible kinetics, simulating the natural calcium leaching process for concrete specimens with a size suitable for further evaluation of the mechanical and physical properties of the specimens are needed. In order to comply with this requirement a new method for accelerated leaching of cementitious materials is developed and utilized in this study. This paper presents the results from the study of the changes in mechanical and physical properties of solid cementitious specimens which have been depleted in calcium. The following properties have been studied: tensile strength, elastic modulus, permeability and water absorption. In addition, the chloride diffusion coefﬁcient of concrete specimens has been studied in order to give an indication of the transport properties of the specimens. 2. Materials and Methods 2.1 Specimen Preparation The paste specimens were cast from a mixture of Swedish structural Portland cement for civil engineering (CEM I 42.5N MH/SR3/LA) and deionized water at a water-cement ratio of 0.5. The chemical composition of the cement is listed in Table 1. Fresh cement paste was cast in acrylic cylinders with an internal diameter of 50 mm and a length of 250 mm. The cylinder’s ends were sealed with silicone rubber stops. The cylinders containing fresh paste were rotated longitudinally at a rate of 12–14 rpm for the ﬁrst 18–24 h of hydration in order to produce specimens with a homogeneous composition and structure. Afterwards the rubber stops were removed and the ends of the cylinders were sealed with plastic tape. The specimens were stored for over 6 months in a moist plastic box and then cut to cylinders with the size of Ø50 9 75 mm for use as specimens in the experiments. In order to prevent carbonation, saturated lime water was used at the bottom of the plastic box as absorbent for carbon dioxides during the storage of specimens. To further ensure that the specimens used in the leaching experiments were not carbonated, the paste portions about 10–20 mm near the ends of the cylinders was cut off prior to the specimen cutting. The mortar specimens at water- cement ratio of 0.5 and a cement: sand ratio of 1:2, were cast from mixtures of Swedish structural Portland cement for civil engineering (Table 1), deionized water and a siliceous natural sand with maximum particle size of 1 mm. Similar casting procedure as of paste specimens was followed. The concrete specimens were cast from mixtures of Swedish structural Portland cement for civil engineering, natural sand (the sand was a type of siliceous gravel with size of 0–8 mm and ﬁneness modulus of 3.82, in accordance with European standard EN 933-1 (EN 933-1 Tests for geometrical properties of aggregates)) and crushed coarse aggregate with maximum size of 16 mm. 65 % of total aggregate content was ﬁne aggregate (0–8 mm) and the rest was the course aggregate (8–16 mm). The course aggregate was an equal bland of (50 %), 8–12 mm and 12–16 mm of crushed aggregates. The specimens were cast in cylinders in two different dimensions of Ø100 9 200 mm and Ø50 9 250 mm with two different water cement W/C-ratios (according to the properties of the concrete used in the Final Repository for Short-lived Radioactive Waste, SFR, in Sweden (Emborg et al. 2007; Höglund 2001)). The observations from the slump test prior to casting was 25 mm for the concrete with W/C = 0.48 and 35 mm for W/C = 0.62. The specimens were demolded 24 h after casting and then cured in the saturated lime water for more than 3 months after which they were stored for over 3 months in a moist plastic box and then cut to cylinders with the dimensions of Ø50 9 75 and Ø100 9 50 mm to be depleted in Ca in the leaching experiments. The speciﬁcations of all the specimens such as mix proportions as well as cast sizes and cut sizes are presented in Table 2. It should be noted that the W/C-ratios for paste and mortar specimens were chosen as to obtain better homogenized mixes. However, as noted the concrete specimens were cast according to the used W/C-ratios used in repository of nuclear waste in Sweden, SFL. 296 \| International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) Table 1 Chemical characteristics of Swedish CEM I 42.5N MH/SR3/LA. Chemical formulation CaO SiO2 Al2O3 Fe2O3 MgO Na2O K2O SO3 Cl Percentage 64 22.2 3.6 4.4 0.94 0.07 0.72 2.2 0.01 Table 2 Cast specimen’s speciﬁcations. Specimens Cement type W/C Aggregate fraction Cement content (kg/m3) Size of cast specimens (mm) Size of specimens after cutting (mm) Paste CEM I 42.5 N MH/SR3/LA 0.5 – 1225 Ø50 9 250 Ø50 9 75 0.5 0.5 635 0.48 0.7 350 0.62 0.7 300 Ø50 9 250 and Ø100 9 200 Ø50 9 75 and Ø100 9 50 Mortar Concrete 2.2 Electrochemical Acceleration Method Concrete specimens were depleted in calcium by the use of a newly developed method for accelerated calcium leaching of solid specimens of ﬂexible size (Babaahmadi et al. 2015). As illustrated in Fig. 1, a cementitious specimen acting as a porous medium for ion migration is placed between two electrolyte solutions. The electrical migration enables faster transport of calcium ions, whilst application of ammonium nitrate as catholyte enables higher rate of dissolution of calcium hydroxides. As of using lithium hydroxide solution as anolyte, the pH of the anolyte was always kept at 13.5–14 and as of using ammonium nitrate solution as catholyte, the pH of catholyte was around a pH level of around 9. The alkaline characteristic of anolyte solution prevented acid attach due to production of H? ions at the anode. As it is concluded by Babaahmadi et al. (Babaahmadi et al. 2015), with a current density of 125–130 A/m2 an approximately 53 days of experimental time is predicted to reach to complete leaching of Portlandite for a paste specimen of Ø50 9 75 mm and W/C-ratio of 0.5. In this study similar experimental time (53 days) was chosen for the specimens. It should be noted that depending on the W/Cratio the Portlandite content varies in the specimens. In this study the concrete specimens have the same volume of aggregate, implying the same volume of CSH, Portlandite and capillary pores. Which means with a higher W/C- ratio, the capillary pore volume is larger, implying the CSH and Portlandite volume is smaller. Accordingly under the same degree of hydration, Portlandite in the concrete with higher W/C is less. As a consequence the experimental time for concrete specimens with W/C-ratio of 0.62 to reach to + dU, V DC 0.3 M ammonium nitrate as Catholytic solution Anode( titanium mesh) Asphalte tape as Sealant 2 M LiOH as Anolytic solution Plastic space Specimen Silicon rubber Plastic box Plastic support Cathode (stainless steel) Fig. 1 Electrochemical migration set-up design. International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) \| 297 complete leaching of Portlandite should be shorter than that of specimens with W/C-ratio of 0.48. However, the prolongation of experimental time after leaching of Portlandite only affects the phase changes in CSH gel and as it was concluded by Carde et al. the changes in CSH phases due to leaching is not affecting the mechanical properties (Carde et al. 1996). 2.3 Natural Leaching Test The natural immersion test involved the immersion of the test specimen in groundwater to account for the leaching of calcium ions under relatively natural condition. This method is chosen as most of the reference natural leaching test methods introduced in the literature involve an immersion test with frequent exchanges of the leaching solution (Adenot and Buil 1992; Carde and François 1997; Faucon et al. 1996, 1998; Gustafson 2008; Haga et al. 2005; Heukamp et al. 2001; Langton and Kosson 2009; Mainguy et al. 2000; Maltais et al. 2004; Peyronnard et al. 2009; Revertegat et al. 1992; Ryu et al. 2002; Saito et al. 1992; Wittmann 1997). In this study the specimens of Ø46 9 100–250 mm, cured for 6 months, were coated with thick (3–4 mm) epoxy, sealing all surfaces except one for exposure. The experiment was carried out in the Äspö laboratory in Oskarshamn, Sweden. The laboratory is located 400 m deep under the ground which could facilitate access to the groundwater. The specimens were placed in a container and immersed in the groundwater. The groundwater was exchanges continuously as it was ﬂowing into the container with a tube, Fig. 2. After 2 and 3.5 years of exposure, samples were chemically analyzed in order to obtain their leaching proﬁles. 2.4 Assessments of the Changes in Hydrated Phases Due to Decalciﬁcation Line scans quantifying the axial (longitudinal) changes in Ca/Si ratios of solid samples were performed by Laser Ablation- Inductive Coupled Plasma- Mass Spectrometry (LA-ICP-MS). Laser Ablation analysis was performed using a New Wave NWR213 laser ablation system coupled to an Agilent 7500a quadrupole ICP-MS (upgraded with shield torch and a second rotary vacuum pump). A 30 l laser spot size, beam energy density of ca. 6 J/cm2 and repetition rate of 10 Hz was used in line scan mode. Each analysis included background measurement for 30 s, before switching on the laser. Figure 3, illustrates the cut samples used for the measurements. The surface of the cut sample was polished with a sand paper after saw cutting and later the sample was vacuum dried. An average of three line scans was used to report the results. In order to characterize the changes in hydrated phases, especially Portlandite, in the leached specimens produced in this study thermogravimetry analysis (TGA) were performed with a Netzsch, STA 409 PC/PG. To perform the measurements powder samples were prepared out of leached specimens. For electrochemically leached specimens a longitudinal section as shown in Fig. 3 was cut from the specimen (paste, mortar and concrete) and hand crushed in a mortar and vacuum dried afterwards. However for the case Fig. 2 Natural leaching test set-up. of naturally leeched specimens the powders were prepared from the outermost 2 mm of the leaching front. The samples were placed in a crucible and heated in pure nitrogen (inlet ﬂow rate of 20 ml/min) to a set temperature of 900 C. The heating rate was a linear ramp of 10 C/min. 2.5 Transport Properties The gas permeability and capillary water absorption tests were performed according to state of the art report of RILEM Technical Committee 189-NEC (Torrent 2007). The measurements were performed on concrete specimen of the size Ø100 9 50 mm. The specimens were preconditioned for 2 weeks according to recommended procedures stated in RILEM technical committee 189-NEC (Torrent 2007) prior to the measurements. The step by step procedure for the preconditioning, measurements and instrument to carry out the measurements of gas permeability and capillary absorption of water is explained in 189-NEC standard. The chloride diffusion coefﬁcient of the pristine and leached specimens were studied by means of the rapid chloride migration test according to NT BUILD 492 described by Tang (NT BUILD 492, Concrete, Mortar and Cement-based Repair Materials: Chloride Migration Coefﬁcient from Nonsteady-state Migration Experiments 1999; Tang 1996). For the calcium depleted specimens prediction of the duration of the experimental time was difﬁcult due to that the increased porosity of the specimens as well as the reduced 298 \| International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) Electro-Chemical Migration Natural immersion Sealed surface Anode Cathod: Leaching front Exposed surface Fig. 3 Sample preparation for ICP-MS analysis. concentration of chloride in the pore solution dramatically alters the chloride diffusion rate. Here the duration of the experiments were reduced to 15 h compared to fresh concrete specimens for which a duration of 24 h is normally used (NT BUILD 492, Concrete, Mortar and Cement-based Repair Materials: Chloride Migration Coefﬁcient from Nonsteady-state Migration Experiments 1999; Tang 1996). However, in spite of the large reduction in experimental time, chloride ions penetrated through the entire thickness of the specimens during this experiment. For that reason, no exact value of the chloride diffusion rate could be obtained but instead the minimum chloride diffusion coefﬁcient was calculated for the calcium depleted concrete specimens. 2.6 Mechanical Properties The tensile strength of the leached and reference concrete specimens of size Ø100 9 50 mm was measured using the splitting test on a Toni-Technik compression testing machine with a maximum capacity of 100 kN. The test procedure is in accordance with American standard ASTM C49 (ASTM C496/C496M-11, Standard Test Method for Splitting Tensile Strength of Cylindrical Concrete Specimens). The compressive strength of concrete, mortar and paste specimens of size Ø50 9 75 mm was measured with same compression testing machine and according to ASTM C39 standard (ASTM C39/C39M-14a, Standard Test Method for Compressive Strength of Cylindrical Concrete Specimens). According to this standard a correction factor of 0.96 was applied to the compressive strength results to account for the length:dimater ratio which is lower than 1.75. The calcium depleted specimens were kept in 100 % RH until being tested in order to avoid any internal cracks. Elastic modulus of the concrete specimens of size Ø50 9 75 mm was obtained as the slope of stress–strain curves recorded by means of an ALPHA compression testing machine. The load cell had a maximum capacity of 50 kN and was loaded with a mechanical press at a constant rate of 0.01 kN/ms. The vertical strain was measured utilizing a calibrated Linear Variable Differential Transformers (LVDT) sensor. The end surfaces of the specimens, perpendicular to the longitudinal axis of specimen, were cut with a diamond saw and polished in order to create a smooth surface. The concrete specimens of the size Ø50 9 75 mm were placed between two platens and positioned under the load cells. 4 LVDT sensors were used to measure the displacement of the bottom platen and three more sensors were employed to measure the displacement of the upper platen. The sensors were connected to a data-log system to record the gradient of strain as a function of stress. An impact resonant apparatus was used to measure the fundamental longitudinal frequency of concrete, mortar and paste specimens of the size Ø50 9 75 mm. One end of the horizontally placed concrete cylinder was vibrated with an impactor and the other end was connected to an accelerometer. The accelerometer was connected to an ampliﬁer and the set-up was connected to a wave form analyzer. The waveform analyzer shall have a sampling rate of at least 20 kHz and should record at least 1024 points of the wave form. By utilizing the fact that the elastic modulus is proportional to the square root of the resonant frequency the elastic modulus of each specimen can be calculated according to American standard ASTM C215. The dynamic elastic modulus was calculated according to Eq. (1). E ¼ D M ðn0 Þ2 ð1Þ where E is the dynamic elastic modulus, M is the mass and n0 is the fundamental longitudinal frequency and D is a factor deﬁned according to Eq. (2) for cylindrical specimens. D ¼ 5:093 ðL=d 2 Þ ð2Þ The values from this non-destructive test can be compared with those obtained according to stress–strain curves. 3. Results and Discussions 3.1 Characterization of Degraded Specimens The LA-ICP-MS results, presenting the electrochemicalmigration-induced longitudinal change in the Ca/Si ratio of the leached paste specimen are illustrated in Fig. 4. As can be seen, ICP-MS line scans representing the composition of International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) \| 299 obtained when comparing the specimen size and leaching duration in electro-chemical migration test and leaching experiments reported in the literature. Haga et al. (2005) have shown that a depletion depth of a maximum of 1.25 mm can be expected after 100 days of a natural immersion test. Faucon et al. (1998) reported that up to 60 days of natural leaching leads to 0.7 mm of dissolved thickness in exposed specimens. Lagerblad (2001) and Trägårdh and Lagerblad (1998) reported a leaching depth equal to 5–10 mm can be expected after up to 100 years. Heukamp et al. (2001) used specimens with the size of Ø11.5 9 60 mm for a leaching period of 45 days. Nguyen et al. (2007) have reported on the application of specimens of Ø32 9 100 mm and Ø110 9 220 for an experimental time of up to 547 days and Choi and Yang (2013), have used leaching front in degraded specimens leached with electrochemical migration and natural immersion test methods are illustrated. It is shown that the propagation of leaching front (shape of leaching front) obtained by the two methods are relatively comparable and the longitudinal changes in the Ca/Si ratio propagates towards a homogeneous leaching state in time, indicating a complete leaching of Portlandite. Moreover, as shown after approximately 53 days of experimental time (1.2 9 106 Coulombs), a Ca/Si ratio close to complete leaching of Portlandite is obtained in electrochemically leached specimens, while the naturally leached specimens indicate up to 10 mm of leaching front in 3.5 years of leaching. This indicates that considerable acceleration rate is gained with application of electrochemical migration method. Similar outcome can be 3.5 3 Ca/Si Ref Ca/Si ~10 days 2.5 Ca/Si CH leached 2 Ca/Si CSH 1.5 1 0.5 0 10 20 30 40 50 60 Length (Cathode side towards Anode side) 70 80 0 10 20 30 40 50 60 Length (Cathode side towards Anode side) 70 80 3.5 3 Ca/Si 2.5 ~ 50 days Electro-Chemical Migration 0 2 1.5 1 0.5 0 3.5 Ca/Si ~ 70 days 3 2.5 2 1.5 1 0.5 0 0 10 20 30 40 50 60 Length (Cathode side towards Anode side) 70 80 3.5 Ca/Si 2 years 3 2 years leaching 2.5 2 Natural leaching 1.5 1 0 1 2 3 4 5 6 7 8 Length (mm) 9 10 11 12 13 14 Line scans for ICP ~ 20 mm 15 3.5 3.5 years leaching Ca/Si 3.5 years 3 2.5 2 1.5 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Length (mm) Fig. 4 LA-ICP-MS analysis for naturally and accelerated leached samples. 300 \| International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) (Adenot and Buil 1992). This is also an indication that the electrochemical migration method enables leaching of cementitious specimens even when containing aggregates. The average changes in the mass of concrete specimens representing the changes in the bulk density and eventually changes in porosity are presented in Table 3. As shown the specimens with a lower W/C-ratio are indicating higher degree of mass changes after leaching. Moreover, the changes in porosity according to total leached content of Portlandite are also presented. The results are in accordance with explanations in Sect. 2.2, stating that with a lower W/C-ratio a larger Portlandite content is expected and moreover, it is shown that the mass depletion due to leaching is mainly attributed to leaching of Portlandite. Accordingly a 100 0.4 100 Leached paste with ElectroChemical migartion 0.2 Portlandite 0.1 70 60 400 800 600 80 0.2 70 0.1 60 0 1000 0 200 400 100 0.2 70 60 Weight % 80 Derivative Weight (%/°C) Weight % 100 0.4 Naturally leached paste for 2 years 0 0 200 400 90 0.3 80 0.2 70 0.1 0 60 600 100 0 200 100 0.02 400 Temp 0.01 97 96 95 400 600 800 98 0.01 97 96 95 0.00 1000 0 200 400 600 800 0.04 Reference mortar Leached mortar with Electro-Chemical migartion 100 0.02 0.01 90 200 400 600 Temp 800 0.00 1000 Weight % 95 0.04 0.03 Derivative Weight (%/°C) Weight% 0.03 0 0.00 1000 Temp Temp 100 0.02 99 Weight% 98 Derivative Weight (%/°C) Weight % 99 600 Leached concrete with Electro-Chemical migartion Reference concrete 200 0 1000 0.4 Naturally leached paste for 3.5 years Temp 0 800 Temp Temp 90 600 Derivative Weight (%/°C) 200 0.3 Deivative Weight (%/°C) 0 0.4 95 0.02 0.01 90 0 200 400 600 800 Derivative Weight (%/°C) Weight % 80 90 Weight % 0.3 90 Derivative Weight (%/°C) Reference paste Derivative Weight (%/°C) cylindrical concrete samples of Ø100 9 100 mm for an experimental time of up to 365 days. Further, the TGA analyses results are presented in Fig. 5 comparing temperature-induced mass changes for reference, accelerated leached and naturally leached paste samples. It should be noted that for the case of natural leaching the samples were taken from 0 to 2 mm of the outer most part of the exposed surface. As can be seen, the reference sample exhibits a mass change at approximately 400 C, which is the assigned peak to the Portlandite (Zhang and Ye 2012). However for all degraded samples no matter if leached naturally or with the acceleration method the Portlandite peaks are vanished. The preferential leaching of Portlandite due to decalciﬁcation has been also stated in literature 0.00 1000 Temp Fig. 5 TGA analysis for reference and degraded paste samples. International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) \| 301 higher degree of mass change will be attained. It should be noted that the specimens were pre-conditioned according to the state of the art report of RILEM Technical Committee 189-NEC (Torrent 2007), before weighting to have a comparable moisture condition. 3.2 Transport Properties The capillary water absorption results representing the changes in pore structures of the specimens are presented on the left side of Fig. 6. Considering the presented results it can be inferred that the capillary water absorption in 24 h is increased by a factor of 4 and 2 for calcium depleted specimens with W/C-ratios of 0.48 and 0.62, respectively. As can be seen that the difference in rate of capillary water absorption of aged specimens with different water cement ratios, is much less than that of reference specimens. This indicates that a very similar pore structure is expected in aged specimens no matter which initial W/C-ratio the specimens had. Further, the change in gas permeability coefﬁcient due to degradation is presented in the right side of Fig. 6. As illustrated the gas permeability in the calcium depleted specimens is more than 10 times higher than in the pristine material. This can be explained by the larger porosity after decalciﬁcation. Furthermore, the changes in chloride migration coefﬁcient of the concrete specimens due to degradation are presented in Fig. 7. As it can be seen, at least three times higher chloride migration coefﬁcient is expected after calcium depletion. It should be noted that the presented results regarding the calcium depleted specimens are the minimum chloride migration coefﬁcient due to the full penetration through the specimen thickness as it was pointed out previously. The results are in good agreement with the results presented Choi and Yang (2013). 3.3 Mechanical Properties The average tensile strength of concrete specimens of size Ø100 9 50 mm are presented in Fig. 8. As shown the average tensile strength for the specimens with a W/C-ratio of 0.48 is reduced by up to approximately 70 % due to calcium depletion whereas the reduction in tensile strength for the specimen with a W/C-ratio of 0.62 is about 55 %. Further, the average compressive strength results for specimens of size Ø50 9 75 mm is presented in Fig. 9. As it can be seen the reduction in compressive strength due to leaching is 70 % for concrete specimens with W/C = 0.48, 59 % for concrete specimens with W/C = 0.62, 74 % for mortar specimens and 56 % for paste specimens. Interestingly the residual strength of the Ca-depleted concrete specimens is very similar regardless of the water cement ratios of the original specimens. This can also be seen for paste and mortar specimens. This indicates that when leaching propagates towards an approximate complete leaching of the Portlandite, comparable strength properties can be expected for samples with different original W/Cratios. This is due to comparable pore structures that are obtained in the specimens after leaching. It has also been shown in literature that the changes in pore structure have a detrimental inﬂuence on the strength properties of cementitious materials (Carde et al. 1996; Haga et al. 2005; Mainguy et al. 2000). The ﬁndings presented above are in good agreement with the conclusions presented by (Carde et al. 1996). In their study they showed that the loss of strength due to complete removal of Portlandite can be up to 70 %. Similar results have also been presented by (Choi and Yang 2013). Moreover, rate of change in the strain of the concrete specimens as a function of applied stress representing stress–strain curves is presented in Fig. 10. As shown the elastic modulus is represented by the slope of the stress–strain curves is reduced by up to 40 % after leaching for both types of specimens. In addition, to conﬁrm the credibility of the results from stress–strain curves, elastic modulus was also estimated through measuring the natural frequency of the specimens. The results which are presented in Table 4 are comparable with the results presented in Fig. 10. 4. Conclusions In this paper a study of mechanical and physical properties of concrete specimens which are depleted in calcium by a newly developed acceleration method is presented. Providing such results requires calcium depleted concrete Table 3 Mass changes due to leaching for concrete specimens of size Ø100 9 50 mm. REF (g) Aged (g) Mass depletion V depletiona Porosity changeb W/C = 0.48 927 881 46.00 20.81 5.3 44.4 5.12 W/C = 0.62 907 878 29.00 31.12 3.3 29.6 3.41 a Leached Porosity changed c portlandite (g) Calculated assuming that total mass change is attributed to Portlandite depletion (V depletion = mass change/Portlandite density). Calculated according to V depletion and considering the dimension of the specimens, Ø100 9 50 mm (Porosity Change = Vdepletion/total volume of specimen). c Leached Portlandite is according to measured leached calcium content in catholyte solution. d Calculated according to total leached Portlandite and considering the dimension of the specimens (Ø100 9 50 mm). b 302 \| International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) W/C=0.62 REF 1E-15 6000 W/C=0.48 Aged 5000 W/C=0.62 Aged W/C=0.48 REF 1E-16 4000 W/C=0.62 REF K (m2) Water adsorption per unit area (g/m2) W/C=0.48 REF 7000 3000 W/C=0.48 Aged 1E-17 2000 W/C=0.62 Aged 1000 1E-18 0.12 0 0 50 100 150 200 250 300 0.17 0.22 0.27 P (MPa) √t (√seconds) Fig. 6 Capillary water absorption for concrete specimens for W/C ratios of 0.48 and 0.62 before and after leaching of calcium (left) and Gas permeability coefﬁcient K as function of applied absolute gas pressure P (right). Compressive Strength (MPa) Chloride diffusion coefficient ( ×10-12) 37.2±0.3 140 120 100 80 60 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 9.1±0.1 9.5±0.3 12.1±0.1 12.1±0.1 Paste (W/C=0.5) Mortar (W/C=0.5) Concrete( W/C=0.62) Concrete (W/C=0.48) Aged 40 Fig. 9 Compressive strength results. 20 W/C=0.62 0 W/C=0.48 Aged Fig. 7 Chloride diffusion coefﬁcient for reference and aged concrete specimens. The calculated diffusion coefﬁcient for the case of aged specimens is the least possible value. Measuring the highest possible value is not practically possible due to very high porosity of the specimens. Tensile Strength (MPa) 20.6±0.2 40.0 REF Ref 12.0 45.0 41.2±0.3 28.8±0.1 compressive strength, elastic modulus, gas permeability as well as chloride diffusion coefﬁcient are taken into consideration. One of the direct applications of this study is to provide experimental approaches to be able to validate the risk assessment analyses regarding the functionality of engineered cementitious barriers. Based on the results presented in this study the following conclusions can be drawn: • • 10.2±0.1 10.0 5.5±0.5 8.0 6.0 2.5±0.1 4.0 2.8±0.1 2.0 Concrete (W/C=0.62) 0.0 Concrete (W/C=0.48) • REF (± 0.7) Aged (± 0.3) Fig. 8 Tensile strength results. specimens of proper sizes. The presented method is shown to be effective producing decalciﬁed concrete specimens of ﬂexible size. The changes in properties such as tensile and • The electrochemically-induced leaching is not affected by the aggregate content in mortar or paste specimens. Leaching of Portlandite causes considerable changes in physical and mechanical properties of concrete specimen, primary due to the increase in pore volume. It was concluded that larger pore volume due to complete leaching of portlandite can be expected which would cause up to 70 % decrease in mechanical strength and 40 % decrease in elastic modulus. Larger pore volume after degradation causes more than 10 times increase in gas permeability and at least 3 times higher chloride diffusion rate. The residual strength properties of the concrete specimens after complete leaching of portlandite are shown to be relatively similar no matter which initial water cement ratios the specimens have. This is to great extent due to similar pore structures in concrete specimens after leaching. International Journal of Concrete Structures and Materials (Vol.9, No.3, September 2015) \| 303 REF. W/C=0.62 30 y = 51.629x - 2.9151 20 15 20 15 y = 32.255x - 1.3883 10 10 5 5 0 Aged. W/C=0.62 25 Stress (MPa) 25 Stress (MPa) 30 0 0 0.1 0.2 0.3 0.4 0.5 0 0.6 0.1 Strain (%) Stress (MPa) Stress (MPa) 15 20 15 y = 35.19x - 1.8704 10 10 5 5 0.2 0.3 0.4 0.6 25 20 0.1 0.5 30 y = 55.921x - 0.6167 0 0.4 Aged. W/C=0.48 REF. W/C=0.48 0 0.3 Strain (%) 30 25 0.2 0.5 0.6 0 0 0.1 0.2 0.3 0.4 0.5 0.6 Strain (%) Strain (%) Fig. 10 Stress-strain curves for concrete specimens. Table 4 Natural frequency and calculated E-modulus of the reference and aged specimens. Mass (g) Ref Aged Length (m) Ref Aged F (kHz) E (GPa) Ref Aged Ref Aged W/C = 0.48 390 325 0.078 0.078 27.4 21.6 46.5 24.1 W/C = 0.62 350 330 0.076 0.075 29.3 22.1 46.5 24.6 Mortar W/C = 0.5 323 326 0.072 0.076 25.7 17.6 30.4 15.18 Paste W/C = 0.5 230 220 0.076 0.08 17.6 13.2 11.4 6.24 Concrete Acknowledgments The authors greatly appreciate financial support from Swedish Nuclear Fuel and Waste Management Company (SKB). Assistance with mechanical tests, BET and MIP analysis by Lars Wahlström, Ann Wendel and Liu Wei respectively are hereby appreciated and acknowledged. 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W3215113499.txt	https://www.frontiersin.org/articles/10.3389/fimmu.2021.774177/pdf	en		Increased Ratio of CD14++CD80+ Cells/CD14++CD163+ Cells in the Infrapatellar Fat Pad of End-Stage Arthropathy Patients	Frontiers in immunology	2,021	cc-by	7,509	ORIGINAL RESEARCH published: 26 November 2021 doi: 10.3389/fimmu.2021.774177 Increased Ratio of CD14++CD80+ Cells/CD14++CD163+ Cells in the Infrapatellar Fat Pad of End-Stage Arthropathy Patients Shuhe Ma 1, Kosaku Murakami 2, Rintaro Saito 1, Hiromu Ito 3,4, Koichi Murata 5, Kohei Nishitani 3, Motomu Hashimoto 5,6, Masao Tanaka 5, Masahi Taniguchi 1, Koji Kitagori 1, Shuji Akizuki 1, Ran Nakashima 1, Hajime Yoshifuji 1, Koichiro Ohmura 1, Akio Morinobu 1 and Tsuneyo Mimori 1,7 1 Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School of Medicine, Kyoto, Japan, Center for Cancer Immunotherapy and Immunobiology, Kyoto University Graduate School of Medicine, Kyoto, Japan, 3 Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan, 4 Department of Orthopaedic Surgery, Kurashiki Central Hospital, Okayama, Japan, 5 Department for Advanced Medicine for Rheumatic Disease, Kyoto University Graduate School of Medicine, Kyoto, Japan, 6 Department of Clinical Immunology, Osaka City University Graduate School of Medicine, Osaka, Japan, 7 Ijinkai Takeda General Hospital, Kyoto, Japan 2 Edited by: Matthew William Grol, Western University, Canada Reviewed by: Berta Cillero-Pastor, Maastricht University, Netherlands Alexander Knights, University of Michigan, United States Correspondence: Kosaku Murakami kosaku@kuhp.kyoto-u.ac.jp Specialty section: This article was submitted to Inﬂammation, a section of the journal Frontiers in Immunology Received: 11 September 2021 Accepted: 08 November 2021 Published: 26 November 2021 Citation: Ma S, Murakami K, Saito R, Ito H, Murata K, Nishitani K, Hashimoto M, Tanaka M, Taniguchi M, Kitagori K, Akizuki S, Nakashima R, Yoshifuji H, Ohmura K, Morinobu A and Mimori T (2021) Increased Ratio of CD14++CD80+ Cells/CD14++CD163+ Cells in the Infrapatellar Fat Pad of End-Stage Arthropathy Patients. Front. Immunol. 12:774177. doi: 10.3389/fimmu.2021.774177 Objectives: This study sought to identify the ratio of M1/M2 cells in the infrapatellar fat pads (IFP) and subcutaneous fat tissues (SC) of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. The clinical features of OA and RA patients treated with or without biological disease-modifying anti-rheumatic drugs (bDMARDs) were also assessed. Methods: IFP and SC were collected from patients with OA and RA who are undergoing total knee arthroplasty (TKA). CD14-positive cells were then isolated from these samples. Flow cytometry was used to determine the number of CD14++CD80+ cells and CD14++CD163+ cells. The expression levels of lipid transcription factors, such as sterol regulatory element-binding protein 1 (SREBP1) and liver X receptor alpha (LXRA), and inﬂammatory cytokines were also evaluated. Results: Twenty OA patients and 22 RA patients were enrolled in this study. Ten of the RA patients (45.4%) received bDAMRDs before TKA. On average, a ﬁvefold increase in the number of CD14-positive cells and lower expression levels of SREBP1C and LXRA were observed in OA IFP relative to OA SC; however, these results were not obtained from the RA samples. The median ratio of CD14++CD80+ cells/CD14++CD163+ cells of OA IFP was 0.87 (0.76–1.09, interquartile range), which is higher to that of OA SC with a lower ratio (p = 0.05835). Conclusions: The quantity and quality of CD14-positive cells differed between IFP and SC in arthropathy patients. To our knowledge, this is the ﬁrst study to characterize the ratio of M1/M2 cells in the IFP and SC of end-stage OA and RA patients. The increased ratio of CD14++CD80+ cells/CD14++CD163+ cells in the IFP from patients with OA and RA treated with bDMARDs indicated that inﬂammation was localized in the IFP. As adipose tissue-derived innate immune cells were revealed as one of the targets for regulating Frontiers in Immunology \| www.frontiersin.org 1 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP inﬂammation, further analysis of these cells in the IFP may reveal new therapeutic strategies for inﬂammatory joint diseases. Keywords: osteoarthritis, rheumatoid arthritis, monocytes/macrophage, inﬂammation, infrapatellar fat pad (Hoffa’s) the IFP is involved in shock absorption, lubrication, and synovial ﬂuid secretion. Furthermore, it is an important site for inﬂammation in patients with pain due to OA. Notably, serious inﬂammation in the IFP is often associated with severe pain in such patients (12, 13). In this study, subcutaneous fat tissue (SC) is deﬁned as fat tissues located under the epidermis and dermis but not found in the IFP tissues located in the lesion parts of the knees. Adipose tissue comprises the stromal vascular fraction (SVF) and adipocytes, and adipose tissue macrophages and monocytes can be collected from the SVF (10). Adipose tissue macrophages (ATMs) in the IFP demonstrate aspects of both pro- and antiinﬂammatory phenotypes in vitro, with most cells expressing the M2 marker, CD206, but secreting interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a), and relatively low amounts of interleukin-10 (IL-10), similar to M1 cells (13–16). ATMs have been revealed to play important roles in both physiological processes and pathological mechanisms in adipose tissue—for instance, the proportion of ATM in lean mice was estimated to be less than 10% of all adipose tissues; however, in leptindeﬁcient mice, this proportion increased to over 50% (17). Unlike ATMs that are distributed throughout uninﬂamed adipose tissue and provide limited inﬂammatory activity in the lean state (18), ATMs in obese adipose tissue surround and consume dead adipocytes and exhibit a pro-inﬂammatory phenotype (19, 20). Furthermore, bigger amounts of TNF-a and IL-6 are secreted by ATMs in obese people (9), and a twofold increase in the release of IL-6 was observed in the IFP compared to the SC in patients with OA (21). However, the differentiation INTRODUCTION Osteoarthritis (OA) and rheumatoid arthritis (RA) affects millions of people worldwide, with one in three people over the age of 65 having OA (1), and every ﬁve out of a thousand people suffer from RA (2). OA and RA are characterized by joint pain; however, in some cases, they are characterized by deformity owing to inadequate therapeutic strategies. The progression of OA can be seen as multiple reasons, such as mechanical stress to the cartilage (3, 4), subchondral bone remodeling (5), biochemical cascades (6), and inﬂammation (5, 6). On the other hand, RA is an autoimmune disease that causes inﬂammation in the synovial tissue (3, 7). Disease-modifying anti-rheumatic drugs (DMARDs), such as biological DMARDs (bDMARDs), are used to treat RA (8); however, currently, no disease-modifying therapeutics is available for OA patients. Thus, elderly patients with OA or RA sometimes opt for total knee arthroplasty (TKA) to restore joint function. Adipose tissue participates in inﬂammatory processes because it is a source of cytokines, chemokines, and adipokines (9). The infrapatellar fat pad (IFP or Hoffa’s fat pad) is an adipose tissue depot located within the knee joint and is surrounded by synovium, cartilage, and bone (10). The anatomical location of the IFP is displayed in Figure 1A (11). As described by Jiang et al., the IFP is sandwiched between the patellar retinacula and the patellar tendon anteriorly and the trochlear surface of the femur posteriorly on the horizontal perspective. In contrast, on the vertical perspective, the IFP is located between the patella, femoral condyle, and intercondylar notch (12). Functionally, A B FIGURE 1 \| (A) The anatomical location of infrapatellar fat pads. (B) Flow chart of the experimental method. Frontiers in Immunology \| www.frontiersin.org 2 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP patterns of M1 and M2 cells in the IFP of arthropathy patients have not been studied. To our knowledge, this is the ﬁrst study to reveal the differentiation pattern of CD14++CD80+ cells and CD14++CD163+ cells in the IFP and SC of OA and RA patients. Furthermore, as IFP and SC are adipose tissues in the knee joints (Figure 1A) and IFP is considered to have a great potential to participate in inﬂammatory response (13), qRT-PCR was performed to identify the lipid transcription factors related to inﬂammation to clarify the signature expression pattern of these genes in the IFP and SC of arthropathy patients. Gating Strategy The gating strategy and ﬂuorescence minus one are presented in Supplementary Figures S1, S2. Representative APC-FSC twoparameter dot plots of gate 1 among unstained, isotype control, and fully stained samples were presented. Cells gathered in neither unstained nor isotype controls in fully stained samples were allotted to gate 2. In gate 2, representative two-parameter dot plots of PE-APC and PerCP/Cyanine5.5-APC of samples stained by isotypes and antibodies were presented. Compared with the isotype control, positive plots were counted as “CD14+, CD80+” and “CD14+, CD163+” to calculate the proportions of CD14++CD80+ cells and CD14++CD163+ cells in CD14positive cells. MATERIALS AND METHODS Quantitative Real-Time Polymerase Chain Reaction Sample Collection From Patients With OA and RA Total RNA was isolated with Isogen (#315-02504, Nippon Gene) and RNeasy Mini Kit (#74104, Qiagen). After DNAse treatment (#M6101, Promega), cDNA was prepared using an iScript cDNA Synthesis Kit (#1708890, BIO-RAD) according to the protocol of the manufacturer. qPCR was performed using TB Green Premix Ex Taq GC (#RR071A, TaKaRa) in a 7500 Real Time PCR System (Applied Biosystems). The following qPCR conditions were employed: one cycle for the initial denaturation stage at 95°C for 30 s, 70 cycles for the PCR stage with denaturation at 95°C for 10 s, and annealing at 60°C for 34 s; the melt curve stage was set according to the instructions for Takara TB Green for the 7500 Real-Time PCR System. The cell counts of the operation samples used in RNA extraction and qPCR are displayed in Supplementary Table S1. The sequences of the primers are listed in Supplementary Table S2 (22–25). Patient Information This study was approved by the Kyoto University Graduate School and Faculty of Medicine Ethics Committee (approval number G0502-1). Samples of the IFP and the SC were collected during TKA of patients with OA and RA admitted at Kyoto University Hospital from February 2018 to April 2020. SVF Collection and Human CD14 Magnetic Cell Separation Brieﬂy, 2 g of fat tissue from each sample was digested in 5 ml of collagenase medium [0.1 mg collagenase IA (#C2674, Sigma Japan) in 1 ml RPMI medium (#52400, Gibco)] at 37°C for at least 1 h with rotation. The digested tissue was mashed through a 70-µm cell strainer with 2 ml syringe plungers and centrifuged at 18°C 620 × g for 3 min. The obtained pellets, labeled as the SVF, were washed twice in phosphate-buffered saline (PBS) via centrifugation at 18°C 620 × g for 3 min. Thereafter, they were rewashed in a magnetic cell separation (MACs) buffer solution of 0.5 M ethylenediaminetetraacetic acid (EDTA) in PBS containing 0.1% bovine serum albumin (BSA) via centrifugation at 18°C 300 × g for 10 min and subjected to MACs with human CD14 MicroBeads (#130-050-201, Miltenyi Biotec). Statistical Analyses Patient information is presented as median values with interquartile ranges (IQR). For qPCR, the 2−DDCT method was used to quantify the data. As the collected data did not follow normal distribution, Mann–Whitney U-test was utilized for statistics to describe differences between the groups. Furthermore, as sample sizes were the same within the same patients or the same SVF, paired-sample Wilcoxon signed-rank test was utilized to perform a more rigorous analysis. The statistical data were calculated using Origin 9.0 software (Originlab). Flow Cytometry Analysis Staining Strategy CD14-positive cells were labeled with anti-CD14, anti-CD80, and anti-CD163 antibodies in 0.1% BSA and 0.5 M EDTA in PBS, with isotype and unstained controls. The following conjugated antibodies were used: anti-human CD14 (HCD14, APC, BioLegend), anti-human CD80 (2D10, PE, BioLegend), and anti-human CD163 (RM3/1, PerCP/Cyanine5.5, BioLegend). The following isotype controls were used: mouse IgG1, k isotype control (FC) antibody (MOPC-21, APC, BioLegend); mouse IgG1, k isotype control antibody (MOPC-21, PE, BioLegend); and mouse IgG1, k isotype control antibody (MOPC-21, PerCP/ Cyanine5.5, BioLegend). After washing, the cells were analyzed with a LSRFortessor cytometer (BD), and data were analyzed using FlowJo 10.4 software. Frontiers in Immunology \| www.frontiersin.org RESULTS The IFP of Arthropathy Patients Has a Higher Proportion of CD14+ Cells Than the SC The patients included in this study were arthropathy patients who underwent TKA. These patients were divided into OA, RA treated without bDMARDs, and RA treated with bDMARDs groups, as shown in Table 1. Twenty OA patients (group A) [15 females; median (IQR) age, 77.4 (74.7–81.3) years], 12 RA 3 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP TABLE 1 \| Patient characteristics. Patient groups Total patients (n) Female patients (%) Age (years) Height (cm) Body weight/total (kg) Body weight/F (kg) Body weight/M (kg) BMI (kg/m2) % of Obesity CRP (mg/dl) ESR (mm/h) TC (mg/dl) HDL (mg/dl) Disease duration (years) % of RF positive Group A Group B Group C OA RA without bDMARDs RA with bDMARDs 20 75% 12 75% 10 100% 77.4 (74.7– 81.3) 153.3 (149.7– 160.7) 63.5 (55.4– 70.3) 60.9 (52.8– 68.1) 70.4 (70.2– 75.8) 26.1 (24.3– 28.0) 65.0% 0.0 (0.0–0.13) 23.0 (10.8– 30.3) 193.0 (185.8– 224.0) 63.0 (57.3– 73.8) 67.6 (66.5–70.5) 158.3 (154.3– 162.0) 51.8 (47.8–55.4) 48.8 (45.4–53.3) 60.1 (55.1–63.6) A and B Age (years) Height (cm) 68.2 (46.5–76.0) Body weight/total (kg) 153.8 (152.0– Body weight/F (kg) 156.7) 59.8 (56.6–63.7) Body weight/M (kg) 59.8 (56.6–63.7) BMI >24.99 Not applicable 19.9 (19.5–23.6) 71.4 100 42.9 66.7 0 (0–8.5) 0.6628 0.1855 0.0040 0.9741 0.0023 0.0358 0.5661 0.0087 0.0101 0.8244 0.0736 Not assessed Not assessed Not assessed Not assessed 0.3929 0.4574 ESR (mm/h) 0.1327 0.5278 0.4539 TC (mg/dl) HDL (mg/dl) Disease duration (years) % of RF positive 0.7958 0.9892 0.1273 0.3053 0.2702 0.0926 0.1498 221.5 (209.0– 234.0) 77.0 (64.0–83.0) % of ACPA positive 15.1 (12.0–22.5) % MTX use 100 0.0033 0.3658 0.5942 197.0 (173.8– 225.5) 62.0 (51.0–76.0) 78.6 A and C 0.1717 70.0% 0.05 (0.0–0.4) 26.5 (6.0–44.0) 11.7 (5.6–19.2) B and C CRP (mg/dl) 8.3% 0.1 (0.0–1.6) 27.0 (18.5–50.5) 0.2530 0.1273 0.4003 MTX dose (mg/ week) % PSL use 0.4205 0.7277 6.0 (0–11.0) PSL dose (mg/ day) TJC 0.0659 28.6 0 (0–2.5) 33.3 0 (0–4.5) SJC PtGA VAS (cm) 0.8313 0.8847 2.0 (1.0–2.0) 1.0 (1.0–2.0) 3.6 (2.1–6.9) 3.3 (1.9–3.9) 11.5 (8.0–15.7) 1.0 (0–2.0) 1.0 (1.0–2.0) 4.0 (1.9–6.7) 2.0 (1.2–3.6) 10.7 (6.2–11.3) PhGA VAS (cm) CRP (mg/dl) SDAI 0.2461 0.0755 0.2043 % of ACPA positive % MTX use MTX dose (mg/ week) % PSL use PSL dose (mg/ day) TJC SJC PtGA VAS (cm) PhGA VAS (cm) SDAI p-values between groups 1.0000 Data are presented as medians and interquartile ranges. Statistics between groups were assessed using Mann–Whitney U-tests or Fisher’s exact tests. Bold values indicates signiﬁcance. OA, osteoarthritis; RA, rheumatoid arthritis; bDMARDs, biological disease-modifying anti-rheumatic drugs (tocilizumab, n = 4; etanercept, n = 3; golimumab, n = 2; sarilumab, n = 1); F, female; M, male; BMI, body mass index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; TC, total cholesterol; HDL, high-density lipoprotein; RF, rheumatoid factor; ACPA, anti-citrullinated protein antibodies; MTX, methotrexate; PSL, prednisolone; TJC, tender joint count; SJC, swollen joint count; PtGA, patient’s global assessment; VAS, visual analog scale; PhGA, physician’s global assessment; SDAI, simpliﬁed disease activity index. signiﬁcance found between OA and both RA groups. The median body weight (kg) of patients who underwent surgery in these groups was 63.5, 51.8, and 59.8 kg, respectively, with a signiﬁcance found between the group of RA patients who were not treated with bDMARDs and the other two groups. To gain more insights on whether gender is a factor in this signiﬁcance, female and male patients were divided within each group. As shown in Table 1, female body weight had the same signiﬁcance as that found for total patients, revealing a bias in body weight between genders. As the body mass index (BMI) of all members of the “RA without bDMARDs treatment” group patients who were not treated with bDMARDs (group B) [9 females; 67.6 (66.5–70.5) years], and 10 RA patients who were treated with bDMARDs (group C) [10 females; 68.2 (46.5–76.0) years] were included in the study. Information regarding Creactive protein (CRP), erythrocyte sedimentation rate, total cholesterol, high-density lipoprotein, rheumatoid factor, anticitrullinated protein antibodies, tender joint count, swollen joint count, patient’s and physician’s global assessment visual analog scale, and simpliﬁed disease activity index and the use of methotrexate (MTX) and prednisolone were described. OA patients who opted for surgery tended to be older, with a Frontiers in Immunology \| www.frontiersin.org 4 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP CD14++CD80+ and CD14++CD163+ Cell Ratio Increased by 1.36-Fold in OA IFP was below 25 kg/m2, the p-values of the overweight population between this group and the other two groups could not be calculated. CD14-positive cells from IFP and SC were assessed. The strategies to obtain CD14-positive cells and a schematic for ﬂow cytometry and qPCR are displayed in Figure 1B. The amount of CD14-positive cells per gram of IFP was compared to that of SC for the same patients in each group (Figure 2). In the OA group, the amount of CD14-positive cells (×105) in the IFP of median (IQR) was 2.57 (0.96–4.13); however, this amount decreased to 0.28 (0.067–0.55) in the SC, with a p-value less than 0.0001 depicting signiﬁcance. In RA patients who were not treated with bDMARDs, the number of CD14-positive cells (×105) in the IFP was 1.64 (0.61–4.84); however, this number decreased to 0.42 (0.20–0.58) in the SC, with signiﬁcance found (p = 0.00792). In RA patients treated with bDMARDs, the number of CD14-positive cells (×105) in the IFP was 1.89 (0.69–2.49); however, this number decreased to 0.39 (0.13– 1.74) in the SC. Statistics on the mean (standard deviation, SD) of CD14-positive cells per gram of fat tissue in each group are displayed in Supplementary Figure S3A. CD14-positive cells from the IFP and SC of all three groups were assessed. CD14++CD80+ cells and CD14++CD163+ cells were considered as M1 and M2 macrophages. As described in Figure 3A, the ratio [median (IQR)] of CD14++CD80+ cells/ CD14++CD163+ cells of OA IFP was 0.87 (0.76–1.09); this ratio was higher to that of OA SC with a lower degree (p = 0.05835). In the group of RA patients who were not treated with bDMARDs, the ratio of CD14++CD80+/CD14++CD163+ from both the IFP and SC was as high as 0.79 (0.65–0.90) and 0.86 (0.43–0.98) (Figure 3B). The RA bDMARDs group also had an increased ratio from 0.65 (0.44–0.86) in the SC to 0.94 (0.82–1.12) in the IFP (Figure 3C). Figure 3D shows that both diseases even had differentiation of CD14++CD80+ cells and CD14++CD163+ cells in the IFP. Statistics on the mean (SD) of M1/M2 ratio as well as their proportions in CD14-positive cells in each group are shown in Supplementary Figures S3B, C. The cell numbers in each group are presented in Table 2. However, the M1/M2 ratio was 0.59 (0.31–1.11) and was found to be biased to the M2 phenotype in peripheral blood mononuclear cell from RA patients (26). A B C FIGURE 2 \| The infrapatellar fat pads (IFP) of arthropathy patients has a higher proportion of CD14-positive cells than the subcutaneous fat tissues. CD14-positive cells per gram of SC or IFP in the osteoarthritis (OA) (A), rheumatoid arthritis (RA) without bDMARDs (B), and RA with bDMARDs (C). Samples were obtained during total knee arthroplasty, digested in collagenase medium to obtain the stromal vascular fraction, and stained with human CD14 antibody to separate adipose-tissueresident CD14-positive cells. p < 0.01 and p < 0.001, as determined by the paired-sample Wilcoxon signed-rank test to identify differences between SC and IFP OA. n = 20; RA without bDMARDs, n = 12; RA with bDMARDs, n = 10. Frontiers in Immunology \| www.frontiersin.org 5 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP A B D C FIGURE 3 \| CD14++CD80+ and CD14++CD163+ cell ratio increases by 1.36-fold in osteoarthritis (OA) infrapatellar fat pads (IFP). (A–C) Ratio of CD14++CD80+/CD14+ CD163+ cells in the infrapatellar fat pads (IFP) and subcutaneous fat tissues (SC) from the OA, rheumatoid arthritis (RA) without bDMARDs, and RA treated with bDMARDs groups. To calculate ratios and proportions, adipose-resident CD14-positive cells were stained with anti-human CD80, CD163, and CD14 antibodies and evaluated using ﬂow cytometry. (D) Proportions of CD14++CD80+ and CD14++CD163+ cells in the SC and IFP tissues of OA and RA patients (SC OA, n = 4; SC RA, n = 6; IFP OA, n = 11; IFP RA, n = 13). The x-axis displays samples from IFP and SC grouped by OA and RA, and the y-axis displays the proportions of CD14++CD80+ cells or CD14++CD163+ cells in CD14-positive cells. Lines connecting the dots between SC and IFP indicate samples from the same patients. Gating strategies for obtaining CD14++CD80+ cells and CD14++CD163+ cells as well as ﬂuorescence minus one are presented in Supplementary Figures S1 and S2. Statistics were calculated by the paired-sample Wilcoxon signed-rank test between CD14++CD80+ and CD14++CD163+ cells from the same patients. The Mann–Whitney U-test was used for analysis between different groups. + TABLE 2 \| CD14++CD80+ cells and CD14++CD163+ cells from the IFP and SC in each disease group. IFP ++ CD14 CD80 OA + SC ++ CD14 CD163 9.3 × 104 (7.7 × 104) + 1.1 × 105 (1.1 × 105) ++ CD14 CD80 + 3.9 × 103 (4.7 × 103) N = 11 RA without bDMARDs 3.2 × 104 (2.8 × 104) 4.4 × 104 (3.3 × 104) 5.7 × 104 (4.0 × 104) 5.4 × 103 (5.5 × 103) N=4 4.9 × 103 (4.7 × 103) N=8 RA with bDMARDs CD14++CD163+ 4.8 × 103 (4.6 × 103) N=3 7.4 × 104 (6.3 × 104) N=4 672 (76) 1,646 (948) N=2 Cell numbers were determined using ﬂow cytometry and are displayed as mean (SD). IFP, infrapatellar fat pad; SC, subcutaneous fat tissue. Frontiers in Immunology \| www.frontiersin.org 6 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP The Expression of Lipid Transcription Factors Related to Inﬂammation Signiﬁcantly Decreases in the IFP were quantiﬁed by qRT-PCR (Figures 4, 5). Statistics on mean (SD) of the expression levels of these genes in CD14-positive cells in each group are presented in Supplementary Figure S4. The samples analyzed by qRT-PCR were magnetically sorted CD14positive cells in the SVF from each SC and IFP sample. Lines connecting the dots from the SC to IFP indicated samples from the same patients. Consequently, as described in the left panel of Figure 4A, the relative expression level [median (IQR)] of As IFP and SC are fat tissues, and IFP is found in an environment related to inﬂammation, lipid transcription factors related to inﬂammation, such as SREBPs and LXRs, were of interest in this study. The expression levels of these genes and the inﬂammatory cytokines produced by CD14-positive cells from the IFP and SC A B C FIGURE 4 \| Expression levels of lipid transcription factors related to inﬂammation decrease in the infrapatellar fat pads (IFP). Expression levels of SREBP1A (A), SREBP1C (B), and LXRA (C) in the IFP and subcutaneous fat tissues (SC) from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. The panels to the left contain results comparing the expression levels between SC and IFP without division by diseases. The panels to the right contain results comparing the expression levels of SC and IFP in patients with OA, RA treated with bDMARDs, and RA without bDMARDs treatment. Lines connecting the dots from SC to IFP indicate samples from the same patients. The samples were labeled and separated into CD14-positive cells from the stromal vascular fraction of the IFP and SC from each patient. The median (interquartile range, IQR) expression levels of SREBP1A, SREBP1C, and LXRA in the SC from all patients were 0.31 (0–5.25), 0.98 (0.22–2.98), and 0.69 (0.34–2.12); these expression levels changed to 1.1 (0.09–4.81), 0.30 (0.16–0.71), and 0.039 (0.0085–0.50) in the IFP. The median (IQR) expression levels of SREBP1C and LXRA in the SC from OA patients were 1.03 (0.03–3.69) and 0.69 (0.34–2.14); these expression levels changed to 0.31 (0.23–0.71) and 0.039 (0.018–0.50) in the IFP. The median (IQR) expression levels of LXRA in the SC and IFP from RA patients who were not treated with bDMARDs were 1.04 (0.065– 20.89) and 0.017 (0.0067–0.81). Signiﬁcant differences are displayed in each ﬁgure. OA, n = 15; RA without bDMARDs, n = 7; RA with bDMARDs, n = 9, respectively. The 2−DDCT method was used to quantify the qPCR data. GAPDH was used as the reference gene. The average GAPDH Ct values from all SC samples were used to normalize the Ct values. The Y-axes indicate the relative expression levels of mRNA, which were normalized by GAPDH. p < 0.05, p < 0.01, and *p < 0.0001, as determined using the Mann–Whitney U-test for differences between the IFP and SC groups and the paired-sample Wilcoxon signed-rank test for differences between IFP and SC within the OA, RA without bDMARDs, and RA with bDMARDs groups. Frontiers in Immunology \| www.frontiersin.org 7 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP A B C FIGURE 5 \| Expression levels of inﬂammatory cytokines decrease in the IFP. Expression levels of CXCL10 (A), IL1B (B), and IL6 (C) in the infrapatellar fat pads (IFP) and subcutaneous fat tissues (SC) from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. The panels to the left contain results comparing the expression levels between SC and IFP without division by diseases. The panels to the right contain results comparing the expression levels of SC and IFP in patients with OA, RA treated with bDMARDs, and RA who were not treated by bDMARDs. Lines connecting the dots from SC to IFP indicate samples from the same patients. The samples were labeled and separated into CD14-positive cells from the stromal vascular fraction of the IFP and SC of each patient. The median (interquartile range) expression levels of CXCL10 in the SC and IFP from patients were 1.13 (0.23–2.33) and 0.33 (0.10–0.55); the expression level in the SC was 0.98 (0.07–2.33) and changed to 0.17 (0.05–0.42) in the IFP of OA patients. Signiﬁcant differences are displayed in each ﬁgure. OA, n = 15; RA without bDMARDs, n = 7; RA with bDMARDs, n = 9, respectively. The 2−DDCT method was used to quantify the qPCR data. GAPDH was used as the reference gene. The average GAPDH Ct values from all SC samples were used to normalize the Ct values. The Y-axes indicate the relative expression levels of mRNA, which were normalized by GAPDH. p < 0.05, as determined using the Mann–Whitney U-tests for differences between the IFP and SC groups and the paired-sample Wilcoxon signed-rank test for differences between the IFP and SC within the OA, RA without bDMARDs, and RA with bDMARDs groups. Data for the sample whose CXCL10 2−DDCT value was 69.2-fold higher than the average were removed from the plot. 1.03 (0.03–3.69), 0.69 (0.34–2.14), and 0.98 (0.07–2.33) in the SC, which decreased to 0.31 (0.23–0.71), 0.039 (0.018– 0.50), and 0.17 (0.05–0.42) in the IFP, with p-values less than 0.05 depicting signiﬁcance, as shown in the right panels of Figures 4B, C, 5A. In addition, the expression levels of LXRA in the RA without bDMARDs treatment group were 1.04 (0.065– 20.89) and 0.017 (0.0067–0.81) in the SC and IFP, respectively, which indicated signiﬁcance. No signiﬁcant relationship was found between IL1B and IL6 within or among each group (Figures 5B, C). SREBP1A in the CD14-positive cells of SC in all patients was 0.31 (0–5.25); however, in the IFP, this value changed to 1.1 (0.09–4.81), without any signiﬁcance. As displayed in the left panels of Figures 4B, C, 5A, the respective expression levels of SREBP1C, LXRA, and CXCL10 in the CD14-positive cells of SC from whole patients were 0.98 (0.22–2.98), 0.69 (0.34–2.12), and 1.13 (0.23–2.33); however, these levels respectively decreased to 0.30 (0.16–0.71), 0.039 (0.0085–0.50), and 0.33 (0.10–0.55) in the IFP, with p-values less than 0.05 depicting signiﬁcance. These differences were also apparent in patients with OA, with Frontiers in Immunology \| www.frontiersin.org 8 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP not treated by bDMARDs groups. However, in the age groups divided into below or above median age in each disease, the signiﬁcance of LXRA in RA patients who were not treated by bDMARDs was missing (Supplementary Table S3), which illustrated the accuracy of age classiﬁcation in Table 3. Overall, inﬂammation was found to be inﬂuenced by BMI and age in arthropathy patients. Inﬂammation Status Is Inﬂuenced by BMI and Age in Arthropathy Patients The impact of BMI and age on CD14-positive cells, the ratio of CD14 ++ CD80 + cells/CD14 ++ CD163 + cells, and the gene expression levels in each disease are highlighted in Table 3. Some of the results were not covered in the analysis among whole samples—for instance, in OA patients whose BMI was >25, IL6 expression was signiﬁcantly lower in the IFP than the SC; however, in OA patients whose BMI was <25 and in the whole group, this signiﬁcance disappeared. Furthermore, some results in the whole group showed bias to a certain group in BMI—for example, in the RA without bDMARDs treatment group with BMI >25, CXCL10 expression was signiﬁcantly lower in the IFP than SC, while in the RA with bDMARDs treatment group with BMI <25, LXRA expression was signiﬁcantly lower in the IFP than SC. Moreover, patients with BMI >25 in both the OA and RA with bDMARDs treatment groups tended to have higher proportions of CD14-positive cells in the fat tissue. To determine whether age impacts these results, the patients were divided into age groups below or above 75 (Table 3), which is indicated as senior people, or were divided into age groups below or above the median age (Supplementary Table S3). In Table 3, signiﬁcant differences of CD14-positive cells per gram of fat tissues were found in OA patients who were over 75 years old as well as in the group of RA patients who were not treated by bDMARDs and under 75 years old. On the other hand, the expression levels of LXRA and SREBP1C had signiﬁcant differences between SC and IFP in patients under 75 years old of both OA and RA who were DISCUSSION In the present study, samples obtained from patients with OA and RA who underwent knee surgery revealed that the number of CD14-positive cells per gram of IFP was signiﬁcantly higher than that of the SC. The ratios of CD14 ++ CD80 + cells/ CD14++CD163+ cells increased between IFP and SC within the OA group and the RA treated with bDMARDs group (p = 0.05835 and 0.8170, respectively). In addition, the expression levels of lipid transcription factors related to inﬂammation were signiﬁcantly lower in OA IFP than OA SC. The IFP is demonstrated as an active inﬂammatory site in arthritis (27). Although the IFP is an adipose tissue, its characteristics differ from other “classic” adipose tissues, such as SC (21). Thus, we compared the features of IFP to those of SC to assess the varying differentiation pattern of macrophages in the IFP in the inﬂammatory environment. In our study, an average increase of ﬁvefold was found for the number of CD14-positive cells in the IFP relative to that in the SC (Figure 2). Therefore, SC and IFP TABLE 3 \| Impact of body mass index (BMI) and age category of osteoarthritis (OA) and rheumatoid arthritis (RA) patients on the related gene expression. BMI <25 CD14-positive cells (105)/grams of fat tissue M1/M2 ratio SREBP1A SREBP1C 0.0156 N=7 0.0068 N = 12 0.2500 N=3 4.88E-04 N = 13 Not assessed N=1 0.0156 N=7 0.0625 N=6 0.0195 N = 11 0.4375 N=6 1.22E-04 N = 14 0.33333 N=2 0.1250 N=4 Not assessed N=1 1.0000 N=3 Not assessed N=0 0.0952 N=3 Not assessed N=0 1.0000 N=2 0.1143 N=4 0.8333 N=3 Not assessed N=1 Not assessed N=0 Not assessed N=0 Not assessed N=1 0.6250 0.7500 0.1563 0.15623 0.6953 0.1094 0.4375 0.5625 0.6875 0.0313 0.2188 0.2188 1.0000 0.3125 0.5703 0.4961 0.5000 0.7500 OA RA without bDMARDs RA with bDMARDs BMI ≥25 OA RA without bDMARDs RA with bDMARDs Age <75 OA RA without bDMARDs RA with bDMARDs Age ≥75 OA RA without bDMARDs RA with bDMARDs LXRA CXCL10 0.5000 1.0000 N=4 0.0313 1.0000 N=6 Not assessed N=2 0.0977 0.0098 N = 11 Not assessed N=1 0.4375 0.0313 N=6 0.0313 0.0938 N=6 0.0313 0.5625 N=6 0.0625 0.3125 N=5 0.0742 0.1953 N=9 Not assessed N=1 1.0000 0.2500 N=3 IL1B IL6 0.2500 0.8750 0.5625 0.6875 0.2402 0.0420 0.8438 0.3125 0.5625 0.5625 0.5625 0.8438 0.6250 0.1875 0.7344 0.0742 0.7500 1.0000 The impact of BMI and age in the OA and RA without bDMARDs and RA with bDMARDs groups. The patients were classiﬁed into groups with BMI below or over 25 or into age groups below or over 75 years old. The statistics of CD14-positive cells and the gene expression levels in each group were determined by paired-sample Wilcoxon signed-rank test, while the statistics for the M1/M2 ratio in each group were determined using Mann–Whitney U-tests. Data are expressed as p-values between subcutaneous fat tissue and infrapatellar fat pad. Data in bold font indicate signiﬁcance. Frontiers in Immunology \| www.frontiersin.org 9 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP were categorized as “inﬂammation-low tissue” and “inﬂammationhigh tissue”, respectively. Furthermore, the ratio of CD14++CD80+ cells/CD14++CD163+ cells and the expression levels of the genes related to lipid regulation and inﬂammation were compared to investigate the role of adipose-tissue-resident innate immune cells in the pathological process of arthropathy diseases. However, more samples are needed for this analysis. Furthermore, in addition to staining cells with anti-CD14, CD80, and CD163 antibodies, the quantiﬁcation of CD68, inducible nitric oxide synthase, and arginase-1 in the adipose-tissue-resident CD14-positive cells by qRT-PCR might provide more reliable results. As different groups of arthropathy patients have varying characteristics, a comparison between the groups was not performed. The number of CD14-positive cells and ratios of CD14++CD80+ cells/CD14++CD163+ cells were increased in the IFP in both the OA group and the RA treated with bDMARDs group. The CRP results based on blood tests (in Table 1) revealed a lower inﬂammation level in the OA [0.0 (0.0–0.13)] and RA treated with bDMARDs [0.05 (0.0–0.4)] groups than the RA without bDMARDs treatment [0.2 (0.0–1.7)] group. Such ﬁndings indicate that both OA and RA treated with bDMARDs patients had a lower inﬂammation level in their blood. However, a higher ratio of CD14++ CD80 + cells/CD14 ++ CD163 + cells appeared in the IFP of these patients, implying inﬂammation localization in the IFP. The expression levels of LXRA and SREBP1 displayed different patterns between the IFP and SC in OA and RA patients, and the effects of concurrent or historical administration of MTX could serve as one of the reasons; this is because MTX was revealed to inhibit the amounts of inﬂammatory signals via the JAK1-STAT3 and JAK2-STAT5 transcriptional pathways in human macrophage cell lines (28, 29). In the right panel of Figure 5A, the expression levels of CXCL10 in CD14-positive cells signiﬁcantly decreased from the SC to IFP in OA; however, in both RA groups, this signiﬁcance disappeared. As CXCL10 responds to TNF-a, IFN-a/b/g, IL-1b, or LPS (30), anti-TNF inhibitors, such as etanercept and golimumab used as bDMARDs (31), can alter CXCL10 expression. The lower expression of LXRA in the IFP can be considered as an inhibitory effect of cytokines (32). The ratios of CD14++CD80+ cells/CD14++CD163+ cells in the IFP were relatively higher than those in the SC, which indicated that the IFP was in an inﬂammatory state. The p-value was displayed to show importance, but as the tendency is strong, if more samples can be obtained in the future, the interpretive power is supposed to increase. To understand the mechanism of LXR downregulation in the IFP, besides inﬂammatory response genes, the expression levels of genes encoding enzymes involved in unsaturated fatty acid biosynthesis (e.g., SCD2, FADS1, ACOX3, and ELOVL5) and the concentrations of omega-3 fatty acids (e.g., docosahexaenoic acid and eicosatetraenoic acid) and omega-7 fatty acids (e.g., 9Zpalmitoleic acid) need to be evaluated (33). Based on the theory that lipid transcription factors participate in the inﬂammation process, these results would be the ﬁrst step to see the panorama of “localized lipid immunization.” This study revealed that, in the IFP of arthropathy patients, abundant CD14-positive cell stores and the ratio of CD14++CD80+ Frontiers in Immunology \| www.frontiersin.org cells/CD14++CD163+ cells were elevated, implying the localization of inﬂammation in the IFP. Therefore, targeting adipose-tissueresident innate immune cells in the IFP can be considered as a new therapeutic strategy for inﬂammatory arthritis. 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. ETHICS STATEMENT The studies involving human participants were reviewed and approved by the Kyoto University Graduate School and Faculty of Medicine Ethics Committee (approval number G0502-1). The patients/participants provided their written informed consent to participate in this study. AUTHOR CONTRIBUTIONS SM and KosM were responsible for the study design. SM, RS, and MTani collected samples during total knee arthroplasty. SM and RS carried out the experiments. HI, KoiM, and KN performed the surgery. SM wrote the ﬁrst draft of the manuscript. MH, MTana, KK, SA, RN, HY, KO, AM, and TM supervised the draft of the manuscript. All authors contributed to the article and approved the submitted version. FUNDING This work was supported by Nagahama City, Shiga, Japan; Toyooka City, Hyogo, Japan; and ﬁve pharmaceutical companies (Mitsubishi Tanabe Pharma Co., Chugai Pharmaceutical Co. Ltd., UCB Japan Co. Ltd., AYUMI Pharmaceutical Co., and Asahi Kasei Pharma Co.). The KURAMA cohort study is supported by grants from Daiichi Sankyo Co. Ltd. This study was conducted as an investigatorinitiated study. The companies had no role in the design of the study, the collection or analysis of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. ACKNOWLEDGMENTS The authors would like to specially thank Atsuko Tamamoto for collecting samples in March of 2020 and Editage (www.editage. com) for English language editing. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2021.774177/ full#supplementary-material 10 November 2021 \| Volume 12 \| Article 774177 Ma et al. M1/M2 Differentiation in Arthritis IFP REFERENCES 1. Hawker GA. Osteoarthritis is a Serious Disease. Clin Exp Rheumatol (2019) 37 Suppl;120:3–6. 2. Aletaha D, Smolen JS. Diagnosis and Management of Rheumatoid Arthritis: A Review. JAMA (2018) 320:1360–72. doi: 10.1001/jama.2018.13103 3. Penatti A, Facciotti F, De Matteis R, Larghi P, Paroni M, Murgo A, et al. Differences in Serum and Synovial CD4+ T Cells and Cytokine Proﬁles to Stratify Patients With Inﬂammatory Osteoarthritis and Rheumatoid Arthritis. Arthritis Res Ther (2017) 19:103. doi: 10.1186/s13075-017-1305-1 4. Harasymowicz NS, Clement ND, Azfer A, Burnett R, Salter DM, Simpson AHWR. 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Cell Metab (2017) 25:412–27. doi: 10.1016/j.cmet.2016.11.009 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. 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 © 2021 Ma, Murakami, Saito, Ito, Murata, Nishitani, Hashimoto, Tanaka, Taniguchi, Kitagori, Akizuki, Nakashima, Yoshifuji, Ohmura, Morinobu and Mimori. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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https://openalex.org/W2080834130	http://dm5migu4zj3pb.cloudfront.net/manuscripts/0/375/JCI9800375.pdf	English	null	Various mechanisms cause RET-mediated signaling defects in Hirschsprung's disease.	The Journal of clinical investigation/The journal of clinical investigation	1,998	cc-by	8,089	Introduction Hirschsprung’s disease (HSCR) is a common congenital malformation characterized by the absence of intramural ganglion cells of the hindgut. Recently, mutations of the RET tyrosine kinase receptor have been identified in 50 and 15–20% of familial and sporadic HSCR, respectively. These mutations include deletion, insertion, frameshift, nonsense, and missense mutations dispersed throughout the RET cod- ing sequence. To investigate their effects on RET function, seven HSCR missense mutations were introduced into ei- ther a 1114–amino acid wild-type RET isoform (RET51) or a constitutively activated form of RET51 (RET-MEN 2A). Here, we report that one mutation affecting the extracyto- plasmic cadherin domain (R231H) and two mutations lo- cated in the tyrosine kinase domain (K907E, E921K) im- paired the biological activity of RET-MEN 2A when tested in Rat1 fibroblasts and pheochromocytoma PC12 cells. However, the mechanisms resulting in RET inactivation differed since the receptor bearing R231H extracellular mu- tation resulted in an absent RET protein at the cell surface while the E921K mutation located within the catalytic do- main abolished its enzymatic activity. In contrast, three mu- tations mapping into the intracytoplasmic domain neither modified the transforming capacity of RET-MEN 2A nor stimulated the catalytic activity of RET in our ligand-inde- pendent system (S767R, P1039L, M1064T). Finally, the C609W HSCR mutation exerts a dual effect on RET since it leads to a decrease of the receptor at the cell surface and converted RET51 into a constitutively activated kinase due to the formation of disulfide-linked homodimers. Taken to- gether, our data show that allelic heterogeneity at the RET locus in HSCR is associated with various molecular mecha- nisms responsible for RET dysfunction. (J. Clin. Invest. 1998. 101:1415–1423.) Key words: RET • tyrosine kinase • Hirschsprung’s disease • mutations • genetics Hirschsprung’s disease (HSCR,1 aganglionic megacolon) is a frequent congenital malformation (1/5,000 live births) respon- sible for intestinal obstruction in neonates, and characterized by the absence of parasympathetic intrinsic ganglion cells in the terminal hindgut (1). Since enteric neuroblasts are derived from neural crest cells, HSCR is regarded as a neurocristopa- thy (2, 3). According to segregation analysis suggesting incom- pletely penetrant dominant inheritance in long-segment HSCR (4), we and others have mapped a dominant gene for HSCR to chromosome 10q11.2 (5, 6) and ascribed the disease to het- erozygous mutations of the RET protooncogene (7, 8) that en- codes a tyrosine kinase receptor (9). Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease Anna Pelet,* Olivier Geneste,‡ Patrick Edery,* Andrea Pasini,‡ Sophie Chappuis,‡ Tania Attié,* Arnold Munnich,* Gilbert Lenoir,‡ Stanislas Lyonnet,* and Marc Billaud‡ Unité de Recherches sur les Handicaps Génétiques de l’Enfant INSERM U-393 et Département de Génétique, Hôpital Necker-Enfants Malades, 75743, Paris Cedex 15, France; and ‡Laboratoire de Génétique, CNRS UMR 5641, Domaine Rockefeller, 69373 Lyon Cedex 08, France Gilbert Lenoir,‡ Stanislas Lyonnet, and Marc Billaud‡ Unité de Recherches sur les Handicaps Génétiques de l’Enfant INSERM U-393 et Département de Génétique, Hôpital Necker-Enfants Malades, 75743, Paris Cedex 15, France; and ‡Laboratoire de Génétique, CNRS UMR 5641, Domaine Rockefeller, 69373 Lyon Cedex 08 France J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/98/03/1415/09 $2.00 Volume 101, Number 6, March 1998, 1415–1423 http://www.jci.org Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease Methods Biotin labeling of cell surface proteins. NIH3T3 cells stably ex- pressing mutant RET proteins were washed with biotinylation buffer (10 mM Hepes, pH 8.8, 150 mM NaCl) then biotin (Biosys, Com- piègne, France) was added to a final concentration of 150 mg/ml of biotinylation buffer and incubated 15 min at 208C. The biotinylation reaction was stopped by the addition of Tris-HCl, pH 7.4, to a final concentration of 50 mM. Cells were washed with 10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl (21, 26). Cell pellets were precipitated by streptavidin agarose (20347; Pierce Chemical Co., Rockford, IL) and analyzed on an 8% SDS-PAGE gel. Site-directed mutagenesis and production of ecotropic retroviruses. The cDNA coding for the 1,114–amino acid isoform of human RET (RET51) was cloned into the pBabe Puro retroviral vector (23, 30). HSCR mutations were introduced by site-directed mutagenesis (Unique Site Elimination Mutagenesis kit; Pharmacia Biotech, Piscataway, NJ) with the following primers: R231H: 59-CTGGGCCCTGGAC- CACGAGCAGCGGGAGAAGTACG-39; C609W: 59-GGCTATG- GCACCTGGAACTGCTTCCCT-39; S767R: 59-GAACGCCTCCCC- GAGGGAGCTTCGAGACCTG-39; K907E: 59-GAGGATTCCTA- CGTGGAGAGGAGCCAGGGTC-39; E921K: 59-GTTAAATG- GATGGCAATTAAATCCCTTTTTGATCAT-39; P1039L: 59-GAG- GAGGAGACACTGCTGGTGGACTGT-39; M1064T: 59-CAAA- CTCTATGGCACGTCAGACCCGAAC-39. Each mutation was con- firmed by DNA sequencing on both strands. Ecotropic retroviruses were obtained by transient transfection of the pBabe Puro retroviral vectors into the BOSC 23 packaging cell line according to the proto- col described by Pear et al. (31). Titrations of the virus supernatants and infections were performed as described previously (23). y g Analysis of Shc phosphorylation. NIH3T3 cells stably expressing the various RET alleles were starved overnight in medium containing 0.5% of FBS and then lysed as described previously (23). Shc proteins were immunoprecipitated with a polyclonal anti-Shc antibody (Trans- duction Laboratories). Immunoprecipitates were subjected to 12% SDS-PAGE and transferred to a polyvinylidene difluoride mem- brane (Immobilon P; Millipore Corp., Bedford MA). To analyze the phosphotyrosine content of Shc, immunoblots were incubated with the antiphosphotyrosine monoclonal antibody 4G10 (Upstate Bio- technology Inc.), then incubated with anti–mouse antibody conju- gated to horseradish peroxidase (HRP) (Amersham France, Les Ulis, France). The immunoreactive complexes were visualized using che- miluminescence (ECL Western blotting system; Amersham France). The membranes were then stripped by incubation in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8) and treated at 558C for 30 min. Introduction Recently, it has been shown that RET is the signaling component of a multisubunit complex acting as a receptor for the glial cell line–derived neu- rotrophic factor (GDNF) (10–13) and neurturin (14) two neu- rotrophic factors related to the TGF-b family, both requiring GPI-linked glycoprotein coreceptors to allow RET dimeriza- tion (GDNFRa and GDNFRb) (15). Germline mutations of RET are also responsible for multiple endocrine neoplasia type 2 (MEN 2), a group of dominantly inherited cancer syn- dromes (for reviews see references 16–18). MEN 2 mutations are missense mutations located in the cysteine-rich region of the extracellular domain of RET and cause the replacement of one of five clustered cysteines by a different amino acid (MEN 2A) or within the catalytic tyrosine kinase domain (MEN 2B) (19). Recently, we and others have demonstrated that MEN 2A mutations convert the RET protooncogene in a dominantly acting transforming gene due to covalent dimerization of RET monomers via disulfide bond, resulting in ligand-independent constitutive activation of the tyrosine kinase (20–23). Con- versely, several lines of evidence support the view that HSCR mutations are inactivating and cause the loss of RET function, namely: (a) the nature of mutations which include both trunca- tion and missense mutations scattered throughout the RET gene coding sequence (24); (b) the ability of HSCR missense mutations to abolish the transforming effects of oncogenic forms of RET (25–27); and (c) the targeted homozygous dis- ruption of RET that results in intestinal aganglionosis in mice (28). However, some observations are difficult to reconcile with a loss of function model such as the cosegregation of MEN 2A and HSCR in several families (29). To better under- stand the molecular mechanisms underlying RET dysfunction, we have analyzed the biological consequences of seven inde- Address correspondence to Marc Billaud, Laboratoire de Génétique, CNRS UMR 5641, Domaine Rockefeller, 8, avenue Rockefeller, 69373 Lyon Cedex 08, France. Phone: 33-478-77-72-13; FAX: 33-478- 77-72-20; E-mail: billaud@univ-lyon1.fr Olivier Geneste’s present ad- dress is Transcription Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London W2CA 3PX, United Kingdom. Received for publication 10 April 1997 and accepted in revised form 7 January 1998. 1. Abbreviations used in this paper: GDNF, glial cell line–derived neu- rotrophic factor; HRP, horseradish peroxidase; HSCR, Hirsch- sprung’s disease; MBP, myelin basic protein; MEN, multiple endo- crine neoplasia. 1415 Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease tein (MBP). Introduction The reaction was stopped by addition of Laemmli buffer (20 mM Tris-HCl, pH 6.8, 2 mM EDTA, 2% SDS, 20% glycerol, and 20 mg/ml of bromophenol blue) in the presence of 1.4 M of 2-mercap- toethanol. Proteins were boiled 3 min, then loaded on a 15% SDS polyacrylamide gel. 32P-labeled bands were revealed by autoradiogra- phy of the dried gel and quantified on an imaging densitometer (model GS 670; Bio-Rad Laboratories, Richmond, CA). To detect the disulfide-linked RET homodimer, the same immunokinase assay protocol was used, except that 2-mercaptoethanol was omitted at the last step. Proteins were boiled 3 min and separated on a 6% SDS polyacrylamide gel. pendent HSCR mutations. Here we report that three HSCR mutations inhibited the biological action of a constitutively ac- tive form of RET while one mutation transformed the RET gene into an oncogene. Finally, three HSCR mutations had no detectable effect on RET function in our system. Thus, we demonstrate that RET dysfunction in HSCR could be ascribed to various mechanisms depending on the location of missense mutation in the RET protein. Methods To study the Shc proteins, the filters were sequentially reacted with the polyclonal anti-Shc serum (Trans- duction Laboratories) for 2 h at room temperature, then with biotiny- lated protein A (Amersham France) for 1 h and finally with HRP conjugated to streptavidin (Pierce, Interchim, France) for 1 h. The ECL system was used for the revelation of immunoreactive bands. 2 mg of Shc antibody was used for each immunoprecipitation experi- ment and the same serum was diluted at a final concentration of 1 mg/ ml for Western blot; biotinylated protein A was diluted 1:500; strepta- vidin-HRP was used at a final concentration of 1 mg/ml. Anchorage-independent growth of Rat1 cells. Retroviral infected Rat1 cells were mass-selected with puromycin and seeded into cul- ture medium containing 10% FCS and 0.3% agar as described in Ros- sel et al. (23). Colonies were counted 3 wk after plating. Effects of the expression of mutant RET proteins in PC12 cells. PC12 cells were maintained in DME supplemented with glucose (4.5 g/liter), 6% horse serum (Sigma Chemical Co., St. Louis, MO), 6% FCS, and antibiotics (32). PC12 cells were plated at 3 3 105 cells per 60-mm culture dish. The day after plating, cells were incubated with 106 infectious particles in 3 ml of the culture medium containing 8 mg/ml of polybrene. 7 h after infection, the medium was removed and re- placed by fresh medium. Micrographs were taken 4 d after infection. RET protein studies. The antiserum anti-RET was raised against a peptide corresponding to the 20 amino acids of the carboxy-termi- nal part of the short RET isoform (prepared by Biocytex, Marseille, France). This antibody reacts with both the short (RET9) and long RET (RET51) isoforms (23). Other antibodies used in this study are commercially available: the monoclonal antiphosphotyrosine 4G10 and the rabbit polyclonal anti-Shc were purchased from Upstate Bio- technology Inc. (Lake Placid, NY) and Transduction Laboratories (Lexington, KY), respectively. RET protein analyses were performed as described by Rossel et al. (23). Results The various domains of the RET protein are indicated: SP, sig- nal peptide; wavy line, cadherin-domain; shaded oval, cysteine-rich domain; TM, transmembrane domain; striped box, ty- rosine kinase domain. tations S767R, P1039L, and M1064T did not substantially modify the transforming capacity of RET-634R, we could not formally exclude that they might be activating. To address this question, these mutations were introduced in RET wild-type. Rat1 cells transduced with retroviruses expressing these mutants did not produce colonies in agar (data not shown). Finally, HSCR mutation C609W on a wild-type RET back- ground gave rise to RAT1 cell colonies, although the trans- forming effect was weaker than the one observed with RET- 634R (Table I). the effects of HSCR mutations on RET intrinsic kinase activ- ity, the mutant RET proteins were immunoprecipitated and analyzed by Western blot technique. As previously described, two immunoreactive bands migrating at 150 and 170 kD were detected. These two RET species are assumed to correspond to an incompletely glycosylated RET product present in the endoplasmic reticulum (150 kD) and to the fully glycosylated protein expressed at the plasma membrane (170 kD) (21, 33). Fig. 3 shows that the amount of 170-kD RET isoform is de- creased in cells expressing RET-609W and RET-634R/231H as compared with cell lines expressing wild-type RET or other RET mutants. Conversely, the level of the 150-kD product is markedly increased in cell lines expressing RET-609W and RET-634R/231H (Fig. 3 A). To test the catalytic activity of RET HSCR mutants, we measured the level of steady-state phosphorylation on tyrosine residues. In accordance with pre- vious reports, the RET-634R mutant showed a significant in- crease of phosphotyrosine content (20–23). Three RET HSCR mutants were not phosphorylated (RET-609W, RET-634R/ 231H, and RET-634R/921K, Fig. 3 B). Conversely, RET-634R protein carrying each of the four HSCR mutants K907E, S767R, 1039L, and M1064T was phosphorylated on tyrosine, although at a reduced level for the three latter mutants. Fi- nally, mutations S767R, P1039L, and M1064T did not activate the RET wild-type catalytic activity (Fig. 3 B). Previous reports have shown that the expression of MEN 2 forms of RET is transforming in rodent fibroblasts and induces neuronal differentiation of rat pheochromocytoma PC12 cells (20–23). PC12 cells infected with retroviruses expressing RET- 634/767R, RET-634R/1039L, RET-634R/1064T, and RET- 609W elaborated neurites and displayed morphological changes similar to those observed with RET-634R. Results Six HSCR mutations were introduced into a cDNA encoding a constitutively activated MEN 2A mutant form of RET (RET51-C634R, Fig. 1). The C609W HSCR mutation leads to the replacement of a cysteine involved in MEN 2A (19). This mutation was likely to result in RET activation and thus was introduced into the RET51 wild-type cDNA. y ( ) Immunokinase assay and studies of the RET covalent dimer. Mass-selected NIH 3T3 cells stably expressing mutant RET proteins were rinsed with PBS, scraped into 1 ml of lysis buffer (20 mM Tris- HCl, pH 7.8, 150 mM NaCl, 1 mM sodium orthovanadate, 1% Nonidet P-40, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin and 2 mM EDTA), and incubated on ice for 15 min. Lysates were precleared with the rabbit preimmune serum, then subjected to immunoprecipi- tation with the anti-RET serum. Immune complexes were absorbed on protein A coupled to Sepharose beads (protein A–Sepharose CL4B; Pharmacia Biotech) and successively washed twice in immu- noprecipitation buffer and twice in immunokinase assay buffer (50 mM Hepes, pH 7.2, 5 mM MnCl2, 1 mM PMSF). The immunoprecipi- tates were then incubated for 20 min at room temperature in 30 ml of immunokinase buffer containing 4 mCi of [g-32P]ATP (5,000 Ci/ mmol; ICN Biomedicals Inc., Irvine, CA) diluted with unlabeled ATP to a final concentration of 26 pmol with 7 mM myelin basic pro- yp Consequences of HSCR mutations on RET-induced bio- logical effects. To investigate the biological effects of HSCR mutations, we assessed the anchorage-independent growth ca- pacity of Rat1 cells stably expressing various RET mutants. 3 wk after seeding in soft agar, Rat1 cells expressing RET-634R containing mutations S767R, P1039L, and L1064T gave rise to colonies (Table I). Conversely, the R231H, HSCR mutant sig- nificantly reduced the number of colonies in transduced RAT1 cells, and mutations K907E and E921K produced a number of colonies not significantly different from the background levels observed with the wild-type RET control (Table I). Since mu- 1416 1416 Pelet et al. Pelet et al. Figure 1. Schematic representation of the HSCR mutations introduced into RET wild-type or RET-MEN 2A. (Top) HSCR missense mutations analyzed in this study. (Bottom) The RET-MEN 2A mutant C634R was used as a constitutively acti- vated form of RET. Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease 1417 Results On the contrary, PC12 cells expressing RET-634R with HSCR mutations R231H, K907E, and E921K did not show any phenotypic change as compared with PC12 cells expressing wild-type RET (Fig. 2). These results indicate that several HSCR mutations abolish (K907E and E921K) or dramatically reduce (R231H) the biological activity of RET-634R, whereas HSCR mutations S767R, P1039L, and M1064T did not substantially modify the capacity of RET-634R to transform fibroblasts and to induce neuronal differentiation of PC12 cells. Finally, the RET-609W HSCR mutant exhibited the same transforming and neural dif- ferentiation abilities as the RET MEN 2 mutants, although to a weaker extent. We next examined the catalytic activity of HSCR mutants with an immunocomplex-kinase assay using the MBP as an ex- ogenous substrate (Fig. 4). Quantification of the reactive bands showed that the level of MBP phosphorylation correlated to RET autophosphorylation allowing further classification of the kinase activity of the various RET mutants (RET-634R; Biochemical analyses of RET HSCR mutants. To address Table I. Transformation Effects of RET51 Wild-Type and Mutant Protein on RAT1 Cell Lines WT 634R 634R/231H 609W 634R/767R 634R/907E 634R/921K 634R/1039L 634R/1064T First assay , 3 113 41 40 130 6 3 100 , 3 100 50 62 100 19 10 130 ND , 3 110 52 60 112 26 3 P , 1029 P , 1029 NS P , 1029 P , 1023 NS Second assay , 3 130 25 45 180 7 4 207 134 , 3 130 29 67 175 11 2 180 113 , 3 170 55 59 165 8 2 110 P , 1029 P , 1028 P , 0.04 P , 1023 P , 1023 P , 1022 NS Two independent infection assays were carried out on 103 RAT1 cells. Foci were scored 3 wk after plating. The number of foci is indicated for each plate. ND, Not determined. A x2 test was performed. Student’s t test was performed due to the very low number of colonies. Table I. Transformation Effects of RET51 Wild-Type and Mutant Protein on RAT1 Cell Lines Table I. Transformation Effects of RET51 Wild-Type and Mutant Protein o Effects of RET51 Wild-Type and Mutant Protein on RAT1 Cell Lines Two independent infection assays were carried out on 103 RAT1 cells. Foci were scored 3 wk after plating. The number of foci is indicated for each plate. ND, Not determined. A x2 test was performed. Two independent infection assays were carried out on 103 RAT1 cells. Foci were scored 3 wk after plating. The number of foci is indicated for each plate. ND, Not determined. A x2 test was performed. Student’s t test was performed due to the very low number of colonies. Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease 1417 Results *Student’s t test was performed due to the very low number of colonies. 1417 Figure 2. Effects of RET carrying HSCR mutations on PC12 cell morphology. The PC12 pheochro- mocytoma cell line was infected with retro- viruses expressing nor- mal RET51 (RET WT), mutant RET alleles, and control (RET51/634R, RET-918T, and LacZ NIH3T3 expressing b galactosidase). Micro- graphs were taken 4 d after infection. Neuronal differentiation was ob- served for RET-609W, RET-634R/767R, RET- 634R/1039L, and RET- 634R/1064T. Figure 2. Effects of RET carrying HSCR mutations on PC12 cell morphology. The PC12 pheochro- mocytoma cell line was infected with retro- viruses expressing nor- mal RET51 (RET WT), mutant RET alleles, and control (RET51/634R, RET-918T, and LacZ NIH3T3 expressing b galactosidase). Micro- graphs were taken 4 d after infection. Neuronal differentiation was ob- served for RET-609W, RET-634R/767R, RET- 634R/1039L, and RET- 634R/1064T. Figure 2. Effects of RET carrying HSCR mutations on PC12 cell morphology. The PC12 pheochro- mocytoma cell line was infected with retro- RET-609W . RET-634R/767R; RET-634R/907E; RET- 634R/1064T . RET-634R/1039L . RET-634R/231H . RET- WT . . RET-634R/921K). These results are consistent with the analysis of the steady-state phosphorylation of RET HSCR mutants, although it should be noted that the immu- nokinase assay allowed detection of a clear increase of RET- 609W kinase activity. RET-609W . RET-634R/767R; RET-634R/907E; RET- 634R/1064T . RET-634R/1039L . RET-634R/231H . RET- WT . . RET-634R/921K). These results are consistent with the analysis of the steady-state phosphorylation of RET HSCR mutants, although it should be noted that the immu- nokinase assay allowed detection of a clear increase of RET- 609W kinase activity. tivation. The last four HSCR mutants do not significantly im- pair RET, MBP, or Shc phosphorylation. p , , p p y HSCR mutations affecting the extracytoplasmic domain re- sult in a reduced level of RET protein expressed at the cell sur- face. The reduction of the 170-kD RET product observed in NIH3T3 cells expressing RET-634R/231H or RET-609W sug- gested that inhibition of RET transport to the cell surface might account for the disease-causing effects of HSCR muta- tion (Fig. 6). To test this hypothesis, NIH3T3 cells expressing equal amounts of RET protein were biotinylated in vivo. Western blot analysis shown in Fig. 6 A revealed that the RET-634R/231H and RET-609W 170-kD isoforms were dra- matically reduced at the cell surface. Fig. Results 6 A, these findings support the view that the C609W HSCR mu- tation exerts a dual effect on RET resulting in both a reduced expression at the cell surface and the formation of a covalent dimer. assay. The 340-kD product corresponding to RET homodimer and specific to RET MEN 2 mutants carrying a cysteine muta- tion was detected in NIH3T3 cells expressing RET-609W and RET-634R (Fig. 7). Together with the results showed in Fig. 6 A, these findings support the view that the C609W HSCR mu- tation exerts a dual effect on RET resulting in both a reduced expression at the cell surface and the formation of a covalent dimer. Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease 1419 Results 6 B shows that RET wild-type proteins bearing mutation affecting the intracyto- plasmic domain (S767R, P1039L, M1064T, as well as RET51- 634R/907E and RET-634R/921K) were readily detected at the plasma membrane, thus supporting the view that HSCR muta- tions located in the extracytoplasmic domain decrease the amount of mature receptor present at the cell surface. y We then studied the tyrosine phosphorylation of Shc, a transduction protein that bridges receptor tyrosine kinases to the Ras-mitogen activated protein kinase pathway (34) and that has been shown to be phosphorylated by MEN 2 forms of RET (35–37). Fig. 5 shows that equivalent amounts of the three Shc isoforms were present in each lane (46, 52, and 66 kD). How- ever, the phosphotyrosine content of p52Shc, p46Shc and, to a lesser extent, of p66Shc was markedly increased in NIH3T3 cells expressing RET-609W as well as RET-634R carrying HSCR mutations S767R, K907E, P1039L, and M1064T. Conversely, the tyrosine phosphorylation of Shc was comparable to that of RET wild type in cell lines expressing RET-634R/231H or RET-634R/921K (Fig. 5). Therefore, both sets of data indicate that two RET HSCR mutants inactivate RET-634R catalytic functions (R231H, E921K), while the C609W leads to RET ac- HSCR mutation C609W has a dual effect on RET. To in- vestigate whether the C609W HSCR mutation might result in RET dimerization, we performed an immunocomplex kinase 1418 Pelet et al. 8 Pelet et al. 1418 Figure 2 (Continued) Figure 2 (Continued) Figure 2 (Continued) Figure 2 (Continued) Figure 2 (Continued) segment (total colonic aganglionosis for mutations S767R and E921K) and the associated malformations (unilateral renal agenesis and congenital central hypoventilation syndrome for mutation K907E and P1039L, respectively). This study has re- vealed that three mutations disrupted the biological activity of the MEN 2A form of RET (R231H, K907E, E921K), while one mutation resulted in the oncogenic activation of RET due to the constitutive stimulation of the tyrosine kinase (C609W). Finally, in our system three HSCR mutations did not signifi- cantly change the biological effects mediated by RET-MEN 2A (S767R, P1039L, M1064T). assay. The 340-kD product corresponding to RET homodimer and specific to RET MEN 2 mutants carrying a cysteine muta- tion was detected in NIH3T3 cells expressing RET-609W and RET-634R (Fig. 7). Together with the results showed in Fig. Discussion Recent work has suggested that HSCR point mutations in the RET gene are invariably inactivating mutations although through distinct molecular mechanisms (25–27). In this report, we have investigated the effects of seven distinct HSCR mis- sense mutations on RET function. Three mutations were se- lected owing to their location in the RET protein, namely, the cadherin-like domain (R231H), the cysteine-rich motif at a cysteine codon involved in MEN 2A (C609W), and the car- boxy-terminal region specific for the long RET isoform (M1064T). Four mutations were chosen based on the pheno- type of HSCR patients including the length of the aganglionic Arginine 231 is located in the RET cadherin domain. It is one of the four residues of the motif Leu-Asp-Arg-Glu (LDRE) that is highly conserved in most cadherins and forms one of the loops involved in calcium-binding (38, 39). How- ever, while the RET cadherin domain can be superimposed to the amino-terminal cadherin domains of E-cadherin (Legrand, P., and M. Billaud, unpublished results) its function is not yet defined, especially as RET does not display clear Ca21-depen- dent homophilic adhesive properties (40). The substitution of arginine 231 for histidine leads to a drastic reduction of the mutant RET protein present at the cell surface. Similarly, mu- 1419 Figure 3. Expression and tyrosine phosphorylation of mutant RET proteins. Comparable amounts of RET proteins were immunoprecipitated from mass populations of puromycin-resistant NIH3T3 cells expressing the various RET mutants. Controls were NIH, RETWT, RET-634R, and LacZ. (A) RET monomer detection. Samples were fractionated on an 8% SDS polyacrylamide gel and analyzed by Western blotting with an anti-RET polyclonal serum. The two RET species of 170 and 150 kD are indicated. (B) Tyrosine phosphorylation of RET. Western blots were reprobed with the monoclonal antiphosphotyrosine antibody 4G10. The 170-kD phosphorylated band was absent for three constructions, namely RET-634R/231H, RET-609W, and RET-634R/921K and was decreased for RET-634R/1039L and the RET-634R/1064T. Figure 3. Expression and tyrosine phosphorylation of mutant RET proteins. Comparable amounts of RET proteins were immunoprecipitated from mass populations of puromycin-resistant NIH3T3 cells expressing the various RET mutants. Controls were NIH, RETWT, RET-634R, and LacZ. (A) RET monomer detection. Samples were fractionated on an 8% SDS polyacrylamide gel and analyzed by Western blotting with an anti-RET polyclonal serum. The two RET species of 170 and 150 kD are indicated. (B) Tyrosine phosphorylation of RET. Western blots were reprobed with the monoclonal antiphosphotyrosine antibody 4G10. Discussion The 170-kD phosphorylated band was absent for three constructions, namely RET-634R/231H, RET-609W, and RET-634R/921K and was decreased for RET-634R/1039L and the RET-634R/1064T. tations of either codon Asp 264 (D264K) or Asp 300 (D300K) which are part of the putative Ca21-binding site of the RET cadherin domain, and mutation of arginine 287 (R287Q) which is also located in the cadherin domain resulted in the intracel- lular accumulation of the immature 150-kD product (21) (Chappuis, S., and M. Billaud, unpublished results). Other HSCR mutations affecting the extracytoplasmic domain, out- side the cadherin domain, have the same disruptive effect as mutation R231H. Thus, these data therefore support the idea that most extracellular missense mutations of RET might alter the protein folding and cause inappropriate intracellular traf- ficking (26, 27). Figure 4. Immunocomplex kinase assay. Equal amounts of RET pro- teins were immunoprecipitated from lysates of NIH3T3 cells stably expressing various RET mutants. The immunocomplexes were sub- jected to an in vitro kinase assay using MBP as an exogenous sub- strate. Bands corresponding to autophosphorylated RET and to phosphorylated MBP are indicated. Among HSCR mutations located in the RET tyrosine ki- nase domain tested in this study, two out of three inhibited the biological activity of the MEN 2A form of RET (K907E, E921K). Although the functional consequences of each muta- tion were the same, biochemical analyses suggested that the underlying molecular mechanisms were different. Mutation K907E did not abrogate the tyrosine kinase activity of RET- MEN 2A since this mutant was still able to efficiently phos- phorylate substrates such as MBP and Shc. These results indi- cate that the K907E mutation might interfere with one of the RET-signaling pathways required for mitogenesis. Since Lys 907 is located in the close vicinity of Tyr 905 (41), a residue whose autophosphorylation creates a putative docking site for Grb10 and possibly Grb7, it is tempting to speculate that the K907E mutation might uncouple Grb7 and/or Grb10 from RET, thus dampening down the RET signal (27, 42–44). The negative effect of mutation E921K on RET-MEN 2A enzy- matic activity implies that Glu 921, an amino acid strictly con- served in all serine/threonine and tyrosine kinases identified thus far, participates in the folding of the RET kinase domain (45). Similarly, three other HSCR mutations located in the ty- rosine kinase domain have been shown to abolish or decrease the kinase activity of oncogenic forms of RET (25). without any clinical manifestation involving either thyroid or adrenals (24) while other missense mutations at codon 609 have been identified in 8% of MEN 2A (19) . Thus, it ap- peared likely that the replacement of cysteine 609 by a tryp- tophan exerted a disruptive effect on RET. Yet, the C609W mutation was found to cause RET covalent dimerization simi- lar to the one observed with MEN 2A cysteine mutations. However, the amount of the mature 170-kD form of RET present at the cell membrane was significantly decreased as compared with RET wild type or RET-634R, thereby indicat- ing that mutation C609W also impaired the RET maturation. Consequently, the transforming capacity of RET-609W was approximately twofold less than the one displayed by RET- 634R. It is thus tantalizing to hypothesize that the weak level of the RET dimer cannot compensate for the marked decrease of RET at the plasma membrane of enteric neuroblasts result- ing in the HSCR phenotype. We cannot exclude the possibility that the covalent dimerization of RET-609W requires the overexpression of RET, the same as achieved in NIH3T3 cells and that, under physiological conditions, the dimer is not formed in enteric neuroblasts. Along the same lines, it is worth noting that two mutations, previously reported in MEN 2A pa- tients have been identified in HSCR patients without evidence of MEN 2 phenotype (C609Y, C620R) (46). Finally, the same dual impact (the formation of disulfide-bound RET ho- modimer and inhibition of RET maturation) (47) might ex- plain the occurrence of both HSCR and MEN 2A in rare fami- lies with RET mutation at codons 618 or 620. Finally, three HSCR mutations did not substantially mod- ify the biological activity of RET-MEN 2A in our systems (S767R, P1039L, M1064T). Mutation S767R was found in a sporadic case, while the two other mutations were inherited Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease 1421 Figure 6. Cell surface expression of mu- tant RET proteins. NIH3T3 cells stably expressing equivalent amount of mutant RET proteins were labeled with biotin and immunoprecipitated with streptavi- din conjugated to agarose. The resultant immunoprecipitates were separated on an 8% SDS polyacrylamide gel and Western blotted with an anti-RET poly- clonal serum. The immunoreactive band corresponds to the fully glycosylated RET product of 170 kD present at the cellular membrane. Discussion Figure 4. Immunocomplex kinase assay. Equal amounts of RET pro- teins were immunoprecipitated from lysates of NIH3T3 cells stably expressing various RET mutants. The immunocomplexes were sub- jected to an in vitro kinase assay using MBP as an exogenous sub- strate. Bands corresponding to autophosphorylated RET and to phosphorylated MBP are indicated. The RET C609W mutation occurred in an HSCR family 1420 1420 Pelet et al. Figure 5. Tyrosine phosphorylation of Shc by RET mutants. Retroviral infected NIH3T3 cells stably expressing the various RET mutants were starved overnight in 0.5% FBS and then whole cell lysates were prepared. (A) Shc proteins were immuno- precipitated with a rabbit polyclonal anti- Shc antibody (Transduction Laboratories) and the resultant immunoprecipitates were subjected to Western blot analysis using a monoclonal antiphosphotyrosine antibody (4G10; Upstate Biotechnology Inc.). (B) Filters were reprobed with the anti-Shc an- tibody; the three Shc isoforms of 66, 52, and 46 kD are indicated. Figure 5. Tyrosine phosphorylation of Shc by RET mutants. Retroviral infected NIH3T3 cells stably expressing the various RET mutants were starved overnight in 0.5% FBS and then whole cell lysates were prepared. (A) Shc proteins were immuno- precipitated with a rabbit polyclonal anti- Shc antibody (Transduction Laboratories) and the resultant immunoprecipitates were subjected to Western blot analysis using a monoclonal antiphosphotyrosine antibody (4G10; Upstate Biotechnology Inc.). (B) Filters were reprobed with the anti-Shc an- tibody; the three Shc isoforms of 66, 52, and 46 kD are indicated. Acknowledgments We thank Dr. W.S. Pear for providing us with the BOSC 23 cells and Dr. H. Land for the pBabe Puro retroviral vector. We wish to thank L. Fournier for her excellent technical help, Mrs. M. Billaud and S. Balter for the graphic work, and S. Lefebvre, J. Amiel, and R. Salomon for helpful discussion. from an unaffected parent (24). These mutations have not been identified in 100 control chromosomes and it seems un- likely that they are rare polymorphisms, although this possibil- ity cannot be completely ruled out. However, it is possible that these mutations can reduce the activation of RET in response to GDNF, a hypothesis that should be tested in cells that stably express the RET coreceptor GDNFRa (data not shown). It is also possible that the signaling pathways activated from RET in enteric neurons depend on an array of transduction effec- tors which might not be expressed in fibroblasts or PC12 cells, while these mutations would indeed interfere with the ability of RET to mediate either the GDNF or the neurturin signal. Our data emphasize the necessity to develop fibroblast and/or neuronal cell cultures expressing RET coreceptors (GDNFRa and/or GDNFRb) providing better insights into the conse- quences of these mutations on RET function. This study was supported by the Ligue Nationale Contre le Can- cer (Axe Oncogénèse and Comité Départemental de Saône et Loire), the Association de la Recherche sur le Cancer (ARC), the Associa- tion Française contre la Myopathie (AFM), the Assistance Publique- Hopitaux de Paris (AP-HP, PHRC94), and the Ministère de la Re- cherche (ACC-SV2). During this study, O. Geneste was the recipient of a Fellowship from the Ligue Nationale Contre le Cancer, Comité Départemental de l’Indre. S. Chappuis and A. Pasini are the recipi- ents of a fellowship from the Association de Recherche sur le Cancer and from the Ligue Nationale contre le Cancer, respectively. depend on the location of the mutation in the protein. We then propose to classify HSCR missense mutations of RET into four groups. Class I mutations are located in the extracytoplas- mic domain, causing impairment in RET maturation and pre- venting the protein from reaching the cell surface. Class II mu- tations in the cysteine-rich domain might be responsible for both the formation of covalently linked RET dimers and the transport defect of the protein. Class III mutations are located in the tyrosine kinase domain altering either the catalytic activ- ity or the stability of the enzyme structure. Finally, class IV mutations are found in the intracytoplasmic domain and might prevent the interaction between RET and cytosolic proteins which would be critical to signaling events. However, the ab- sence of correlation between the nature or the position of the RET mutations and the clinical phenotype as well as the low penetrance of the disease indicates that other genetic factors might contribute to the HSCR phenotype. The recent demon- stration that germline mutations in the gene encoding GDNF might either be responsible for or modify the HSCR clinical manifestations strengthens the view that the role of the multi- component RET/GDNFR-a receptor is crucial during enteric neurogenesis (48–50). The identification of such components of the RET-signaling pathways should extend our understand- ing of the molecular bases of HSCR. Note added in proof: Since the submission of this manuscript, Ta- kahashi and co-workers reported on the biochemical analysis of the C609W mutation with results paralleling our data regarding C609W HSCR mutation (51). Figure 7. Dual effect of HSCR mutation C609W. Mutation C609W promotes RET covalent dimerization. NIH3T3 cells stably expressing equivalent amount of mutant RET proteins were lysed and the RET receptor was immunoprecipitated and subjected to an in vitro immu- nokinase assay using [g-32P]ATP. 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A survey of these results allow us to draw several conclusions: (a) the vast majority of HSCR mis- sense mutations (12/16) result in RET inactivation, therefore implying that HSCR can be merely ascribed to the loss of RET function or a reduced dosage of RET protein; (b) however, rare HSCR-causing mutations could be activating (e.g., C609W); and (c) the molecular mechanisms of RET dysfunction highly g g 4. Badner, J., W. Sieber, K. Garver, and A. Chakravarti. 1990. A genetic study of Hirschsprung disease. Am. J. Hum. Genet. 46:568–580. 5. Lyonnet, S., A. Bolino, A. Pelet, L. Abel, C. Nihoul-Fékété, M. Briard, V. Mok Siu, H. Kääriäinen, G. Martucciello, M. Lerone, et al. 1993. A gene for Hirschsprung disease maps to the proximal long arm of chromosome 10. Nat. Genet. 4:346–350. 6. Angrist, M., E. Kauffman, S.A. Slaugenhaupt, M. Cox Matise, E.G. Puffenberger, S.S. Washington, A. Lipson, D.T. Cass, T. Reyna, D.E. Weeks, et al. 1993. A gene for Hirschsprung disease (megacolon) in the pericentromeric region of human chromosome 10. Nat. Genet. 4:351–356. g 7. Edery, P., S. Lyonnet, L. Mulligan, A. Pelet, E. Dow, L. Abel, S. Holder, C. Nihoul-Fekete, B. Ponder, and A. Munnich. 1994. Mutations of the RET 1422 Pelet et al. proto-oncogene in Hirschsprung’s disease. Nature. 367:378–380. verse phenotypes associated with exon 10 mutations of the RET proto-onco- gene. Hum. Mol. Genet. 3:2163–2167. 8. Romeo, G., P. Ronchetto, Y. Luo, V. Barone, M. Seri, I. Ceccherini, B. Pasini, R. Bocciardi, M. Lerone, H. Kääriäinen, et al. 1994. Point mutations af- fecting the tyrosine kinase domain of the RET proto-oncogene in Hirsch- sprung’s disease. Nature. 367:377–378. g 30. Morgenstern, J.P., and H. Land. 1990. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18:3587– 3596. 9. Takahashi, M., Y. Buma, T. Iwamoto, Y. Inaguma, and H. Ikeda. 1988. References Asai, N., T. Iwashita, M. Matsuyama, and M. Takahashi. 1995. Mecha- nism of activation of the ret proto-oncogene by endocrine neoplasia 2A muta- tions. Mol. Cell. Biol. 15:1613–1619. 44. Durick, K., R.Y. Wu, G.N. Gill, and S.S. Taylor. 1996. Mitogenic signal- ing by Ret/ptc2 requires association with enigma via a LIM domain. J. Biol. Chem. 271:12691–12694. 22. Borrello, M.G., D.P. Smith, B. Pasini, I. Bongarzone, A. Greco, M.J. Lorenzo, E. Arighi, C. Miranda, C. Eng, L. Alberti, et al. 1995. RET activation by germline MEN2A and MEN2B mutations. Oncogene. 11:2419–2427. 45. Hanks, S.K., A. Quinn, and T. Hunter. 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 241:42–52. 23. Rossel, M., A. Pasini, S. Chappuis, O. Geneste, L. Fournier, I. Schuffe- necker, M. Takahashi, L.A. Van Grunsven, J.L. Urdiales, B.B. Rudkin, et al. 1997. Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations. Oncogene. 14:265–275. 46. Angrist, M., S. Bolk, B. Thiel, E. Puffenberger, R. Hofstra, C. Buys, and A. Chakravarti. 1995. Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease. Hum. Mol. Genet. 4:821–830. 24. Attié, T., A. Pelet, P. Edery, C. Eng, L. Mulligan, J. Amiel, S. Beldjord, C. Nihoul-Fékété, A. Munnich, B. Ponder, and S. Lyonnet. 1995. Diversity of RET proto-oncogene mutations in Hirschsprung’s disease. Hum. Mol. Genet. 4: 2407–2409. 47. Carlomagno, F., G. Salvatore, A.M. Cirafici, G. De Vita, R.M. Melillo, V. De Franciscis, M. Billaud, A. Fusco, and M. Santoro. 1997. The different RET-activating capability of mutations of cysteine 620 or cysteine 634 corre- lates with the multiple endocrine neoplasia type 2 disease phenotype. Cancer Res. 57:391–395. 25. Pasini, B., M.G. Borello, A. Greco, I. Bongarzone, Y. Luo, P. Mondel- lini, L. Alberti, C. Miranda, E. Arighi, R. Bocciardi, et al. 1995. Loss of function effect of RET mutations causing Hirschsprung disease. Nat. Genet. 10:35–40. 48. Salomon, R., T. Attie, A. Pelet, C. Bidaud, C. Eng, J. Amiel, S. Sar- nacki, O. Goulet, C. Ricour, C. Nihoul-Fekete, et al. 1996. Germline mutations of the RET ligand, GDNF, are not sufficient to cause Hirschsprung disease. Nat. Genet. 14:345–347. 26. Carlomagno, F., G. De Vita, M.T. Berlingieri, V. De Franciscis, R.M. Mellilo, V. Colantuoni, M.H. Kraus, P.P. Di Fiore, A. Fusco, and M. Santoro. 1996. Molecular heterogeneity of RET loss of function in Hirschsprung’s dis- ease. EMBO (Eur. Mol. Biol. Organ.) J. 15:2717–2725. Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease References 49. Angrist, M., S. Bolk, M. Halushka, P.A. Lapchak, and A. Chakravarti. 1996. Germline mutations in glial cell line-derived neurotrophic factor (GDNF) and RET in a Hirschsprung disease patient. Nat. Genet. 14:341–343. 49. Angrist, M., S. Bolk, M. Halushka, P.A. Lapchak, and A. Chakravarti. 1996. Germline mutations in glial cell line-derived neurotrophic factor (GDNF) 27. Iwashita, T., H. Murakami, N. Asai, and M. Takahashi. 1996. Mecha- nism of Ret dysfunction by Hirschsprung mutations affecting its extra cellular domain. Hum. Mol. Genet. 5:1578–1580. 50. Ivanchuk, S.M., S.M. Myers, C. Eng, and L.M. Mulligan. 1996. De novo mutation of GDNF, ligand for the RET/GDNFR-a receptor complex, in Hirsch- sprung disease. Hum. Mol. Genet. 5:2023–2026. 28. Schuchardt, A., V. D’Agati, L. Larsson-Blomberg, F. Costantini, and V. Pachnis. 1994. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature. 367:380–383. 51. Ito, S., T. Iwashita, N. Asai, H. Murakami, Y. Iwata, G. Sobue, and M. Takahashi. 1997. Biological properties of Ret with cysteine mutations correlate with multiple endocrine neoplasia type 2A, familial medullary thyroid carci- noma, and Hirschsprung’s disease phenotype. Cancer Res. 57:2870–2872. y p 29. Mulligan, L.M., C. Eng, T. Attie, S. Lyonnet, D.J. Marsh, V.J. Hyland, B.G. Robinson, A. Frilling, C. Verellen-Dumoulin, A. Safar, et al. 1994. Di- 1423 Various Mechanisms Cause RET-mediated Signaling Defects in Hirschsprung’s Disease
https://openalex.org/W3045317941	https://pubs.rsc.org/en/content/articlepdf/2020/ta/d0ta04034g	English	null	Solvothermal synthesis of Sn<sub>3</sub>N<sub>4</sub> as a high capacity sodium-ion anode: theoretical and experimental study of its storage mechanism	Journal of materials chemistry. A	2,020	cc-by	13,220	aSchool of Chemistry, University of Southampton, Southampton SO17 1BJ, UK. E-mail: A.L.Hector@soton.ac.uk bDiamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK cSchool of Physics and Astronomy, University of Exeter, Stocker Road, Exeter EX4 4QL, UK dDeregallera Ltd, Unit 2 De Clare Court, Pontygwindy Industrial Estate, Caerphilly CF83 3HU, UK † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ta04034g Solvothermal synthesis of Sn3N4 as a high capacity sodium-ion anode: theoretical and experimental study of its storage mechanism† ess Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: J. Mater. Chem. A, 2020, 16437 Received 14th April 2020 Accepted 19th July 2020 DOI: 10.1039/d0ta04034g rsc.li/materials-a en Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: J. Mater. Chem. A, 2020, 8, 16437 Samuel D. S. Fitch,a Giannantonio Cibin,b Steven P. Hepplestone,cd Nuria Garcia- Araez a and Andrew L. Hector *a A new simple and scalable method to synthesise spinel-structured Sn3N4 has been developed using SnCl4 and LiNH2 precursors under solvothermal conditions. Nanocrystalline Sn3N4 with a crystallite size < 10 nm was produced and tested as anode material in sodium half cells, demonstrating a very high reversible (de- sodiation) capacity of 850 mA h g1 measured over 50 cycles, the highest reported reversible capacity for a sodium anode apart from sodium itself. Ex situ X-ray absorption spectroscopy and X-ray diﬀraction show that the electrochemical reactions are reversible and that Sn3N4 is recovered upon re-oxidation. X-ray diﬀraction shows that the peaks associated with Sn3N4 reﬂections become narrower during discharge (reduction), evidencing that the smaller Sn3N4 particles are primarily involved in the electrochemical reactions, and broadening of the peaks is reversibly recovered upon oxidation. The analysis of the near edge X-ray absorption data (XANES) shows that the Sn oxidation state decreases during reduction and nearly recovers the initial value during oxidation. DFT calculations suggest that the insertion of Na into the Sn3N4 surface followed by substitution of tetrahedral Sn by Na is energetically favourable, and evidence of the removal of tetrahedral Sn from the spinel Sn3N4 structure is obtained from the analysis of the extended X-ray absorption ﬁne-structure (EXAFS) measurements of reduced electrodes, which also show the recovery of the pristine structure at the end of oxidation. DFT also shows that Sn substitution by Na is only favourable at the Sn3N4 surface (not for bulk Sn3N4), in agreement with the electrochemical characterisation that shows that controlling the nanoparticle size is crucial to achieve full utilisation of Sn3N4 (and thus high capacity). Solvothermal synthesis of Sn3N4 as a high capacity sodium-ion anode: theoretical and experimental study of its storage mechanism† Received 14th April 2020 Accepted 19th July 2020 DOI: 10.1039/d0ta04034g rsc.li/materials-a p y DOI: 10.1039/d0ta04034g rsc.li/materials-a (cathode) materials, including layered transition metal oxides (NaxMO2 where M ¼ Ni, Co, Mn etc.) and polyanionic compounds such as NaFePO4, oen perform well.5–9 PAPER Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16437 Journal of Materials Chemistry A View Article Online View Journal \| View Issue Introduction Use of lithium-ion batteries (LIBs) in electrical energy storage is undergoing rapid growth due to their high energy density and long cycling stability. However, demand for battery grade lithium carbonate has up driven costs for manufacturing LIBs.1,2 The renewed interest in sodium-ion batteries (SIBs) is in part due to the high abundance, low cost and wide geographical distribution of the alkali metal. Sodium is the sixth most abundant element and is found both in sea water and in various mineral forms.3,4 The metal shares physico-chemical properties with lithium, and sodium analogues of LIB positive electrode For SIBs, the anode is still a considerable challenge. For LIBs, the standard anode material is graphite, however the larger ionic radius of sodium ions (1.02 ˚A for Na+, 0.76 ˚A for Li+) leads to sluggish reaction kinetics, lower capacities and poor cycling stability in sodium-ion cells. Other carbon materials in the form of amorphous and hard carbon are able to accom- modate sodium ions, delivering reversible capacities of up to 350 mA h g1.10–12 Tin-based alloying materials have been studied as alternative anodes for SIBs. The full sodiation of Sn to Na15Sn4 has a theoretical capacity of 847 mA h g1.13,14 However, Sn undergoes a volume variation of 420% during alloying/ dealloying with Na. The large volume change imposed on the metallic Sn particles leads to pulverization, delamination and loss of conduction pathways, although nanostructuring does improve stability. Tin-based binary materials (e.g. SnO2, SnS, Sn4P3) can mitigate the stresses imposed on the electrode by the formation of a matrix structure during initial reduction (Sn + NaxMy),15–20 as well as oﬀering possible extra capacity by the J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16437 This journal is © The Royal Society of Chemistry 2020 16437 View Article Online Journal of Materials Chemistry A Paper combination of conversion and alloying processes. The most studied of these is SnO2, owing to its high theoretical capacity (1378 mA h g1), low redox potential and ease of synthesis.21,22 However, the material suﬀers from a large initial irreversible capacity loss, low electronic conductivity and poor cyclabil- ity.23–25 Nano-structuring of conversion materials and formation of composites with carbon structures have yielded signicant improvements in their electrochemical performance. Some of the best of these are summarised in Table 1. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Even higher capacities are, in principle, obtainable with tin nitride (Sn3N4). Sn3N4 has a tuneable band gap and applications in photocatalysis, chemical sensing and solid state LIBs have been suggested.32–35 As an anode material in SIBs, the conver- sion of Sn3N4 to Sn and Na3N followed by full alloying of Sn to Na15Sn4 would result in a capacity of 1512 mA h g1. Previously we produced bulk nanocrystalline Sn3N4 by ammonolysis of a tin dialkylamide and demonstrated a reversible (de-sodiation) capacity of 270 mA h g1 aer 50 cycles at 50 mA g1 with a coulombic eﬃciency close to 95%.36 Ex situ diﬀraction measurements on cycled electrodes suggested that a signicant fraction of the material was not involved in the electrochemical processes. Consequently, it was envisioned that smaller crys- tallite sizes could produce larger capacities. This is now demonstrated in this work, together with a theoretical and experimental study of the reaction mechanism. Herein we report a joint theoretical and experimental study of the application of Sn3N4 as a high capacity anode material in SIB, using a new synthesis method to make Sn3N4 microcrys- talline and nanocrystalline powders. The capacity achieved with nanocrystalline Sn3N4 over 50 cycles exceeds those for anodes reported to date. The underlying mechanism is interrogated using rst principle methods and experimental characteriza- tion (XRD, EXAFS and XANES). Recent years have expanded the list of tools capable of pre- dicting new phases of materials using rst principles calcula- tions.37,38 Their application to the interaction of Na with Sn compounds has attracted considerable theoretical interest. An initial study in 2011 by Chevrier et al. used rst-principle calculations to predict the voltage prole of the process of sodiation of Sn by formation of NaxSny compounds.39 Later experimental studies using X-ray diﬀraction and operando TEM corroborated the formation of Na15Sn4 as the crystalline phase of maximum sodiation.40–42 Further theoretical work combined with M¨ossbauer and X-ray absorption spectroscopies shed light into the reaction mechanism of Sn sodiation.43,44 A decisive in- depth examination of the reaction pathway by Stratford et al. using ab initio random structure searching and operando XRD, This journal is © The Royal Society of Chemistry 2020 Introduction PDF and NMR characterization identied the structure of the intermediate NaxSny compounds and showed that the nal product Na15Sn4 can store additional sodium atoms as an oﬀ- stoichiometry product Na15+xSn4.45 y p Theoretical studies have also been very useful for under- standing the compounds formed with Na and N. The formation of metastable Na3N was predicted from theoretical consider- ations and later demonstrated in practice by synthetic routes using atomic co-deposition or reactions of sodium with active nitrogen, it was also shown that Na3N decomposes at 100 C.46–50 On the other hand, sodium azide (NaN3) is a well- known stable compound of Na and N and it is manufactured at around 250 tonnes per year, with the main application in car airbags.51,52 A theoretical study using global structure optimi- zation identied a new compound, sodium azenide (Na2N2), which was predicted to be stable (that is, it had a negative enthalpy of formation from the respective elements Na and N2),53 but the experimental synthesis of Na2N2 has not been reported. Finally, theoretical and experimental studies on the tertiary compounds of NaxSnyNz are currently limited, but ab initio calculations by Sun et al. showed that a phase of NaSnN is thermodynamically stable54 and this was subsequently demon- strated experimentally.55 Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. p Electrode preparation involved homogenizing the Sn3N4 active material (75%) with acetylene black (20%, Shawingan black, 100%-compressed, Chevron) and sodium alginate (5%, Aldrich) dissolved in deionised water. The slurry was cast onto Cu foil (17.5 mm thick, 99.9% purity, Goodfellow Ltd) using a K bar (wet thickness of 200 mm) and dried at room temperature. The coated foil was punched into circular discs (11 mm diam- eter) and pressed at 10 tonnes to obtain the Sn3N4 electrode. Typical mass loadings of electrodes prepared in this way were 0.5–1.0 mg cm2. Swagelok cells were assembled in an argon- lled glove box. Sodium half-cells were prepared by cutting a fresh face of sodium metal (Aldrich, 99% purity) and rolling to produce sodium foil counter-electrodes, with two microber lter (Whatman, GF/F grade) separators soaked in 120 ml of 1 mol dm3 NaPF6 (Aldrich, anhydrous) in a 47.5 : 47.5 : 5 by wt mixture of ethylene carbonate (EC), diethyl carbonate (DEC) and uoroethylene carbonate (FEC) (all Aldrich, 99%, anhydrous or vacuum distilled before use). Electrochemical testing was per- formed with a BioLogic MPG multi-channel potentiostat. Gal- vanostatic cycling was carried out at 25 C at various rates of charge/discharge within the potential range of 10 mV to 2.5 V vs. the sodium metal counter-electrode. Targeted compounds of known composition and structure were simulated from rst principles as implemented in the Vienna Ab initio Simulation Program (VASP).60 This approach is based upon density functional theory (DFT), and the PBE and HSE06 functionals were considered with the projected augmented waves method used for the pseudopotentials.61,62 An energy cut-oﬀof 600 eV and a minimum of a 4 4 4 (2 2 2) Monkhorst–Pack k-grid were used. The total energy of each system was minimised with respect to the internal coordinates and the lattice parameters, with a force tolerance of 0.01 eV ˚A1. All charge analysis was performed using the Bader scheme.63 Two functionals were considered here. PBE is a highly suitable functional for geometric considerations, whereas for chemical accuracy one would consider HSE06. In general, whilst broad trends can be extrapolated from a PBE result, the chemically accurate results require HSE06. Experimental Synthesis of Sn3N4 was carried out under anaerobic conditions using either a nitrogen-lled glovebox or Schlenk line techniques. Benzene (Aldrich) was reuxed over sodium for 8 h, distilled and stored under nitrogen. SnCl4 (Aldrich) was short-path distilled. LiNH2 was obtained by reacting nBuLi (1.6 mol dm3 in hexane, 500 cm3, Aldrich) with ammonia (distilled from a 100 cm3 sodium/ Table 1 Some high-performing tin-based materials for sodium-ion negative electrodes Material Current (mA g1) First sodiation capacity (mA h g1) First de-sodiation capacity (mA h g1) Final de-sodiation capacity (mA h g1)/no. of cycles Sn/graphite composite26 50 580 410 360/20 C–SnO2–C hollow core/shell/ shell27 100 1200 620 420/30 Amorphous SnO2 on C nanotubes28 100 1110 630 590/50 SnS–acetylene black composite29 100 760 500 530/80 Sn4P3 nanoparticles on reduced graphene oxide30 100 1600 780 640/100 Sn4P3 nanoparticles in a cube-shaped carbon shell31 100 1430 750 700/50 Table 1 Some high-performing tin-based materials for sodium-ion negative electrodes This journal is © The Royal Society of Chemistry 2020 View Article Online Paper liquid ammonia solution) at 0 C, ltering the white solid product and drying in vacuo. Solvothermal synthesis was performed in a 75 cm3 autoclave (Parr 4740CH), with a silica liner reducing the internal volume to 60 cm3. SnCl4 (0.60 g, 2.3 mmol) was placed in the silica liner and covered by 30 cm3 of benzene. LiNH2 (0.21 g, 9.2 mmol) was added and stirred. The autoclave was sealed and heated for 12 hours at temperatures as discussed later (300 to 430 C). Typically, 50 atm pressure was developed during heating. Aer cooling to room temperature, the autoclave was opened and the solid was washed with deionised water (50 ml) and MeOH (20 ml) to remove the LiCl by-product. The powder was then washed further with HCl (3 mol dm3, 30 ml) to remove any tin metal formed by thermal decomposition of the metal nitride. prepared without the homogenizing step show the same reac- tions (see ESI, Fig. S1†). For ex situ measurements before and aer galvanostatic cycling, microcrystalline tin nitride electrodes were cycled in Na half-cells to specic potentials of interest at 200 mA g1. The Swagelok cells were disassembled in an argon-lled glovebox as soon as the target potential had been reached. The electrodes were rinsed with anhydrous DEC solvent and allowed to dry. This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16439 Experimental Ex situ X-ray diﬀraction patterns collected in a sealed XRD sample holder (Bruker, loaded in the glove box) were collected in grazing incidence geometry (1 incidence angle) with Cu-Ka radiation (l ¼ 1.5418 ˚A) using a Rigaku Smartlab with Hypix 2D detector. Near-edge (XANES) and extended ne-structure (EXAFS) X-ray absorption measurements in sealed polyfoil pouches were made at the B18 beamline of Diamond Light Source across the Sn K-edge region from 28.8 to 30.0 keV. Tin standards were produced by preparing inks with Sn, SnO2, SnO (Aldrich) and Na4Sn powders diluted with acetylene black to match the respective mass loadings of the tin nitride electrodes. The Na4Sn standard was prepared in a nitrogen-lled glovebox where a slight excess of Na (4.4 equiv., Aldrich, 99% purity) and Sn (Aldrich) was loaded into a furnace tube and sealed, following a previously reported synthesis method for Na4Sn.58 The tube was placed into a furnace at 300 C and heated for 1 hour under owing N2. Aer heating, the sample was trans- ferred to the glovebox before diluting with acetylene black to produce the Na4Sn standard. The Sn edge positions were ob- tained as the rst inexion point of the respective XANES absorption spectra, and the EXAFS data has been tted using two independent Sn–N coordination shells, similarly to previous studies of Sn3N4.59 Journal of Materials Chemistry A Journal of Materials Chemistry A p reaction is the energy of formation, Ef, of the NaxSn1x and NayN1y compounds from the corresponding elements, which quantify the stability of the diﬀerent compounds (negative formation energies indicate thermodynamic stability). The energies of formation are calculated from: Ef ¼ E(NaxSn1x) xE(Na) (1x)E(Sn) (3) Ef ¼ E NayN1y yEðNaÞ 1 y 2 EðN2Þ (4) (3) (4) Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. where E(NaxSn1x) and E(NayN1y) are the total energy (per atom) of the structure, E(Na) and E(Sn) are the total energies of the bulk elements (per atom) and E(N2) is the total energy of the nitrogen molecule. For Sn, the metallic beta phase was used in the calculations, following previous work,45 since that is the stable phase at room temperature. To study the reaction of Na with Sn3N4, the associated reaction energies were computed: Ef ¼ E Sn3xN4yNaz EðSn3N4Þ þ xEðSnÞ 1 y 2 EðN2Þ zEðNaÞ (5) (5) where E(Sn3xN4yNaz) and E(Sn3N4) are the total energies of the structure with and without the Na atom. These energies are given per Na atom. Raw data associated with gures in the manuscript and ESI† are available from ref. 99. Results and discussion Fig. 1 Powder XRD patterns for the products of solvothermal synthesis at various temperatures. The black pattern denotes the literature Sn3N4 reﬂection positions and intensities.75 Solvothermal synthesis of Sn3N4 Solvothermal synthesis has become an eﬀective, well-controlled route for the preparation of crystalline metal nitrides.65–67 By heating reactants in a solvent medium inside a sealed auto- clave, metathesis reactions can proceed at mild temperatures due to the solvent absorbing heat produced in the exothermic reactions.68–70 Thermal decomposition of the formed metal nitrides is also signicantly reduced under solvothermal conditions, and the products are usually crystalline.20,71,72 Sn3N4 was synthesised via a simple and easily-scalable solvothermal method with dened particle size via a solvothermal metathesis reaction, with benzene employed as the solvent owing to its stability under solvothermal conditions:73,74 430 C, all the XRD peaks are symmetrical and narrow, demonstrating that the sample is made of crystalline particles in the micron size range; this material will be referred to as microcrystalline Sn3N4 in the following text. A Rietveld t to this XRD pattern (Rwp ¼ 5.0%, Rp ¼ 3.4%) yielded a lattice parameter of 9.0549(2) ˚A and an average crystallite size of 310(40) nm (ESI, Fig. S2 and Table S1†). TEM micrographs corroborate this crystallite size (Fig. 2a). In the XRD data for the sample prepared at 300 C, broad asymmetric peaks are present at similar positions to small sharp peaks, the latter resembling the microcrystalline sample obtained at 430 C. The asymmetric broadening feature is due to the fact that the sample is made of a mixture of nano-sized and larger crystallites. The presence of nano-size crystallites could be increased by sonicating the sample in HCl, the asso- ciated XRD pattern shows that the asymmetric broadening of the peaks is accentuated. The material obtained by heating at 300 C followed by sonication will be referred to as nano- crystalline Sn3N4 in all that follows. A two-phase Rietveld t (Rwp ¼ 9.4%, Rp ¼ 7.4%) conrmed the presence of two distinct phases, both tting well as Sn3N4 (ESI, Fig. S2 and Table S1†). The lattice parameters of the two phases were 9.0514(4) and 9.139(2) ˚A, with average crystallite sizes of 137(13) nm (13.2(4)% of the material present) and 5.950(9) nm (86.8(2)%), 3SnCl4 + 12LiNH2 / Sn3N4 + 12LiCl + 8NH3 (6) (6) Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To investigate the reaction pathways, we calculated the energies of the diﬀerent compounds following the standard approach.64 The reactions of Na with the Sn or N2 can be written as: Powder X-ray diﬀraction (XRD) measurements were recorded on a Bruker D2 Phaser using Cu-Ka radiation (l ¼ 1.5418 ˚A) in Bragg–Brentano geometry. The reections present were identi- ed by comparison with the ICSD database and patterns rened using the GSAS package.56 Scale factor, background, lattice parameter, zero point correction, thermal displacement parameters, nitrogen atom positions, crystallite size broadening and isotropic strain. An Al2O3 standard collected in the same geometry was used to dene the Gaussian instrumental peak shape, with the crystallite size obtained from the Lorentzian crystallite size broadening terms.57 In two-phase ts containing two crystallite sizes, the tin thermal displacement parameters were constrained to a single value and the nitrogen thermal displacements were xed. Transmission electron microscopy (TEM) was carried out with a FEI Technai12 (120 kV) on samples that were dispersed into propanol with ultrasound and dropped onto carbon grids. Bulk elemental CHN combustion analysis was obtained from Medac Ltd, and the values are reported in weight percentages. The XRD patterns of the pristine electrodes show some small, unidentied impurity peaks, which are formed during the homogenizing process in electrode prepa- ration, but additional diﬀraction studies with electrodes xNa + (1 x)Sn / NaxSn1x (1) yNa þ 1 y 2 N2 / NayN1y (2) (1) (2) where NaxSn1x and NayN1y are compounds containing Na and Sn, and Na and N, respectively. The associated energy of the This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16439 16439 Fig. 1 Powder XRD patterns for the products of solvothermal synthesis at various temperatures. The black pattern denotes the literature Sn3N4 reﬂection positions and intensities.75 Paper View Article Online Journal of Materials Chemistry A 3SnCl4 + 12LiNH2 / Sn3N4 + 12LiCl + 8NH3 (6 XRD and TEM were employed to characterise the product of the solvothermal reaction. Sn3N4 crystallises with the cubic spinel structure (Fd3m) with a reported lattice parameter of 9.037(3) ˚A.75 Powder X-ray diﬀraction (XRD) patterns of the brown products produced in solvothermal reactions at 300, 350 and 430 C are displayed in Fig. 1, all showing the peak posi- tions of the Sn3N4 reference. The good match of the XRD peak positions of the Sn3N4 reference and the samples here produced, demonstrates the success in the synthesis of phase pure Sn3N4 for all the reaction conditions here explored (the absence of other peaks demonstrate that no other crystalline impurities are present). In addition, for the sample produced at This journal is © The Royal Society of Chemistry 2020 16440 \| J. Mater. Chem. A, 2020, 8, 16437–16450 16440 Journal of Materials Chemistry A View Article Online View Article Online Paper Journal of Materials Chemistry A Journal of Materials Chemistry A Journal of Materials Chemistry A Fig. 2 Magnitude and real part of the Fourier transforms of K3-weighted Sn K-edge EXAFS and TEM images of microcrystalline (a) and nan crystalline (b) Sn3N4. The scale bar in (a) is 200 nm and in (b) it is 100 nm. Paper Journal of Materials Chemistry Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 Magnitude and real part of the Fourier transforms of K3-weighted Sn K-edge EXAFS and TEM imag crystalline (b) Sn3N4. The scale bar in (a) is 200 nm and in (b) it is 100 nm. Fig. 2 Magnitude and real part of the Fourier transforms of K3-weighted Sn K-edge EXAFS and TEM images of microcrystalline (a) and nano- crystalline (b) Sn3N4. The scale bar in (a) is 200 nm and in (b) it is 100 nm. f the Fourier transforms of K3-weighted Sn K-edge EXAFS and TEM images of microcrystalline (a) and nano- ar in (a) is 200 nm and in (b) it is 100 nm. respectively. TEM shows crystallites around both of these sizes, with a greater number of nanocrystalline particles present (Fig. 2b). the ESI, Table S2†), conrming structural similarities of Sn3N4 in both samples. This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16441 3SnCl4 + 12LiNH2 / Sn3N4 + 12LiCl + 8NH3 (6 The experi- mental values of the nitrogen content are somewhat lower (10.1% and 8.2% for the microcrystalline and nanocrystalline samples). The process of washing the samples in an HCl solu- tion aer synthesis can produce a hydrolysis reaction producing a partial oxygen substitution of nitrogen sites at particle surfaces, forming a tin oxy-nitride, and this eﬀect will be more severe for the nanocrystalline sample since it has a higher surface area. The XRD and EXAFS results show that the spinel structure is preserved aer the oxygen-nitrogen substitution. Incomplete combustion of the samples, as observed in other metal nitride studies,76 could also contribute to the lower than expected nitrogen content obtained from the elemental analysis. (benzene) will take place at the lower temperature used for the solvothermal synthesis method.68,69 Taking into account the presence of carbon and hydrogen residues and the nitrogen vacancies (calculated from the tin oxidation number), the ex- pected values of the nitrogen content are 10.9% and 11.1% for the microcrystalline and nanocrystalline samples. The experi- mental values of the nitrogen content are somewhat lower (10.1% and 8.2% for the microcrystalline and nanocrystalline samples). The process of washing the samples in an HCl solu- tion aer synthesis can produce a hydrolysis reaction producing a partial oxygen substitution of nitrogen sites at particle surfaces, forming a tin oxy-nitride, and this eﬀect will be more severe for the nanocrystalline sample since it has a higher surface area. The XRD and EXAFS results show that the spinel structure is preserved aer the oxygen-nitrogen substitution. Incomplete combustion of the samples, as observed in other metal nitride studies,76 could also contribute to the lower than expected nitrogen content obtained from the elemental analysis. we continued using sodium alginate for this work.36 The binder is comparatively more rigid than the commonly used PVDF, a property that increases the electrode's ability to withstand large volume changes during cycling.77–79 Na half-cells were constructed with 1 mol dm3 NaPF6 in EC/DEC with 5 wt% FEC electrolyte, again following previous work.36 FEC was used as an additive that improves the electrochemical performance of tin- based electrodes.80–83 Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Galvanostatic cycling of microcrystalline Sn3N4 in Na half- cells was investigated at 50 and 200 mA g1 (Fig. 3). 3SnCl4 + 12LiNH2 / Sn3N4 + 12LiCl + 8NH3 (6 At 200 mA g1, 671 mA h g1 of capacity was passed on rst reduction (discharge, negative current) to 10 mV, of which 214 mA h g1 was recovered on the rst oxidation (charge, positive current) to 2.5 V. This large initial capacity loss (32%) is tentatively attributed to irreversible processes such as the decomposition of the electrolyte and formation of an SEI.20,36,84,85 Subsequent cycles demonstrated good cycling stability with a capacity fade of 7% between the 10th (268 mA h g1) and 50th (250 mA h g1) oxidations. The coulombic eﬃciency was 98% at the 50th cycle, indicating a stable, reversible electrochemical process. Greater capacities were achieved at a more modest current of 50 mA g1, with 520 mA h g1 of capacity passed aer the 50th oxidation, with a coulombic eﬃciency of 97%. Capacity fade between the 10th (546 mA h g1) and 50th (521 mA h g1) oxidation was just 4%. 3SnCl4 + 12LiNH2 / Sn3N4 + 12LiCl + 8NH3 (6 In addition, the analysis of the Sn K-edge XANES data enables the evaluation of the Sn oxidation state (see more details below), which were found to be 3.7 and 3.3 for the Sn3N4 micro and nanocrystalline samples, respectively. The microcrystalline and nanocrystalline Sn3N4 samples were also characterised by XANES and EXAFS collected at the Sn K-edge. Fig. 2 shows the magnitudes of the Fourier transforms of the K3-weighted EXAFS data. A peak doublet at 1.5 ˚A radial distance is present in both samples and corresponds to the distances between N sites and tetrahedral tin site (Sntet–N) and octahedral tin site (Snoct–N) at 2.05 and 2.20 ˚A, as expected for the spinel Sn3N4 structure and in agreement with previous studies.59 Peaks between 2.5 and 4 ˚A are attributed to other Sn– Sn interactions within the structure.59 While XRD is suitable to characterise the composition of the microcrystalline sample, the nanocrystalline sample gives broad diﬀraction peaks. However, EXAFS shows that both samples have a very similar set of correlations with similar intensities (see results of the t in The estimation of the nitrogen content of the Sn3N4 samples by combustion analysis gives values (10.1% and 8.2% for the microcrystalline and nanocrystalline samples) that are lower than the expected for pure, stoichiometric Sn3N4 (13.6%). This can be ascribed, in part, to the presence of nitrogen vacancies, which is supported by the fact that the Sn K-edge XANES anal- ysis provides values of the oxidation state of tin lower than 4. The elemental analysis also shows that the samples also contain carbon and hydrogen (12.4% C and 2.0% H for the microcrys- talline sample and 1.0% C and 2.0% H for the nanocrystalline sample). The lower content of carbon in the nanocrystalline sample is expected, since less decomposition of the solvent J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16441 This journal is © The Royal Society of Chemistry 2020 16441 View Article Online View Article Online Paper ew Article Online Journal of Materials Chemistry A Paper (benzene) will take place at the lower temperature used for the solvothermal synthesis method.68,69 Taking into account the presence of carbon and hydrogen residues and the nitrogen vacancies (calculated from the tin oxidation number), the ex- pected values of the nitrogen content are 10.9% and 11.1% for the microcrystalline and nanocrystalline samples. Electrochemistry of Sn3N4 in sodium half cells The coulombic eﬃciency at the 50th cycle was 94%. Similar to other conversion anode materials,26–31 the rst cycle coulombic eﬃciency of the Sn3N4 samples studied here (30–45%) is not suitable for commercial applications, but further improvements could be achieved by material develop- ment. For example, the use of a yolk–shell design, where the active material nanoparticles are sealed inside a carbon shell, has produced major performance improvements for other conversion materials, which has been attributed to the fact that the yolk–shell design enables the volumetric expansion associ- ated with the electrochemical reactions while avoiding the direct contact of the active material with the electrolyte.20,90 The investigation of alternative electrolytes is another highly promising route for improving performance and coulombic eﬃciency.91 The capacity of these Sn3N4 electrodes is remarkable considering that other high capacity tin-based materials all incorporate a carbon architecture material such as graphene sheets, hollow spheres or nanotubes (see Table 1). In other Sn- based conversion systems without a secondary support, capacity fading over multiple cycles is a problem. Wang et al. cycled SnO2 at 50 mA g1 with a capacity of 200 mA h g1 achieved aer 50 cycles.86 Ma et al. compared Sn4P3/hollow C core–shell composites (Table 1) with unsupported Sn4P3, and cycling the unsupported material delivered a capacity of just 104 mA h g1 aer the 20th cycle at 100 mA g1.31 The cycling of unsupported SnS electrodes was reported by Yu et al. with 200 mA h g1 achieved aer 50 cycles at 100 mA g1.87 In addition, further improvements could be achieved in full cell studies, since the reactivity of the sodium metal counter electrode has been shown to compromise cycling stability in studies in Na half- cells.88,89 Electrochemistry of Sn3N4 in sodium half cells Sn3N4 exhibits good stability in deionised water, hence water processable binders can be employed in electrode preparation, avoiding toxic organic solvents. In our previous study, Sn3N4 electrodes prepared with sodium alginate as a binder demon- strated the best electrochemical stability during cycling and so Galvanostatic cycling of nanocrystalline Sn3N4 in Na half- cells (Fig. 4) shows greater capacities compared to the Fig. 3 Galvanostatic cycling (a and c) and plot of speciﬁc capacity vs. cycle number (b and d) of microcrystalline Sn3N4 in Na half-cells at 200 mA g1 (a and b) and 50 mA g1 (c and d). Fig. 3 Galvanostatic cycling (a and c) and plot of speciﬁc capacity vs. cycle number (b and d) of microcrystalline Sn3N4 in Na half-cells at 200 mA g1 (a and b) and 50 mA g1 (c and d). Fig. 3 Galvanostatic cycling (a and c) and plot of speciﬁc capacity vs. cycle number (b and d) of microcrystalline Sn3N4 in Na half-cells at 200 mA g1 (a and b) and 50 mA g1 (c and d). This journal is © The Royal Society of Chemistry 2020 16442 \| J. Mater. Chem. A, 2020, 8, 16437–16450 View Article Online Paper Journal of Materials Chemistry A Fig. 4 Galvanostatic cycling (a and c) and plot of speciﬁc capacity vs. cycle number (b and d) of nanocrystalline Sn3N4 in Na half-cells at 200 mA g1 (a and b) and 50 mA g1 (c and d). Paper Journal of Materials Chemistry A p Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Galvanostatic cycling (a and c) and plot of speciﬁc capacity vs. cycle number (b and d) of nanocrystalline Sn3N4 in Na half-cells at 200 mA g1 (a and b) and 50 mA g1 (c and d). microcrystalline sample. At 200 mA g1, aer the initial irre- versible capacity loss, a reversible (oxidation) capacity of 420 mA h g1 was achieved. Capacity fade from the 10th (421 mA h g1) to the 50th (416 mA h g1) oxidation was only 1%. In addition, the coulombic eﬃciency rose to 98%, demonstrating the favourable reversibility of the charge/ discharge reactions on cycling. As expected, more capacity was achieved at the lower current of 50 mA g1. Reversible capacities of 850 mA h g1 were obtained during cycling. This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16443 DFT study of the charge storage mechanism Previously, it was suggested that Sn3N4 electrodes could undergo complete conversion to Sn metal and Na3N with subsequent alloying of Sn with Na to Na15Sn4.36 Here, we exploit ab initio calculations to identify the candidate compounds involved in the reaction mechanism of sodiation of Sn3N4; this is later analysed in view of the XRD, XANES and EXAFS results. In order to construct the possible reaction pathways, we rst investigated the stability of the various phases of NaxSny and NaxNy compounds. We applied a similar approach to AIRRS,45 except that for any stoichiometry where a known experimental Previously, it was suggested that Sn3N4 electrodes could undergo complete conversion to Sn metal and Na3N with subsequent alloying of Sn with Na to Na15Sn4.36 Here, we exploit ab initio calculations to identify the candidate compounds involved in the reaction mechanism of sodiation of Sn3N4; this is later analysed in view of the XRD, XANES and EXAFS results. In order to construct the possible reaction pathways, we rst investigated the stability of the various phases of NaxSny and NaxNy compounds. We applied a similar approach to AIRRS,45 except that for any stoichiometry where a known experimental This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16443 16443 View Article Online Journal of Materials Chemistry A Paper structure exists, we only consider that structure. The PBE functional was used for an initial search, and we then studied the most stable candidates with the HSE06 functional. The HSE06 functional is necessary for considering nitrogen-based structures due to the diﬀerence in the N2 molecule's binding energy in PBE and HSE06 (the binding energy of N2 in HSE06 is 9.54 eV, whereas in PBE it is 8.44 eV and the experimental value is 9.79 eV).92 Aer calculation of the formation energies of the diﬀerent compounds, using eqn (3) and (4), the lines connect- ing the compounds of lower formation energy (known as convex hull) were calculated. When a candidate compound lies above the convex hull, it is concluded that the compound is not thermodynamically stable. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For the elucidation of the energy storage mechanism of Sn3N4 in sodium-ion cells, we studied the reaction of formation of Na interstitials and substitutional sites in bulk and surface Sn3N4 (see ESI, Tables S3 and S4†). For the study of surface reactions, slabs of material with [001] surface terminations were generated. The plane of cleavage was chosen based on the consideration that both surfaces must be (a) symmetric, (b) charge neutral and (c) stoichiometric. From these criteria four surfaces were created and relaxed. The most stable of these (see ESI, Fig. S3†) was then considered for the surface reaction calculations. We considered slab systems with various thick- nesses ranging from 0.3 to 2 nm to converge the energy calculations. The results of the calculations of the formation energies of Sn–Na compounds are shown in Fig. 5a. The calculations made with the PBE functional agree with the results reported by Stratford et al.45 To ensure chemical accuracy, additional calculations were made with the HSE06 functional. Whilst the formation energies are generally slightly smaller, the general trend in the convex hull is in good agreement with the previous PBE results. Our HSE06 calculations also show that all phases are metallic as expected. Our rst principles simulations combined Bader charge analysis which shows that Sn atoms are charge neutral (0.0\|e\|) in the b phase due to charge being evenly distributed. As the fraction of Na increases, the Sn atoms become increasingly negatively charged with the Na15Sn4 phase Sn atoms possessing Bader charges of 3.7\|e\|. Three diﬀerent mechanisms for the bulk sodiation of Sn3N4 were considered: (a) the addition of interstitial Na ions into Sn3N4 (with an associated reduction of the Sn oxidation state, reaction (7)), (b) the substitution of Na onto Sn sites (with an associated formation of Sn metal, reaction (8)), (c) or the substitution of Na onto N sites (with an associated formation of N2, reaction (9)). The calculation of formation energies of Na–N compounds is shown in Fig. 5b. Due to the signicant error in binding ener- gies of the N2 molecule in PBE, only HSE06 results are dis- cussed. Fig. 5b shows that NaN3 is the only candidate compound that lies on the convex hull, which means that the other candidate compounds are not thermodynamically stable. DFT study of the charge storage mechanism the fact that Na3N is metastable does not mean that it cannot be formed and remain stable: Na3N has been successfully syn- thesised by atomic co-deposition and the reaction of sodium with active nitrogen,47–49 and it was stated that Na3N remained stable for weeks unless it was heated to 100 C.48 At high pressures Na3N was also found to remain stable.93 Our results of the metastability of Na3N are also in agreement with previous theoretical calculations.46,50,53,94 This journal is © The Royal Society of Chemistry 2020 Journal of Materials Chemistry A View Article Online 6 and ESI, Table S3†) were found to have a positive formation energy, meaning that they were not thermodynamically stable with respect to bulk Sn3N4 and Na. The energy of formation of Na interstitials ranged between 1.68 and 3.8 eV using PBE, and the values obtained with HSE06 were 0.3 eV lower. However, it is worth noting that the formation energy for inserting two Na interstitials into the structure was 1.28 eV (per Na atom) (compared to 1.68–3.8 eV for the rst interstitial considered here), indicating that the expansion of the structure to accom- modate the rst Na atom results in a lower formation energy for subsequent Na intercalations. The third reaction pathway considered was Na substituting onto the Sn site (reaction (8)), which was also found to be energetically unfavourable. Substituting the Sn tetrahedra (Sntet) had a formation energy of 4.33 eV in PBE, and substitution at the Sn octahedral site (Snoct) was slightly less favourable at 4.47 eV in PBE (see sites in Fig. 6). In summary, ab initio calculations were applied to evaluate the energy of formation of bulk NaxSnyNz compounds, of which only Na0.0625Sn3N4 was found to be stable. Therefore, it can be proposed that the electrochemical sodiation of Sn3N4 will produce the bulk formation Na0.0625Sn3N4. However, the capacity associated with such a process is very small: the theo- retical capacity is only 4 mA h g1. Previous studies have also identied NaSnN as a stable compound, this phase contains Na ions intercalated in between two-dimensional layers of [SnN].54,55 The sodiation of Sn3N4 to form bulk NaSnN has an associated theoretical capacity of 65 mA h g1. However, the bulk conversion of the Sn3N4 material into NaSnN is not sup- ported by our experimental data: XRD data of the electrodes discharged to diﬀerent potentials in Na half-cells (see discus- sion below, Fig. 9b) show that the diﬀraction pattern of the Sn3N4 spinel structure is still clearly visible even aer polari- zation to 10 mV vs. Na+/Na, thus demonstrating that the core of Sn3N4 particles undergoes little structural change. The absence of transformation of bulk Sn3N4 into bulk NaSnN could be attributed to kinetic limitations. On the other hand, the sodia- tion of Sn3N4 to form Na0.0625Sn3N4 involves little structural rearrangement, so it is expected to be facile and the observed XRD patterns of the sodiated (i.e. reduced) electrodes are also consistent with Na0.0625Sn3N4. Journal of Materials Chemistry A View Article Online View Article Online Paper interstitial forming a pseudo-octahedral Na site face linking to two SnN4 tetrahedra and edge linking to four SnN6 octahedra) was then considered in a larger supercell, corresponding to Na0.0625Sn3N4. This structure was found to be energetically favourable with a formation energy of 0.426 eV per Na atom. Apart from the formation of this energetically favourable Na interstitial, other Na interstitial sites considered (see Fig. 6 and ESI, Table S3†) were found to have a positive formation energy, meaning that they were not thermodynamically stable with respect to bulk Sn3N4 and Na. The energy of formation of Na interstitials ranged between 1.68 and 3.8 eV using PBE, and the values obtained with HSE06 were 0.3 eV lower. However, it is worth noting that the formation energy for inserting two Na interstitials into the structure was 1.28 eV (per Na atom) (compared to 1.68–3.8 eV for the rst interstitial considered here), indicating that the expansion of the structure to accom- modate the rst Na atom results in a lower formation energy for subsequent Na intercalations. The third reaction pathway considered was Na substituting onto the Sn site (reaction (8)), which was also found to be energetically unfavourable. Substituting the Sn tetrahedra (Sntet) had a formation energy of 4.33 eV in PBE, and substitution at the Sn octahedral site (Snoct) was slightly less favourable at 4.47 eV in PBE (see sites in Fig. 6). In summary, ab initio calculations were applied to evaluate the energy of formation of bulk NaxSnyNz compounds, of which only Na0.0625Sn3N4 was found to be stable. Therefore, it can be proposed that the electrochemical sodiation of Sn3N4 will produce the bulk formation Na0.0625Sn3N4. However, the capacity associated with such a process is very small: the theo- retical capacity is only 4 mA h g1. Previous studies have also identied NaSnN as a stable compound, this phase contains Na ions intercalated in between two-dimensional layers of [SnN].54,55 The sodiation of Sn3N4 to form bulk NaSnN has an interstitial forming a pseudo-octahedral Na site face linking to two SnN4 tetrahedra and edge linking to four SnN6 octahedra) was then considered in a larger supercell, corresponding to Na0.0625Sn3N4. This structure was found to be energetically favourable with a formation energy of 0.426 eV per Na atom. Apart from the formation of this energetically favourable Na interstitial, other Na interstitial sites considered (see Fig. J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16445 Journal of Materials Chemistry A View Article Online Thus, we conclude that sodiation of Sn3N4 starts with the facile insertion of a small of amount of sodium in the Sn3N4 structure, forming the identied stable compound Na0.0625Sn3N4. On the other hand, the surface of Sn3N4 particles undergoes signicant reactions during cycling in Na half cells. To study the surface reactions, additional calculations were performed to evaluate the energy of formation of surface compounds. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. By performing ab initio calculations with the PBE functional, we found that the insertion of Na into the Sn3N4 surface (reac- tion (7)) was generally energetically favourable, resulting in a signicant energy gain (at the surface the formation energy is 3.5 eV for the adsorption of 1 Na atom). Further below the surface, this energy decreases but remains favourable for all the slabs we have considered (up to 2 nm thick). This indicates that, near the surface, the energetic cost of expansion and strain on Sn3N4 associated with sodiation reactions is less than the energy gained from adsorbing Na. Furthermore, on the top surface layers of Sn3N4, the substitution of a Na into a Sn site, forming NaySn3yN4 + ySn (reaction (8)) becomes favourable, with formation energies of 1.9 eV for surface substitutions and 0.6 eV for subsurface (1 monolayer below) substitutions (both tin tetrahedral sites). As these represent the rst steps of conversion of the Sn3N4 structure, this suggests a potential 1.9 V vs. Na+/Na for the surface process. However, we treat this with caution as it is heavily dependent on surface reconstruc- tions and terminations. These ndings suggest that the elec- trochemical reaction of sodiation of Sn3N4 is likely initiated by the insertion of Na on the surface followed by the substitution of Sn by Na at the Sn3N4 surface, resulting in the formation of a Sn metal coating on top of the Sn3N4 nanoparticle. Then, Sn metal could react further with Na forming diﬀerent NaxSny alloys, with Na15Sn4 as the nal product, as shown in previous studies of the sodiation of Sn in Na-ion cells.39–42,45 It is reasonable to assume that the products of these surface reac- tions will grow as a shell around a core with NaySn3N4 structure (possibly made of Na0.0625Sn3N4, according to our calculations). These ndings can explain the increase in the capacity values observed with the nanocrystalline tin nitride electrode, as the material will have a larger surface area compared with the microcrystalline material. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For example, Na3N produces a positive energy of formation per atom (Ef ¼ 0.29 eV in HSE06), which means that formation of Na3N from bulk Na and N2 gas is not thermodynamically favourable. These ndings agree with the experimental enthalpy of formation of Na3N obtained by diﬀerential thermal analysis: 64 kJ mol1 (or 0.66 eV),48 which would correspond to a formation energy per atom of 0.66 eV/4 ¼ 0.17 eV. However, Na interstitial formation: Sn3N4 + xNa / NaxSn3N4 (7) Substitution of Sn sites: Sn3N4 + yNa / NaySn3yN4 + ySn (8) Substitution of N sites: Sn3N4 + zNa / NazSn3N4z + z/2N2(9) (7) The associated reaction energies, per Na atom, are given in Table S3, ESI.† We found that, unsurprisingly, the least ener- getically favourable of these was Na substituting into the N site (reaction (9)). For the addition of Na interstitial sites (reaction (7)), we considered various sites for interstitial Na as shown in Fig. 6. The lowest energy conguration (shown in yellow: 16c Fig. 5 (a) Formation energy per atom vs. sodium concentration in Na–Sn compounds calculated with the PBE (black) and HSE06 (red) func- tionals. (b) Formation energy per atom vs. sodium concentration in Na–N compounds calculated with the HSE06 functional. In both panels, the convex hull (black and red lines) are constructed by joining the lowest energy point structures obtained by the searches. Fig. 5 (a) Formation energy per atom vs. sodium concentration in Na–Sn compounds calculated with the PBE (black) and HSE06 (red) func- tionals. (b) Formation energy per atom vs. sodium concentration in Na–N compounds calculated with the HSE06 functional. In both panels, the convex hull (black and red lines) are constructed by joining the lowest energy point structures obtained by the searches. 16444 \| J. Mater. Chem. A, 2020, 8, 16437–16450 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Unfortunately, the larger surface also increases the electrolyte breakdown occurring in the rst charge cycle, which can explain the larger irreversible capacity loss witnessed on the initial cycle of the nanocrystalline material.95 In summary, ab initio calculations were applied to evaluate the energy of formation of bulk NaxSnyNz compounds, of which only Na0.0625Sn3N4 was found to be stable. Therefore, it can be proposed that the electrochemical sodiation of Sn3N4 will produce the bulk formation Na0.0625Sn3N4. However, the capacity associated with such a process is very small: the theo- retical capacity is only 4 mA h g1. Previous studies have also identied NaSnN as a stable compound, this phase contains Na ions intercalated in between two-dimensional layers of [SnN].54,55 The sodiation of Sn3N4 to form bulk NaSnN has an Fig. 6 A ball and stick representation of the Sn3N4 structure. The large purple spheres are Sn; the smaller purple spheres are nitrogen. The Na interstitial in its lowest energy conﬁguration is shown as a yellow sphere. Other sites considered are shown with green dots. The two substitutional sites for the substitution of Sn by Na are highlighted with orange dots (upper: octahedral, lower: tetrahedral). The unit cell is highlighted in red. Fig. 6 A ball and stick representation of the Sn3N4 structure. The large purple spheres are Sn; the smaller purple spheres are nitrogen. The Na interstitial in its lowest energy conﬁguration is shown as a yellow sphere. Other sites considered are shown with green dots. The two substitutional sites for the substitution of Sn by Na are highlighted with orange dots (upper: octahedral, lower: tetrahedral). The unit cell is highlighted in red. J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16445 This journal is © The Royal Society of Chemistry 2020 16445 View Article Online View Article Online Journal of Materials Chemistry A Paper Fig. 7 Initial galvanostatic cycle of microcrystalline Sn3N4 depicting where samples were taken for the ex situ XRD, XANES and EXAFS studies. Fig 7 Initial galvanostatic cycle of microcrystalline Sn N depicting some Sn to have already formed. A large, sloping plateau is observed at 0.5 V, that could be ascribed to the alloying reaction (Sn / NaxSn). This alloying process continues to 10 mV, as described in SnO2 and Sn4P3 systems.96–98 Since the sodiation of Sn produces a series of NaxSny compounds at potentials in between 0.5 V and 0 V vs. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Na+/Na, these last processes have been assigned to the Sn alloying reactions (Sn / NaxSny).39–42,45 Sn K-edge XANES analysis was performed to probe changes in the oxidation state of Sn in the cycled electrodes. For that purpose, prior to the measurements, Sn–K edges were cali- brated against Sn standards with dened oxidation states (Sn, SnO2, SnO and Na4Sn with oxidation states of 0, +4, +2 and 4, respectively, see ESI, Fig. S5†). The XANES spectra (Fig. 8) show the edge energy shiing to lower energy values during the rst reduction and a decrease in the intensity of the white line position at the fully reduced state (10 mV vs. Na+/Na), corre- sponding to an average oxidation state of 0.1 (ESI, Table S5†). Whilst a negative oxidation state implies that Na–Sn alloying has taken place, it also suggests incomplete conversion of Sn3N4 to Na–Sn alloys, as we would expect a much lower oxidation state (1.25) for the fully sodiated Na15Sn4 phase. Fig. 8 also shows an increase in the edge energy shiand intensity of the white line position in the XANES spectra during the rst oxidation of the Sn3N4 electrode. The average oxidation state of Sn was 3.5 in the fully charged state (2.5 V vs. Na+/Na), very close to the average oxidation state of the pristine sample (3.7). This provides strong evidence that reformation of Sn3N4 occurs. Open Access Article. Published on 21 July 2020. Downloaded on 10/24/2024 5:48:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 7 Initial galvanostatic cycle of microcrystalline Sn3N4 depicting where samples were taken for the ex situ XRD, XANES and EXAFS studies. Structural and chemical change during cycling In order to elucidate the reactions undergone by Sn3N4 mate- rials in Na half-cells, electrodes were extracted from the cell at potentials chosen from features observed in the potential prole (Fig. 7, also see diﬀerential capacity plots in the ESI, Fig. S4†). These were 1.6, 1.2, 1.0, 0.5 and 0.01 V vs. Na/Na+ during reduction and 0.1, 0.75, 1.5 and 2.5 V vs. Na/Na+ during oxidation. Samples were then characterised by XRD, XANES and EXAFS. These experiments utilised the microcrystalline Sn3N4 in order to obtain more useful diﬀraction data, although it is recognised that the nanocrystalline material has the larger capacity. On rst reduction, there is a sloping plateau over 1.6 V (Fig. 7) followed by a small plateau at 1 V. We attribute this process to the insertion of Na ions into the Sn3N4 structure via surface reactions such as the substitution of Na to Sn sites which produces Sn metal coatings. These reactions were found to be energetically favourable (negative formation energies) at the Sn3N4 surface by DFT calculations. At lower potentials the formation of NaxSny phases is expected, which likely requires Fig. 9b shows the ex situ XRD data of Sn3N4 electrodes cycled to the same set of potentials as in the XANES measurements in Fig. 8. It is observed that the reections corresponding to Sn3N4 become sharper on reduction of the electrode, this suggests that the smaller crystallites are reacting more readily and hence the average crystallite size increases. At 0.5 V, clear reections for Sn metal appear in the pattern. This is in agreement with the ndings from the DFT calculations, which predict that sodia- tion would induce the insertion of Na into the surfaces of the Sn3N4 particles, thus ejecting Sn metal, followed by the growth of a Na–Sn alloy shell around the Sn3N4 particles. Formation of Na–Sn alloys is not clearly visible in the XRD data, indicating poor crystallinity or small particle size of the Na–Sn alloys. Fig. 8 Sn K-edge XANES spectra during: (a) ﬁrst reduction and (b) ﬁrst oxidation of microcrystalline Sn3N4 as a function of cell potential. The insets show the central part of the absorption edge, magniﬁed for clarity. Fig. 8 Sn K-edge XANES spectra during: (a) ﬁrst reduction and (b) ﬁrst oxidation of microcrystalline Sn3N4 as a function of cell potential. The insets show the central part of the absorption edge, magniﬁed for clarity. Structural and chemical change during cycling This journal is © The Royal Society of Chemistry 2020 16446 \| J. Mater. Chem. A, 2020, 8, 16437–16450 View Article Online Paper Paper Journal of Materials Chemistry A Fig. 9 (a) Magnitude of the Fourier transforms of K3-weighted Sn K-edge EXAFS during initial reduction and oxidation of microcrystalline Sn3N4. The data of Na15Sn4, Na4Sn and Sn from the ICSD database is also included for comparison. (b) Corresponding ex situ XRD patterns, with peaks due to the Cu current collector omitted from the pattern. ve Commons Attribution 3.0 Unported Licence. Fig. 9 (a) Magnitude of the Fourier transforms of K3-weighted Sn K-edge EXAFS during initial reduction and oxidation of microcrystalline Sn3N4. The data of Na15Sn4, Na4Sn and Sn from the ICSD database is also included for comparison. (b) Corresponding ex situ XRD patterns, with peaks due to the Cu current collector omitted from the pattern. During the initial stages of the re-oxidation, the intensity of the Sn metal reections diminishes but small Sn diﬀraction peaks are still visible at the end of charge (2.5 V), suggesting incom- plete reversibility of the conversion process. At the end of oxidation, the Sn3N4 peaks become broader, similar to the pristine samples, indicating that the electrochemical reactions are reversible and the small crystallites reoxidise recovering the spinel Sn3N4 structure. The pristine electrode exhibits a reec- tion at 37.5 that disappears upon reduction and it is not recovered aer oxidation. This peak appears due to the use of a homogeniser for the electrode preparation: Fig. S1 in the ESI† shows that electrodes prepared without homogenization do not have this peak and exhibit the same changes in the XRD pattern induced by the electrochemical reactions. Unfortunately, the assignment of this peak is unclear: NiO and CrO2 show reec- tions close to that position, but at higher angles, and they show other reections that are not observed in the experimental patterns (see ESI, Fig. S6†). The pattern of the electrode at the end of oxidation (2.5 V) shows a very small peak at 26.2, which could be ascribed to the formation of small amounts of SnO2, but again, the position of the peak is not exactly as expected for SnO2 and other reections of SnO2 are not observed (ESI, Fig. S7†). Structural and chemical change during cycling decrease in the intensity of the peak related to the tetrahedral Sntet–N bond distance starts at very high potentials (1.6 V), supporting our interpretation of the electrochemical data that the rst sloping plateau centred at 1.6 V is due to sodiation surface reactions of substitution of a Sn tetrahedral site by Na. Then, as the potential is further reduced, a new environment starts to emerge from 1 V to 10 mV which could be assigned to the formation of Na–Sn alloys. The intensity of the tetrahedral Sntet–N peak increases in the EXAFS spectra during the subse- quent reoxidation of the electrode suggesting that the spinel Sn3N4 is reforming. This is in agreement with the increased oxidation state calculated from the XANES analysis (ESI, Table S5†). This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16447 Conclusions Microcrystalline and nanocrystalline Sn3N4 were prepared using solvothermal conditions at 430 C, or at 300 C followed by ultrasound treatment in aqueous HCl. Sn3N4 electrodes of both materials were cycled in Na half-cells, with the nanocrystalline electrode providing a higher capacity and good cycling stability over 50 cycles. Indeed, the capacity exceeds those reported for other tin-based materials, and good cycling stability with other tin-based materials has only been achieved with carbon supports or composite structures. The results presented here bring a deeper understanding of the Sn3N4 reaction mechanism in Na-ion cells. They provide evidence for the formation of Na– Sn alloys at the end of discharge and of reformation of Sn3N4 at the end of charge. DFT calculations combined with ex situ X-ray diﬀraction and X-ray absorption spectroscopy indicate that the N from Sn3N4 remains in the electrode in the form of some sort of Na–N compound. Na insertion into the Sn3N4 nano- particles, ejecting some Sn which forms SnxNay phases on the surface, has been shown to be energetically feasible from DFT calculations and is also consistent with the observed changes in the Sn–N and Na–Sn bond distances obtained from EXAFS spectra and with the change in the oxidation state obtained from XANES. The new solvothermal synthesis here developed is a simple and low cost synthesis route that enables an unprec- edented control of the Sn3N4 particle size, which is critical to achieve full utilization of Sn3N4 and thus high capacity. However, reaction (10) involves the formation of Na3N which is metastable and decomposes at around 100 C. We then considered the possibility that reaction (10) was not reversible due to decomposition of Na3N and the associated loss of N2, if that was the case, the electrochemical reactions would only involve the Sn–Na alloying reaction (reaction (11)), except for the rst discharge (reduction) of the cell which would also include reaction (10). However, reaction (11) of Na alloying of Sn to Na15Sn4 has a theoretical capacity of 732 mA h g1 when referred to the mass of the initial Sn3N4 material. This capacity value is lower than the observed values of the reversible capacities and it is also inconsistent with the high Sn oxidation state obtained by XANES at the end of oxidation (ca. 3.5) and the recovery of Sn3N4 observed in XRD measurements. Conﬂicts of interest There are no conicts of interest to declare. Conclusions All these measurements suggest that the electrochemical reactions on oxidation largely recover the original compound, Sn3N4. That would not be possible if N2 was lost in the form of gas. Insertion of additional Na beyond Na15Sn4 has been suggested from the analysis of operando NMR measurements of Sn reactions in Na- ion cells,45 but such additional Na insertion on the Na–Sn alloy is insuﬃcient to explain the experimental capacities observed in this work. The discharge prole of Sn3N4 in Na-half cells is also distinct from that observed for Sn, which again supports the hypothesis that the electrochemical reactions are not simply Na–Sn alloying (reaction (11)).14 The results of DFT calculations concur that Na3N is not stable against decomposition to Na and N2, in agreement with experimental observations. On the other hand, NaN3 and Na2N2 are stable or potentially stable. Considering the formation of NaN3 or Na2N2 in the rst conversion reaction, the following possible reaction mechanisms can be proposed: Discussion of reaction mechanism Previously we proposed that the electrochemical reactions of Sn3N4 in Na-ion cells could proceed via a conversion and alloying mechanism:36 Conversion: Sn3N4 + 12Na+ + 12e / 4Na3N + 3Sn (10) Alloying of Sn: 4Sn + 15Na+ + 15e / Na15Sn4 (11) (10) (11) The EXAFS spectra during the rst cycle (Fig. 9a) show a peak doublet at 1.5 ˚A radial distance corresponding to Sn–N bonds. The intensity of the lower radial distance peak within the doublet, corresponding to the tetrahedral Sntet–N bond distance, decreases greatly during reduction compared to the octahedral Snoct–N site (Fig. 9a and expanded in ESI, Fig. S8†) suggesting that Na substitution has taken place preferentially on the tetrahedral Sn sites. This is in agreement with the rst principle calculations that showed substitution of Na onto tetrahedral Sn sites to be slightly more favourable than on octahedral sites (ESI, Table S3†). It is worth mentioning that the Combination of reactions (10) and (11) has an associated theoretical capacity of 1512 mA h g1. This reaction mechanism is, thus, consistent with the observed reversible capacities of up to 850 mA h g1. The fact that the experimental capacity is lower than the theoretical capacity could be ascribed to incomplete conversion of Sn3N4 and alloying to produce Na15Sn4. Indeed, many battery materials undergoing conversion reactions do not reach the full theoretical capacity. Nanostructuring of the material (that is, decreasing the particle size) oen results in signicant improvements in the practical capacity, due to a better utilisation of the whole material. This is also observed This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. A, 2020, 8, 16437–16450 \| 16447 16447 View Article Online View Article Online Journal of Materials Chemistry A Journal of Materials Chemistry A Conversion with Na2N2 formation: Sn3N4 + 4Na+ + 4e / 2Na2N2 + 3Sn (14) Conversion with Na2N2 formation: Sn3N4 + 4Na+ + 4e / 2Na2N2 + 3Sn (14) in the present work by comparing the reversible capacities of the microcrystalline and nanocrystalline samples. Furthermore, the XRD characterisation evidences the presence of unreacted Sn3N4 throughout the whole discharge and charge process for the lower capacity microcrystalline sample. (14) Overall with Na2N2 formation: 4Sn3N4 + 61Na+ + 61e / 8Na2N2 + 3Na15Sn4 (15) (15) The analysis of XANES to quantify the variation of the Sn oxidation state following reduction and oxidation is also consistent with reactions (10) and (11). The Sn oxidation state in microcrystalline Sn3N4 is found to become negative (ca. 0.1) aer reduction, which demonstrates the formation of Sn–Na alloys. While the expected Sn oxidation state in Na15Sn4 is more negative, the XANES data provides an average measurement for the whole sample, and some unreacted or partially reacted Sn3N4 at the end of discharge is demonstrated by XRD. On charge, the Sn oxidation state increases to +3.5, which suggests that the original Sn3N4 material is recovered aer charge, and further support is obtained from the XRD characterisation, which shows the re-growth of broad Sn3N4 reections upon oxidation. The theoretical capacity of conversion and alloying is 818 mA h g1 with azide formation (reaction (13)) and 992 mA h g1 with azenide (reaction (15)). Of the phases considered the highest reversible capacity of 850 mA h g1 aer 50 cycles can only be explained by the azenide (Na2N2, reaction (15)). 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Journal of Materials Chemistry A Journal of Materials Chemistry A Journal of Materials Chemistry A acknowledge the use of the EPSRC-funded National Chemical Database service hosted by the Royal Society of Chemistry. acknowledge the use of the EPSRC-funded National Chemical Database service hosted by the Royal Society of Chemistry. acknowledge the use of the EPSRC-funded National Chemical Database service hosted by the Royal Society of Chemistry. 26 M. K. Datta, R. Epur, P. Saha, K. Kadakia, S. K. Park and P. N. Kumta, J. Power Sources, 2013, 225, 316–322. 27 Q. Wu, Q. Shao, Q. Li, Q. Duan, Y. Li and H. Wang, ACS Appl. Mater. Interfaces, 2018, 10, 15642–15651. Acknowledgements Conversion with NaN3 formation: 3Sn3N4 + 4Na+ + 4e / 4NaN3 + 9Sn (12) Conversion with NaN3 formation: 3Sn3N4 + 4Na+ + 4e / 4NaN3 + 9Sn (12) The authors thank EPSRC for funding an early career fellowship to NGA (EP/N024303/1) and the Smartlab diﬀractometer (EP/ K00509X/1 and EP/K009877/1). 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https://openalex.org/W4386864712	https://www.nature.com/articles/s41467-023-41591-1.pdf	English	null	Histidine modulates amyloid-like assembly of peptide nanomaterials and confers enzyme-like activity	Nature communications	2,023	cc-by	10,932	Histidine modulates amyloid-like assembly of peptide nanomaterials and confers enzyme-like activity Ye Yuan1,2,3, Lei Chen2, Lingfei Kong4, Lingling Qiu5, Zhendong Fu1, Minmin Sun2, Yuan Liu3, Miaomiao Cheng3, Saiyu Ma3, Xiaonan Wang2, Changhui Zhao1, Jing Jiang2, Xinzheng Zhang 4, Liping Wang1 & Lizeng Gao 2,3 Received: 12 July 2022 Accepted: 8 September 2023 Check for updates Amyloid-like assembly is not only associated with pathological events, but also leads to the development of novel nanomaterials with unique properties. Herein, using Fmoc diphenylalanine peptide (Fmoc–F–F) as a minimalistic model, we found that histidine can modulate the assembly behavior of Fmoc–F–F and induce enzyme-like catalysis. Speciﬁcally, the presence of his- tidine rearranges the β structure of Fmoc–F–F to assemble nanoﬁlaments, resulting in the formation of active site to mimic peroxidase-like activity that catalyzes ROS generation. A similar catalytic property is also observed in Aβ assembled ﬁlaments, which is correlated with the spatial proximity between intermolecular histidine and F-F. Notably, the assembled Aβ ﬁlaments are able to induce cellular ROS elevation and damage neuron cells, providing an insight into the pathological relationship between Aβ aggregation and Alzheimer’s disease. These ﬁndings highlight the potential of histidine as a modulator in amyloid-like assembly of peptide nanomaterials exerting enzyme-like catalysis. The ability of proteins and peptides to assemble into amyloid ﬁbrils has been recognized as having a strong association with severe neu- rodegenerative diseases1,2. The amyloid-like assembly shares a com- mon cross-β structure, in which the β-strand segments align perpendicular to the long ﬁbril3,4. Such type of assembly is driven by backbone hydrogen bonding and side-chain interactions (e.g., π–π stacking, hydrophobic interaction, and van der Waals)5. The Aβ is one of the typical proteins that can assemble into amyloid aggregates with a cross β pattern and frequently appear in many neurodegenerative diseases such as AD and Parkinson’s disease (PD)6–8. Studies on Aβ assembly facilitate our understanding of the pathogenesis of AD, possibly providing potential targets for diagnosis and intervention9. Aβ1–42, which is more hydrophobic and more prone to aggregation, is recognized as a more important contributor to AD development than Aβ1–40, which shows less toxic effect. However, the fundamental role and implication of amyloid assembly of Aβ fragments have not yet been fully elucidated in AD pathogenesis. The study on amyloid-like assembly in living systems has also promoted the exploration of engineering short peptide assemblies as emerging nanomaterials with exceptional properties10,11. Many small amyloid-forming peptides have been identiﬁed from parent proteins/ polypeptides. Article https://doi.org/10.1038/s41467-023-41591-1 1Key Laboratory for Molecular Enzymology and Engineering, School of Life Sciences, Jilin University, Changchun 130012, China. 2CAS Engineering Laboratory for Nanozyme, Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 3Nanozyme Medical Center, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China. 4National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 5Key Laboratory of Animal Genetics and Breeding and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China. e-mail: wanglp@jlu.edu.cn; gaolizeng@ibp.ac.cn Histidine modulates amyloid-like assembly of peptide nanomaterials and confers enzyme-like activity The assembly has been mostly conducted in organic solvents, such as dimethyl sulfoxide (DMSO), or in a mixed system of water and strong polar solvents, such as hexaﬂuoroisopropanol (HFIP). In contrast, the strategy is currently limited to modulating the assembly behavior of these short peptides in aqueous conditions, which enables the assembly to more closely resemble those occurring in physiological environments. In our study, we report that amino acids have the potential to modulate the assembly behavior of Fmoc–F–F under aqueous condi- tions through a sonication-standing process. Speciﬁcally, His was found to promote the aggregation of Fmoc–F–F dipeptides from nanorods into nanoﬁlaments through electrostatic interactions and hydrogen bonding, leading to the β structure rearrangement in the Fmoc–F–F amyloid-like assembly. Furthermore, the resulting Fmoc–F–F (His) ﬁlaments exhibited peroxidase (POD)-like activity that catalyzes ROS generation, with the imidazole group of His serving as the active site. Building upon the understanding of the interaction mode between His and F–F dipeptide, we found that Aβ1–42 peptide- assembled nanoﬁlaments exhibited POD-like activity which increased as the aggregation time prolonged. Importantly, Alphafold2-assisted structure analysis demonstrated that the intermolecular interaction between His and F–F contributed to the formation of active sites, which may enable Aβ1–42 ﬁlaments as a nanozyme to work in a phy- siological environment. Cellular and in vivo experiments demon- strated that Aβ1–42 ﬁlaments induced ROS toxicity to damage neuron cells, which provides an insight into the causal link between Aβ aggregation and AD pathogenesis. To obtain the optimal co-assembly conditions for Fmoc–F–F (His), we investigated sonication time, dipeptide concentration, amino acid concentration, and dipeptide to amino acid molar ratio. Only a few ﬁlaments were produced from aggregated nanorods with a short sonication time (10 min). The degree of depolymerization was inten- siﬁed under longer sonication time (20 min), but too long sonication time (45 min) resulted in ﬁlament entanglement (Supplementary Fig. 3). Thus, the Fmoc–F–F (His) ﬁlaments showed the highest degree of expansion and uniform dispersion after sonication treatment for 30 min. When the concentration of Fmoc–F–F was ﬁxed to 2 mg mL−1, low concentrations of His (2 mg mL−1) had little impact on the Fmoc–F–F structure, with only a few ﬁlaments peeling from nanorods. At high concentrations (30 mg mL−1), His intensiﬁed the entanglement of ﬁlaments. The His concentration in the range of 18–20 mg mL−1 was suitable for the formation of ﬁlaments (Supplementary Fig. 4). Histidine modulates amyloid-like assembly of peptide nanomaterials and confers enzyme-like activity For example, the diphenylalanine (F–F) as the shortest peptide able to self-assemble is identiﬁed by dissecting the structural information of Aβ1–42 polypeptide12,13. Due to its chemical simplicity and excellent self-assembly capacity, the F–F peptide has been employed in a wide range of nanostructures, such as nanotubes, 1Key Laboratory for Molecular Enzymology and Engineering, School of Life Sciences, Jilin University, Changchun 130012, China. 2CAS Engineering Laboratory for Nanozyme, Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 3Nanozyme Medical Center, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China. 4National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 5Key Laboratory of Animal Genetics and Breeding and Molecular Design of Jiangsu Province, Yangzhou University, Yangzhou, China. e-mail: wanglp@jlu.edu.cn; gaolizeng@ibp.ac.cn Nature Communications\| (2023) 14:5808 Nature Communications\| (2023) 14:5808 Nature Communications\| (2023) 14:5808 1 https://doi.org/10.1038/s41467-023-41591-1 Article Fmoc–F–F into ﬁlaments. However, the ﬁlaments were not as extensive as those produced by His, and some nanorods were still present in the co-assembly systems. Furthermore, the addition of Pro appeared to change the structure and shape (block-shape) of the Fmoc–F–F nanorods. In contrast, Threonine (Thr), Tyrosine (Tyr), and Cysteine (Cys) could not regulate Fmoc–F–F into ﬁlaments, although the nanorods showed orderly aggregation into spheres, as observed in the TEM images of Fmoc–F–F (Tyr) (Supplementary Fig. 2h), Fmoc–F–F (Thr) (Supplementary Fig. 2i), and Fmoc–F–F (Cys) (Supplementary Fig. 2j). Fmoc–F–F (Cys) showed the greatest degree of sphere for- mation. The morphologies of the other Fmoc–F–F-amino acid co- assemblies were very similar to Fmoc–F–F (Supplementary Fig. 2k–s). Taken together, in the Fmoc–F–F-amino acid co-assembly systems, positively charged amino acids regulated nanorods into ﬁlaments due to electrostatic interactions, whereas the polar uncharged amino acids retained the nanorods to varying degrees. The co-assembly conditions are summarized in Supplementary Table 1. As Fmoc–F–F (His) showed the greatest degree of ﬁbrillogenesis, it was used for the following research. nanowires, nanoarrays, nanospheres, and microtubes14. Given their unique mechanical and physical properties, these materials have pro- mising application potential, including as templates, piezoelectrical and optical biosensors, and nanomedicines15–20. In addition, modiﬁca- tion of chemicals or addition of co-assembly molecules regulates F–F assembly. For example, modiﬁcation at the amino terminus of F–F with hydrophobic ﬂuorenylmethoxycarbonyl (Fmoc)21 facilitates the for- mation of β-sheet-based ﬁbrous-hydrogel22. His regulates Fmoc–F–F (His) assembly from nanorods to nanoﬁlaments To understand the general effects of amino acids on Fmoc–F–F assembly, natural amino acids were mixed with Fmoc–F–F under aqueous condition (Fig. 1a). When amino acids absent, the Fmoc–F–F dipeptides self-assembled into stacked nanorods in aqueous solution and existed as milky white turbid liquid without DMSO or HFIP (Fig. 1b, c, f). The short thick nanorods shown in Fig. 1b (scanning electron microscopy, SEM) and Fig. 1c (transmission electron microscopy, TEM) were 3–6 μm long and 200–400 nm wide. In contrast, a clear hydrogel consisting of nanoﬁlaments was formed with the addition of His to the Fmoc–F–F co-assembly (Fig. 1d–f). Nanorods were converted into slender ﬁlaments (~20 nm wide) (Fig. 1d SEM, Fig. 1e TEM). The high- angle annular dark ﬁeld (HAADF)-TEM further conﬁrmed the mor- phology of nanoﬁlaments (Supplementary Fig. 1a). Negative-staining TEM showed that a single ﬁlament was composed of many ﬁner bun- dles (Supplementary Fig. 1b). The smooth surface of the ﬁlaments (Rq of 6.05 nm) was observed via atomic force microscopy (AFM) (Sup- plementary Fig. 1c, d). Rheological measurements of dynamic strain sweeps indicated the occurrence of a phase change from turbid liquid to hydrogel (G” > G’ →G’ > G”), which was invariant with increasing frequency, demonstrating typical hydrogel properties (Fig. 1g). Encouraged by this phenomenon, we systematically studied the co- assembly of Fmoc–F–F with amino acids. pp y g To understand the chemical group by which His affects the Fmoc–F–F structure, different side-chain modiﬁcations of His were used for co-assembly with dipeptides. Fmoc–F–F (Boc-His (Trt)-OH) (Supplementary Fig. 7a) and Fmoc–F–F (N-Acetyl-L-His) (Supplemen- tary Fig. 7b) showed similar structures to Fmoc–F–F. The difference was that Fmoc–F–F (Boc-His (Trt)-OH) formed thicker nanorods than Fmoc–F–F (N-Acetyl-L-His), which may be related to the asymmetric N-ring structure (like Fmoc–F–F (Pro)). Acetyl-modiﬁed His was unable to form ﬁlaments with Fmoc–F–F due to missing amino groups. Nanorod leaves were obtained from Fmoc–F–F (Fmoc–His) (Supple- mentary Fig. 7c, d). These results demonstrated that both imidazole and amino groups played important roles in the formation of ﬁla- ments. Moreover, Fmoc–F–F (His–OMe) (Supplementary Fig. 6e, f) showed reduced formation of ﬁlaments, indicating that the carboxyl group promoted the dissociation of nanorods and formation of ﬁlaments. Histidine modulates amyloid-like assembly of peptide nanomaterials and confers enzyme-like activity When the concentration of His was ﬁxed to 20 mg mL−1, too low (0.5 mg mL−1) or too high (>3 mg mL−1) concentrations of Fmoc–F–F caused the for- mation of denser ﬁlaments (Supplementary Fig. 5). The resistance value of water was also considered (Supplementary Fig. 6). Nature Communications\| (2023) 14:5808 Cryo-EM characterization reveals Fmoc–F–F (His) ﬁlaments composed of spindle structure In particular, thick bundles seemed to be bifurcated into thin bundles, resulting in the bundle winding pitch at about 132 Å. These features indicated that the two ends of the spindle unit might be transitional junctions to connect short spindles into a long nanoﬁlament. The spindle structure of Fmoc–F–F (His) nanoﬁla- ments was conﬁrmed by the negatively stained TEM images (Fig. 2c). Fig. 1 \| Phase change of Fmoc–F–F in His aqueous solution. a Schematic of phase change of co-assembly Fmoc–F–F by His. b, c SEM and TEM of self-assembled Fmoc–F–F in pure water. Stubby nanorods were obtained. d, e SEM and TEM of co- assembled Fmoc–F–F (His). Thin and long ﬁlaments transformed from nanorods were obtained due to the presence of His. f milky white turbid liquid formed by Fmoc–F–F and clear hydrogel formed by Fmoc–F–F (His). g Occurrence of phase transition based on rheological measurements of dynamic frequency sweeps of Fmoc–F–F (His). Three times each experiment was repeated independently with similar results. Representative images are shown. Source data are provided as a Source Data ﬁle. Nature Communications\| (2023) 14:5808 3 Fmoc–F–F and clear hydrogel formed by Fmoc–F–F (His). g Occurrence of phase transition based on rheological measurements of dynamic frequency sweeps of Fmoc–F–F (His). Three times each experiment was repeated independently with similar results. Representative images are shown. Source data are provided as a Source Data ﬁle. Fig. 1 \| Phase change of Fmoc–F–F in His aqueous solution. a Schematic of phase change of co-assembly Fmoc–F–F by His. b, c SEM and TEM of self-assembled Fmoc–F–F in pure water. Stubby nanorods were obtained. d, e SEM and TEM of co- assembled Fmoc–F–F (His). Thin and long ﬁlaments transformed from nanorods were obtained due to the presence of His. f milky white turbid liquid formed by were at 13.2 nm and the middle part was around 18.5 nm in the spindle unit (with the length ~100 nm) in the corresponding circles in Fig. 2a (Fig. 2b). In the red circle of Fig. 2b, 20 bundles which with the dia- meter around 0.58–0.75 nm were displayed, indicating that the nano- ﬁlaments were parallel aligned by tiny bundles in the middle part. In contrast, in the yellow and blue circles of Fig. 2b, the entangled bundles were observed. In particular, thick bundles seemed to be bifurcated into thin bundles, resulting in the bundle winding pitch at about 132 Å. Cryo-EM characterization reveals Fmoc–F–F (His) ﬁlaments composed of spindle structure To further understand the structure of Fmoc–F–F (His) formed nano- ﬁlaments, Cryo-EM was conducted to characterize local features (Fig. 2). As shown in Fig. 2a, the diameter of Fmoc–F–F nanoﬁlaments was around 20 nm. However, when the image was ampliﬁed, the ﬁla- ment showed heterogeneous diameters with a shape-like spindle structure which is narrow at two ends (yellow and blue circles) and wide in the middle part (red circle). Furthermore, two-dimensional structure analysis performed and demonstrated that the two ends In addition to His, Asparagine (Asn) (Supplementary Fig. 2a), Asparticacid (Asp) (Supplementary Fig. 2b), Serine (Ser) (Supplemen- tary Fig. 2c), Lysine (Lys) (Supplementary Fig. 2d), Arginine (Arg) (Supplementary Fig. 2e), Proline (Pro) (Supplementary Fig. 2f), and Leucine (Leu) (Supplementary Fig. 2g) could regulate the assembly of Nature Communications\| (2023) 14:5808 2 Article https://doi.org/10.1038/s41467-023-41591-1 were at 13.2 nm and the middle part was around 18.5 nm in the spindle nit (with the length ~100 nm) in the corresponding circles in Fig. 2a Fig. 2b). In the red circle of Fig. 2b, 20 bundles which with the dia- bundles were observed. In particular, thick bun bifurcated into thin bundles, resulting in the bund about 132 Å. These features indicated that the two ig. 1 \| Phase change of Fmoc–F–F in His aqueous solution. a Schematic of phase hange of co-assembly Fmoc–F–F by His. b, c SEM and TEM of self-assembled moc–F–F in pure water. Stubby nanorods were obtained. d, e SEM and TEM of co- ssembled Fmoc–F–F (His). Thin and long ﬁlaments transformed from nanorods ere obtained due to the presence of His. f milky white turbid liquid formed by Fmoc–F–F and clear hydrogel formed by Fmoc–F–F (His). g transition based on rheological measurements of dynamic Fmoc–F–F (His). Three times each experiment was repeate similar results. Representative images are shown. Source d Source Data ﬁle. were at 13.2 nm and the middle part was around 18.5 nm in the spindle unit (with the length ~100 nm) in the corresponding circles in Fig. 2a (Fig. 2b). In the red circle of Fig. 2b, 20 bundles which with the dia- meter around 0.58–0.75 nm were displayed, indicating that the nano- ﬁlaments were parallel aligned by tiny bundles in the middle part. In contrast, in the yellow and blue circles of Fig. 2b, the entangled bundles were observed. Cryo-EM characterization reveals Fmoc–F–F (His) ﬁlaments composed of spindle structure The ﬂuorescence intensity of Fmoc–F–F in the presence of His showed an upward trend with longer co-assembly period, indicating that His could regulate the formation of ﬁbril (Supplementary Fig. 15). In addition, zeta potential assay showed that Fmoc–F–F was ~−25 mV, while Fmoc–F–F (His) increased to ~+30 mV, proving the strong electrostatic interactions between His and Fmoc–F–F (Supplementary Fig. 16). Lastly, based on the small-angle X-ray scattering (SAXS) characterization of Fmoc–F–F (His) nanoﬁlaments (Supplementary Fig. 17), the average radius (R) value at 8.18 nm was estimated by ﬁtting the data using the cylinder model with SasView, which is consistent with the observation by Cryo-EM. Based on the above characterizations; we speculated that the whole nanoﬁlament is assembled by forming connective spindle units, which consist of the main structure with parallel bundles and entangled bundles at the ends. Overall, Cryo-EM further conﬁrmed that His changed the aggregation of Fmoc–F–F and formed co-existing tangled nanoﬁlaments from stacked nanorods. Cryo-EM characterization reveals Fmoc–F–F (His) ﬁlaments composed of spindle structure These features indicated that the two ends of the spindle unit might be transitional junctions to connect short spindles into a long nanoﬁlament. The spindle structure of Fmoc–F–F (His) nanoﬁla- ments was conﬁrmed by the negatively stained TEM images (Fig. 2c). Nature Communications\| (2023) 14:5808 3 Article https://doi.org/10.1038/s41467-023-41591-1 Fig. 2 \| Cryo-EM characterizations for Fmoc–F–F (His). a Cryo-EM image of Fmoc–F–F (His) showing primitive spindle ﬁlament with heterogeneous diameter. b Two-dimensional classiﬁcation analysis of Fmoc–F–F (His) showing parallel alignment and entangled bundles in spindle structure. The images correspond to three morphologies (dashed yellow circle, dashed red circle, and dashed blue circle) of boundles in a, respectively. c Negatively stained Cryo-EM image of Fmoc–F–F (His) using uranium acetate conﬁrming the spindle structure of nano- ﬁlaments. Three times each experiment was repeated independently with similar results. Representative images are shown. circle) of boundles in a, respectively. c Negatively stained Cryo-EM image of Fmoc–F–F (His) using uranium acetate conﬁrming the spindle structure of nano- ﬁlaments. Three times each experiment was repeated independently with similar results. Representative images are shown. Fig. 2 \| Cryo-EM characterizations for Fmoc–F–F (His). a Cryo-EM image of Fmoc–F–F (His) showing primitive spindle ﬁlament with heterogeneous diameter. b Two-dimensional classiﬁcation analysis of Fmoc–F–F (His) showing parallel alignment and entangled bundles in spindle structure. The images correspond to three morphologies (dashed yellow circle, dashed red circle, and dashed blue (Supplementary Fig. 11). Fourth, the chemical environment of Fmoc was tracked by ﬂuorescence spectroscopy. Neither F–F (H2O) nor F–F (His) showed emission absorption (Supplementary Fig. 12). However, under 280-nm excitation, Fmoc–F–F (His) showed a strong emission peak at ~312 nm, with a slight blue shift compared to Fmoc–F–F (320 nm) (Supplementary Fig. 13). This may be due to His weakening the interaction of π–π stacked Fmoc but enhancing the local effect of dipeptide. To show its ﬂuorescence characteristics more intuitively, three-dimensional (3D) ﬂuorescence analysis was performed (Supple- mentary Fig. 14). Furthermore, the thioﬂavin-T (ThT)-binding assay illustrated the formation of amyloid-like ﬁlaments under the combined action of Fmoc–F–F and His24. When 20 μM ThT was added to different sample solutions and excited at 438 nm, Fmoc–F–F (His) showed strong ﬂuorescence absorption at 485 nm. The time course of ThT intensity during ﬁbril formations was further tested. His remodels intermolecular interactions for Fmoc–F–F assembly into nanoﬁlament To understand the interactions between Fmoc–F–F and His, spectral characterizations were conducted. First, based on ultraviolet-visible spectroscopy (UV–VIS) (Supplementary Fig. 8), Fmoc–F–F (His) showed strong absorption peaks at 260 nm and 300 nm, corre- sponding to the absorption of benzene ring and Fmoc, respectively. The formation of these peaks in aqueous solution was due to the combination of Fmoc–F–F and His. Consistently, Fourier-transform infrared spectroscopy (FTIR) (Supplementary Figs. 9 and 10) identiﬁed that the benzene band at 1450 cm−1, representing the C = C vibration of the benzene ring, signiﬁcantly decreased in the presence of His. In addition, Fmoc–F–F exhibited stronger C–N stretching (1030 cm−1) and N-H (739 cm−1, 3300 cm−1) bending compared with Fmoc–F–F (His) and His23. Weakening of the Fmoc–F–F (His) amines indicated the formation of non-covalent interactions. The red shift of C = O (1630 cm−1) implicated the involvement of –COOH in hydrogen bonding. Furthermore, the redshift of –OH (3400 cm−1) under different amounts of Fmoc–F–F (His), indicated the formation of intermolecular hydrogen bonds. Third, X-ray diffraction (XRD) of Fmoc–F–F (His) was performed to investigate the interaction between His and Fmoc–F–F. The crystal peak diffraction intensity was high, sharp, and scattered, indicating a good crystal state. In addition, the fewer peaks of Fmoc–F–F (His) accompanied by a shift in the diffraction peaks sug- gested that His may enter the original crystal lattice and cause dis- tortion. Strong diffraction peaks at 21° and 24° were formed Nature Communications\| (2023) 14:5808 Co-assembly mechanism of Fmoc–F–F (His) To understand the co-assembly mechanism, experimental validation, and theoretical calculations were conducted. Firstly, the dynamic co- assembly process was demonstrated, as shown in Supplementary Fig. 18. Switching between ﬁlaments and nanorods was reversible, achieved by controlling the concentrations of Fmoc–F–F and His and Nature Communications\| (2023) 14:5808 4 Article https://doi.org/10.1038/s41467-023-41591-1 Fig. 3 \| Hydrogen bonding interactions of His and Fmoc–F–F destroy π–π stacking of F–F peptides and secondary structure. a XPS of Fmoc–F–F (His) and Fmoc–F–F. b C 1s peak of Fmoc–F–F (His) from XPS. c 1H solid nuclear magnetic resonance (NMR) spectra of Fmoc–F–F (His) and Fmoc–F–F. d Percentage of secondary structure (Fmoc–F–F (His)) of amide I region (1600 cm−1 to 1700 cm−1) characterized by FTIR. e Possible interactions in the process of co-assembly between Fmoc–F–F and His. Representative images are shown. Source data are provided as a Source Data ﬁle. roy π–π F–F (His) and ar magnetic ntage of secondary structure (Fmoc–F–F (His)) of amide I region (1600 cm−1 to 1700 cm−1) characterized by FTIR. e Possible interactions in the process of co-assembly between Fmoc–F–F and His. Representative images are shown. Source data are provided as a Source Data ﬁle. secondary structure (Fmoc–F–F (His)) of amide I region (1600 cm−1 to 1700 cm−1) characterized by FTIR. e Possible interactions in the process of co-assembly between Fmoc–F–F and His. Representative images are shown. Source data are provided as a Source Data ﬁle. Fig. 3 \| Hydrogen bonding interactions of His and Fmoc–F–F destroy π–π stacking of F–F peptides and secondary structure. a XPS of Fmoc–F–F (His) and Fmoc–F–F. b C 1s peak of Fmoc–F–F (His) from XPS. c 1H solid nuclear magnetic resonance (NMR) spectra of Fmoc–F–F (His) and Fmoc–F–F. d Percentage of in the presence of His (Fig. 3c). Lastly, FTIR of the stretching of the C = O groups in the amide I region was used to characterize secondary structures of the peptide assembly. Peak separation calculation was conducted through peak ﬁtting of the amide I region, ranging from 1600 to 1700 cm−1. The self-assembled Fmoc–F–F structure had a large number of antiparallel β-sheets (π–π stacking interaction) and a small number of α-helices (in the presence of –COO−) (The 1H of –COOH of Fmoc–F–F at ~14 ppm was disappeared, shown in Fig. 3c) (Supple- mentary Figs. 19b and 20, Supplementary Table 3), which is consistent with the previous research25. Co-assembly mechanism of Fmoc–F–F (His) Thus, the molecular arrangements of Fmoc–F–F and His could be greatly inﬂuenced by the strong hydrogen bonding interactions between Fmoc–F–F and His, respectively, leading to fur- ther structural transitions. The experimental results indicated that an entangled network of Fmoc–F–F and His was observed in the presence of His, which may be attributed to the driving force in intermolecular interactions of hydrogen bonds, as shown in Fig. 2b. Furthermore, compared to those in self-assemblies with Fmoc–F–F alone, the clas- sical β-sheet hydrogen bonds were prevented while the random hydrogen bonds were formed between Fmoc–F–F molecules in the co- Co-assembly mechanism of Fmoc–F–F (His) However, these assembly modes were destroyed by His due to electrostatic interactions and hydrogen bonding and converted to β-sheets (Fig. 3d, Supplementary Table 2). According to the above characterizations, we provided a possible schematic of Fmoc–F–F (His) co-assembly to demonstrate the role of His in driving amyloid-like assembly of the nanoﬁlaments of Fmoc–F–F by hydrogen bonding and electrostatic interactions (Fig. 3e). disrupting the steady states of the co-assembly system. Secondly, X-ray photoelectron spectroscopy (XPS) showed that π–π accumula- tion in Fmoc–F–F was destroyed during co-assembly through C 1s analysis of Fmoc–F–F and Fmoc–F–F (His) (Fig. 3a, b, Supplementary Fig. 19a and Table 1). 1H NMR demonstrated that the chemical shift of C = C–H in Fmoc–F–F (His) was at 6 ppm, while the corresponding shit in Fmoc–F–F was at ~7 ppm, indicating a decrease of π–π accumulation Table 1 \| Fmoc–F–F (His) and Fmoc–F–F XPS C 1s peak splitting results statistics Functional groups types Fmoc–F–F (His) (%) Fmoc–F–F (%) C=C 62.15 75.98 C–N 26.94 13.9 C=O 2.66 4.56 π–π 1.11 3.22 C–O 7.14 2.34 Table 1 \| Fmoc–F–F (His) and Fmoc–F–F XPS C 1s peak splitting results statistics To further understand the inhibitory mechanism of the Fmoc–F–F β-sheet-rich conformation through co-assembly with His, quantum Nature Communications\| (2023) 14:5808 5 Article https://doi.org/10.1038/s41467-023-41591-1 ig. 4 \| Theoretical analyses of Fmoc–F–F and His co-assembly. a Fmoc–F–F and His molecules. b Frontier orbitals of π-π stacking interaction of Fmoc–F–F dimer. Multiple dimers of Fmoc–F–F and His are connected by different hydrogen onding interaction modes. d Different trimers of 2Fmoc–F–F and His. Fmoc–F–F and 2His are connected by hydrogen bonding interaction modes. The atomic coordinates of the optimized computational models were provided in Supple- mentary Data 1. and 2His are connected by hydrogen bonding interaction modes. The atomic coordinates of the optimized computational models were provided in Supple- mentary Data 1. and 2His are connected by hydrogen bonding interaction modes. The atomic coordinates of the optimized computational models were provided in Supple- mentary Data 1. and 2His are connected by hydrogen bonding interaction modes. The atomic coordinates of the optimized computational models were provided in Supple- mentary Data 1. Fig. 4 \| Theoretical analyses of Fmoc–F–F and His co-assembly. a Fmoc–F–F and His molecules. b Frontier orbitals of π-π stacking interaction of Fmoc–F–F dimer. c Multiple dimers of Fmoc–F–F and His are connected by different hydrogen bonding interaction modes. Co-assembly mechanism of Fmoc–F–F (His) d Different trimers of 2Fmoc–F–F and His. Fmoc–F–F Fig. 4 \| Theoretical analyses of Fmoc–F–F and His co-assembly. a Fmoc–F–F and His molecules. b Frontier orbitals of π-π stacking interaction of Fmoc–F–F dimer. c Multiple dimers of Fmoc–F–F and His are connected by different hydrogen bonding interaction modes. d Different trimers of 2Fmoc–F–F and His. Fmoc–F–F assemblies in the presence of His, leading to secondary structure transformation to form nanoﬁlaments. calculations were conducted to assess the binding interactions between Fmoc–F–F and His molecules (Fig. 4a). The various Fmoc–F–F-His dimers were constructed and optimized using the density functional theory (DFT) calculations (Fig. 4c), and the results showed that these dimers were dominated by strong hydrogen bonding interactions, while the Fmoc–F–F-Fmoc–F–F dimers were dominated by π-π stacking interactions (Fig. 4b). The Fmoc–F–F-His dimers connected through double O−H···O−H hydrogen bonds between carboxyl groups in Fmoc–F–F and His showed the strongest binding energy. Of note, Fmoc–F–F molecules can connect two His molecules to form Fmoc–F–F-His-His trimers, and His molecules can connect two Fmoc–F–F molecules to form Fmoc–F–F-His-Fmoc–F–F trimers via different hydrogen bonding interactions (Fig. 4d, Supple- mentary Data 1). Thus, the molecular arrangements of Fmoc–F–F and His could be greatly inﬂuenced by the strong hydrogen bonding interactions between Fmoc–F–F and His, respectively, leading to fur- ther structural transitions. The experimental results indicated that an entangled network of Fmoc–F–F and His was observed in the presence of His, which may be attributed to the driving force in intermolecular interactions of hydrogen bonds, as shown in Fig. 2b. Furthermore, compared to those in self-assemblies with Fmoc–F–F alone, the clas- sical β-sheet hydrogen bonds were prevented while the random hydrogen bonds were formed between Fmoc–F–F molecules in the co- calculations were conducted to assess the binding interactions between Fmoc–F–F and His molecules (Fig. 4a). The various Fmoc–F–F-His dimers were constructed and optimized using the density functional theory (DFT) calculations (Fig. 4c), and the results showed that these dimers were dominated by strong hydrogen bonding interactions, while the Fmoc–F–F-Fmoc–F–F dimers were dominated by π-π stacking interactions (Fig. 4b). The Fmoc–F–F-His dimers connected through double O−H···O−H hydrogen bonds between carboxyl groups in Fmoc–F–F and His showed the strongest binding energy. Of note, Fmoc–F–F molecules can connect two His molecules to form Fmoc–F–F-His-His trimers, and His molecules can connect two Fmoc–F–F molecules to form Fmoc–F–F-His-Fmoc–F–F trimers via different hydrogen bonding interactions (Fig. 4d, Supple- mentary Data 1). Co-assembled Fmoc–F–F (His) nanoﬁlaments exert enzyme-like activity to regulate ROS Source data are provided asa Sour Data ﬁle. Article https://doi.org/10.1038/s41467-023-41591 The signiﬁcant difference was evaluated by a two-tailed unpaired t-test. p < 0.01. Representative images are shown. g Cytosolic ROS (cROS) levels of HT-22 cells treated by different concentrations of Aβ1-42 ﬁlaments. h Lipid ROS levels of HT-22 cells treated by Aβ1-42 ﬁlaments. i Schematic diagram of injecting Aβ1–42 ﬁlaments into the hippocampus of SD rats. j Percentage of hippocampal histiocytes with high cROS level in SD rats after treatment of Aβ1–42 ﬁlaments. n = 6 biologically inde- pendent animals. Mean ± SD is shown. The signiﬁcant difference was evaluated by a two-tailed unpaired t-test. p < 0.01, p < 0.05. Source data are provided asa Source Data ﬁle. Fig. 5 \| Enzyme-like activity of Fmoc–F–F (His) and Aβ assembly. a Peroxidase (POD)-like of Fmoc–F–F (His), Fmoc–F–F, and His, respectively. b Catalase (CAT)- like of Fmoc–F–F (His), Fmoc–F–F, and His, respectively. c POD-like activity of Aβ1–40 and Aβ1–42 ﬁlaments. The signiﬁcant difference was evaluated by a two- tailed unpaired t-test. n = 3 independent samples, bars represent means ± SD, ns means no signiﬁcance, *p < 0.0001, p < 0.01, p < 0.05). d ESR characterization for •OH generation by POD-like activity of Aβ1–42 aggregates (half a year). e Predicted structures of Aβ1–42 and Aβ1–42 (6His→Ala) by alphafold2. f POD-like of Aβ1–42 and Aβ1–42 (6His→Ala). n = 3 independent samples. Mean ± SD is shown. nanoscale assembly. The kinetics assays showed that. Fmoc–F–F (His) nanoﬁlaments followed the typical Michaelis–Menten kinetics, in which the KM values (the Michaelis constant, representing the afﬁnity of an enzyme to the substrate) of Fmoc–F–F (His) nanoﬁlaments were 5.61 mM for H2O2 and 0.919 mM for TMB, respectively (Supplementary Fig. 23). Importantly, incubating Fmoc–F–F (His) nanoﬁlaments with neuron cells signiﬁcantly increased cellular ROS level (Supplemen- tary Fig. 24). the Aβ monomer and oligomers. When incubated for a longer time (half a year) at 37 °C, Aβ 1–42 formed ﬁlaments similar to Fmoc–F–F (His), and its catalytic activity was further enhanced. Similar to Fmoc–F–F (His) nanoﬁlaments, the kinetics assays showed that Aβ 1–42 ﬁlaments also followed the Michaelis–Menten kinetics, in which the KM values were calculated to be 0.40 mM for H2O2 and 0.32 mM for TMB, respectively (Supplementary Fig. 28). To further conﬁrm free radical generation, electron spin resonance (ESR) characterization was used to monitor the spectrum in the mixture of Aβ1–42 aggregates and H2O2. Co-assembled Fmoc–F–F (His) nanoﬁlaments exert enzyme-like activity to regulate ROS Analysis showed that His conferred Fmoc–F–F with catalytic activity. The oxidoreductase and hydrolase activities of Fmoc–F–F (His) were monitored. Figure 5a demonstrated that Fmoc–F–F (His) exhibited high POD-like activity which catalyzes 3,3’,5,5’-tetramethylbenzidine (TMB) colorimetric reaction (the oxidized TMB with absorbance at 652 nm) in the presence of hydrogen peroxide (H2O2), but His exhib- ited low peroxidase (POD)-like activity and Fmoc–F–F exhibited almost no POD-like activity (Fig. 5a). In particular, the nanoﬁlaments of Fmoc–F–F (His) remained intact structure with high catalytic activity after storage at room temperature for 60 days, showing a high stability (Supplementary Fig. 21). Importantly, the POD-like activity increased in Fmoc–F–F (His) assembly with longer term, indicating a positive cor- relation between catalysis and the assembly time (Supplementary Fig. 22). In contrast, neither Fmoc–F–F (His) nor Fmoc–F–F showed catalase (CAT)-like activity which catalyzes the decomposition of H2O2 into oxygen and H2O, despite His showed high CAT-like activity (Fig. 5b). These results indicate that Fmoc–F–F (His) nanoﬁlaments might be a nanozyme with POD-like activity which can be ascribed to Nature Communications\| (2023) 14:5808 6 Article https://doi.org/10.1038/s41467-023-41591-1 Fig. 5 \| Enzyme-like activity of Fmoc–F–F (His) and Aβ assembly. a Peroxidase (POD)-like of Fmoc–F–F (His), Fmoc–F–F, and His, respectively. b Catalase (CAT)- like of Fmoc–F–F (His), Fmoc–F–F, and His, respectively. c POD-like activity of Aβ1–40 and Aβ1–42 ﬁlaments. The signiﬁcant difference was evaluated by a two- tailed unpaired t-test. n = 3 independent samples, bars represent means ± SD, ns means no signiﬁcance, *p < 0.0001, p < 0.01, p < 0.05). d ESR characterization for •OH generation by POD-like activity of Aβ1–42 aggregates (half a year). e Predicted structures of Aβ1–42 and Aβ1–42 (6His→Ala) by alphafold2. f POD-like of Aβ1–42 and Aβ1–42 (6His→Ala). n = 3 independent samples. Mean ± SD is shown. The signiﬁcant difference was evaluated by a two-tailed unpaired t-test. p < 0.0 Representative images are shown. g Cytosolic ROS (cROS) levels of HT-22 cells treated by different concentrations of Aβ1-42 ﬁlaments. h Lipid ROS levels of HT- cells treated by Aβ1-42 ﬁlaments. i Schematic diagram of injecting Aβ1–42 ﬁlamen into the hippocampus of SD rats. j Percentage of hippocampal histiocytes with hig cROS level in SD rats after treatment of Aβ1–42 ﬁlaments. n = 6 biologically inde pendent animals. Mean ± SD is shown. The signiﬁcant difference was evaluated by two-tailed unpaired t-test. p < 0.01, p < 0.05. Nature Communications\| (2023) 14:5808 Co-assembled Fmoc–F–F (His) nanoﬁlaments exert enzyme-like activity to regulate ROS However, if all three His were mutated (6, 13, 14His→Ala), Aβ1-42 would form a sec- ondary structure mainly in random coils without β conformations, indicating that His is important to maintain β conformations. The cytotoxicity of Aβ 1-42 monomers, oligomers, and ﬁlaments were tested, and the result was shown in Supplementary Fig. 30. Their cytotoxicity (HT-22) may be related to their POD-like activity. Fur- thermore, Aβ1–42 ﬁlaments showed the ability to increase reactive oxygen species (ROS) levels (including lipid ROS, cytosolic ROS (cROS), and ROS of mitochondria) in HT-22 cells (hippocampus neuron26) (Fig. 5g, h and Supplementary Figs. 31 and 32). Consistently, the increased cROS level was observed when injecting Aβ 1–42 ﬁla- ments into the bilateral hippocampus of Sprague Dawley rats (SD rats) (Fig. 5i), in which the percentage of cells with detectable cROS in hippocampal neurons signiﬁcantly increased (Fig. 5j). These results indicate that Aβ1–42 ﬁlaments might be a ROS generator due to its POD activity. H2O2 + 2H + + 2TMB ! 2H2O + 2oxTMB H2O2 + H + + TMB + Fmoc F F ðHisÞ ! H2O* 2 + ðH + + TMBÞ* ð1Þ H2O* 2 + ðH + + TMBÞ * ! HO* + H2O* + oxTMB* ð2Þ HO* + ðH + + TMBÞ * ! H2O* + oxTMB* ð3Þ Asterisk () marks species adsorbed on Fmoc–F–F (His). We then studied the POD-like activities of the oxidation reactions of the two TMB molecules oxidized by H2O2 under acidic conditions. The pro- posed reaction pathway is presented in Fig. 6, as well as the structural parameters for key intermediates in the POD-like catalytic cycle. The relative energies of the key intermediates are shown in Supplementary Fig. 33. As seen in Fig. 6; there were seven steps from the adsorption of H2O2 to the oxidation of the second TMB molecule, including six stable states and a transition state in the proposed reaction pathway. The dominant elementary reactions are presented in Eqs. 1–3, corre- sponding to the three steps in Supplementary Fig. 33, respectively: (1) adsorption of H2O2 and (H+ + TMB) molecules on Fmoc–F–F (His); (2) oxidation reaction of the ﬁrst TMB molecule oxidized by H2O2 under acidic conditions, producing HO, H2O, and oxTMB; and (3) oxidation of second TMB molecule by second HO under acidic conditions, producing H2O* and oxTMB. Co-assembled Fmoc–F–F (His) nanoﬁlaments exert enzyme-like activity to regulate ROS 1–3) serve as a plausible mechanism for the POD- like activities: intermolecular distance between 6His and 19Phe–20Phe was 10.28 Å in tetramer model, which was much shorter than that of 13His and 14His (15.58 Å), indicating that 6His is more important for the construction of catalytic center of peroxidase-like activity (Supplementary Fig. 29). Such mutant Aβ1–42 assembly showed no POD-like activity compared to wild Aβ1–42 (Fig. 5f), indicating that POD activity of Aβ has strong relationship with His. In contrast, the predicted structure of the mutants of 13His→Ala and 14His→Ala showed no much inﬂuence on the distance between 6His and 19Phe–20Phe, indicating that both His may have less contribution on catalysis of Aβ1–42 assembly. However, if all three His were mutated (6, 13, 14His→Ala), Aβ1-42 would form a sec- ondary structure mainly in random coils without β conformations, indicating that His is important to maintain β conformations. The cytotoxicity of Aβ 1-42 monomers, oligomers, and ﬁlaments were tested, and the result was shown in Supplementary Fig. 30. Their cytotoxicity (HT-22) may be related to their POD-like activity. Fur- thermore, Aβ1–42 ﬁlaments showed the ability to increase reactive oxygen species (ROS) levels (including lipid ROS, cytosolic ROS (cROS), and ROS of mitochondria) in HT-22 cells (hippocampus neuron26) (Fig. 5g, h and Supplementary Figs. 31 and 32). Consistently, the increased cROS level was observed when injecting Aβ 1–42 ﬁla- ments into the bilateral hippocampus of Sprague Dawley rats (SD rats) (Fig. 5i), in which the percentage of cells with detectable cROS in hippocampal neurons signiﬁcantly increased (Fig. 5j). These results indicate that Aβ1–42 ﬁlaments might be a ROS generator due to its POD activity. intermolecular distance between 6His and 19Phe–20Phe was 10.28 Å in tetramer model, which was much shorter than that of 13His and 14His (15.58 Å), indicating that 6His is more important for the construction of catalytic center of peroxidase-like activity (Supplementary Fig. 29). Such mutant Aβ1–42 assembly showed no POD-like activity compared to wild Aβ1–42 (Fig. 5f), indicating that POD activity of Aβ has strong relationship with His. In contrast, the predicted structure of the mutants of 13His→Ala and 14His→Ala showed no much inﬂuence on the distance between 6His and 19Phe–20Phe, indicating that both His may have less contribution on catalysis of Aβ1–42 assembly. Co-assembled Fmoc–F–F (His) nanoﬁlaments exert enzyme-like activity to regulate ROS As shown in Fig. 5d, the featured peaks for hydroxyl radical (•OH) demonstrated that Aβ1–42 aggregates performed POD-like activity to generate ROS in the presence of H2O2). Since Aβ polypeptide also contains F–F (19, 20) dipeptide and rich in His (6, 13, 14), it is hypothesized that its aggregates may perform similar catalytic properties due to the formation of nanoﬁlaments. To prove our hypothesis, we prepared Aβ series aggregates (forms of Aβ shown in Supplementary Figs. 25–27) and checked their catalytic activity. We found that both Aβ1–40 and Aβ1–42 aggregates exhibited POD-like activities, in which Aβ1–42 aggregates showed higher activity than Aβ1–40 (Fig. 5c). In addition, Aβ incubated for 14 days (similar TEM to Fmoc–F–F) possessed higher POD-like activity compared with To conﬁrm the contribution of His to catalysis, the mutant with 6His replaced by Ala was prepared(Aβ1–42 (6His→Ala)), and the structure was predicted with Alphafold2 (Fig. 5e). In a tetramer assembly model, the secondary structure in wild-type Aβ1–42 is mainly β conformations (β sheet, β turn) and random coils. The Nature Communications\| (2023) 14:5808 7 https://doi.org/10.1038/s41467-023-41591-1 Article Fig. 6 \| Proposed mechanism of POD-like catalysis of Fmoc–F–F (His) and key structural parameters. Seven reaction states mediated by POD-like catalysis of Fmoc–F–F (His) was proposed. The whole process can be divided into three major steps: The adsorption of H2O2 and (H+ + TMB) molecules on Fmoc–F–F (His) (states 1–3); Oxidation reaction of the ﬁrst TMB molecule oxidized by H2O2 under acidic conditions, producing HO, H2O, and oxTMB* (states 4–5); Oxidation of second TMB molecule by second HO* under acidic conditions, producing H2O* and oxTMB* (states 6–7). The corresponding binding energy of each step can be seen in Supplementary Fig. 33. conditions, producing HO, H2O, and oxTMB* (states 4–5); Oxidation of second TMB molecule by second HO* under acidic conditions, producing H2O* and oxTMB* (states 6–7). The corresponding binding energy of each step can be seen in Supplementary Fig. 33. Fig. 6 \| Proposed mechanism of POD-like catalysis of Fmoc–F–F (His) and key structural parameters. Seven reaction states mediated by POD-like catalysis of Fmoc–F–F (His) was proposed. The whole process can be divided into three major steps: The adsorption of H2O2 and (H+ + TMB) molecules on Fmoc–F–F (His) (states 1–3); Oxidation reaction of the ﬁrst TMB molecule oxidized by H2O2* under acidic three reactions (Eqs. Co-assembled Fmoc–F–F (His) nanoﬁlaments exert enzyme-like activity to regulate ROS To elucidate the POD-like mechanism of Fmoc–F–F (His), the Fmoc–F–F and two His molecule trimer was selected as the peroxidase catalytic reaction site (Fig. 6). We used DFT to calculate the reactants, products, intermediates, and transition states of the possible reaction pathway of the POD-like activities. As shown in Fig. 6, the imidazole rings of the two His molecules formed a catalytic center. The following Our calculations indicated that the H2O2 molecule binds to the imidazole rings via hydrogen bonding with a binding energy of 0.63 eV Nature Communications\| (2023) 14:5808 8 https://doi.org/10.1038/s41467-023-41591-1 Article most widely espoused theories of AD etiology, as Aβ aggregation and deposition exhibit strong associations with neurotoxicity and neurodegeneration31. In such a hypothesis, oxidative stress is con- sidered a potential key pathway for Aβ to generate toxicity, which is usually ascribed to the aggregated form of Aβ coordinating with metal ions such as copper, iron, or zinc or enzyme cofactors such as heme. For example, positions 6, 13, and 14 of His can chelate copper ions, thereby generating ROS and causing neurotoxicity32. In addition, Aβ may bind to heme to form Aβ-heme complexes with POD activity, further generating ROS and neuronal damage33. However, we dis- covered that Aβ-aggregated ﬁlaments themselves exhibited POD-like activity and acted as an ROS initiator. In addition, the POD-like activity of Aβ1–42 ﬁlaments may be correlated with its aggregated form and deposition period. This phenomenon is consistent with the observa- tion that when Aβ1–42 oligomers are converted to ﬁbrils in plaques, the damage is increased and causes chronic neurodegeneration with subsequent cognitive impairment and dementia34,35. Thus, the dis- covery of intrinsic catalytic activity of Aβ aggregates may provide a new aspect of understanding the Aβ cascade hypothesis in AD etiol- ogy, i.e., assembled Aβ serves as a nanozyme to catalyze ROS gen- eration and cause long-acting oxidative stress. In particular, it has been recently reported that Aβ can be easily aggregated in lysosomes36, in which the acidic environment may proﬁt them to perform POD-like activity, thus presenting a feasible way to generate ROS. in the catalytic center, as shown in state (2) in Fig. 6. The hydrogen bonding interactions between H2O2* and imidazole rings facilitate the rapture of the O–O bond in H2O2. Co-assembled Fmoc–F–F (His) nanoﬁlaments exert enzyme-like activity to regulate ROS The adsorbed H2O2 molecule in the catalytic center ﬁrst breaks the O–O bond and one of the dissociated OH radicals binds to the H atom from TMB to form an H2O molecule and oxTMB* molecule. After the escape of H2O* and oxTMB, the remaining OH radical in the catalytic center oxides the next TMB molecule. As shown in Supplementary Fig. 33, under acidic conditions, the ﬁrst TMB molecule is easily oxidized by H2O2* with an energy barrier of 1.41 eV in step 2, while the second TMB molecule is easily oxidized by the OH* radical, as shown in step 3. From DFT analysis, the POD-like catalytic center of the co-assembly of Fmoc–F–F (His) effec- tively catalyzes the oxidation of TMB by H2O2 under acidic conditions due to the arrangement of His molecules in Fmoc–F–F (His) facilitating POD-like activity. Ethical regulations All research complied with all relevant ethical regulations. All animal studies were performed following the protocols approved by the Institutional Animal Care and Use Committee of the Institute of Bio- physics, Chinese Academy of Sciences. In addition, Aβ1–42 ﬁlaments also exhibited POD-like activity, in which His may play an important role in the catalysis, as POD-like activity disappeared when 6His was mutated in Aβ1–42. These features demonstrated that Aβ1–42 ﬁlaments might also be a nanozyme. Importantly, Alphafold2 predication demonstrated that the active site of His/F–F may derive from intermolecular assembly, indicating that Aβ1–42 ﬁlaments are a catalytic unit with multiple active sites in the assembled format, which is distinct from traditional enzymes that often have single active center inside the protein framework. Accord- ing to enzymatic kinetics assays, a single active site of His/F–F in Aβ1–42 may not be as efﬁcient as that in natural peroxidase, which is composed of hemin coordinated with His residues29. However, one Aβ1–42 nanoﬁlament contains multiple active sites in the assembled format, thus, the whole catalytic effect can not be negligible. In addi- tion, the assembled Aβ1–42 nanoﬁlaments are quite stable, hard to degrade, and can exist steadily for a long time, thus readily achieving durable catalytic activity. Therefore, different from traditional enzymes, Aβ1–42 ﬁlaments might act in the catalytic mode of nano- zymes under a physiological environment. Discussion In this study, we demonstrated that His plays a critical role in mod- ulating amyloid-like assembly and building active sites for Fmoc–F–F and Aβ aggregates. Distinct from the studies that most F–F assemblies have been conducted in organic solvents (e.g. DMSO, HFIP)27,28, we demonstrated that the presence of an amino acid, particularly His, allowed Fmoc–F–F assembly from nanorods to nanoﬁlaments under aqueous condition. In addition, the Fmoc–F–F (His)-assembled nano- ﬁlaments exhibit POD-like activity, demonstrating that His makes a major contribution to catalysis, which is similar to His residue in the active center of natural peroxidase29. These features demonstrate that F–F (His)-assembled nanoﬁlaments are a type of POD-like nanozyme. Nanozymes refer, in particular, to the nanomaterials that can catalyze biochemical substrates of enzymes under physiological conditions, following similar enzymatic kinetics and thus performing enzyme-like catalysis, which has been recognized as next-generation artiﬁcial enzymes30. One of the major features is that nanozymes have multiple active sites on the surface, and thus the catalysis is determined by their nanostructures. Distinct from inorganic nanozymes, the Fmoc–F–F (His) nanoﬁlaments performed POD-like activity depending on His mediated peptide assembled ﬁlament nanostructure, which contained a large number of active sites of His/F–F counterparts along the nanoﬁlament. Therefore, Fmoc–F–F (His) nanoﬁlaments are a nano- zyme ascribed to their assembled nanostructure and catalysis, which is different from the structure–activity relationship of natural enzymes, although both of them are composed of amino acids. Overall, His modulation of Fmoc–F–F amyloid-like assembly and enhancement of catalytic activity demonstrated that introducing an amino acid into a peptide assembly is an effective strategy to program nanostructures and design peptide-based nanozymes. Aβ1–42 ﬁla- ments were found to perform POD-like activity to enhance oxidative stress, which might also be ascribed to the interaction mode of His and F–F. Future work will focus on deciphering the precise structure of the assembled Fmoc–F–F (His) ﬁlaments and the role of His in Aβ assembly and catalysis, experimentally verifying whether His residues may be the targets for controlling Aβ aggregation and reducing neurotoxicity in AD. Reagents The dipeptides and thioﬂavin-T (ThT) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (China). Amino acids were purchased from Shanghai Macklin Biochemical Co., Ltd. (China). 3,3’,5,5’-Tetra- methylbenzidine (TMB), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), BODIPY 581/591 C11 probe and p-nitrophenyl acetate (pNPA) were purchased from Sigma-Aldrich Co., Ltd. (USA). M5 HiPure MitoSOX Red Mitochondrial Superoxide Indicator (MitoSOX probe) was pur- chased from Mei5 Biotechnology Co., Ltd. (China). DCFH-DA ROS detection kit was purchased from Beijing Fluorescence Biotechnology Co. Ltd. (China). The Acetylcholinesterase (AchE) Assay Kit was pur- chased from Beijing Solarbio Science & Technology Co., Ltd. (China). The Aβ peptides (purity > 95%) were purchased from GL Biochem (Shanghai, China). Nature Communications\| (2023) 14:5808 Cellular and in vivo ROS assessment Cellular and in vivo ROS assessment HT-22 cells (SCC129, Sigma Aldrich/Merck) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (PS). 1 × 105 cells per well were plated in 12- well plates and cultured with 5% CO2 at 37 °C for 24 h. HT-22 was treated with different concentrations of Aβ1–42 ﬁlaments for 24 h. For oxidative stress assessment, cells were incubated with 200 µL of 2 µM BODIPY 581/591 C11 probe, 4 µM MitoSOX probe, or 10 µM DCFH-DA working solution for 30 min at 37 °C (except MitoSOX probe for 10 min). To measure intracellular lipid peroxidation, ROS of Mito- chondria, and cytosolic ROS (cROS), respectively. After digestion with trypsin, 1 × 104 cells were collected and analyzed using ﬂow cytometry (BD FACSCalibur) to measure the ﬂuorescence intensity of these ROS levels. For cytotoxicity assay, HT-22 cells were planted in 96-well plates (6 × 103 cells per well) and cultured with 5% CO2 at 37 °C overnight. Then the culture medium was replaced by fresh DMEM medium with 10% FBS and 1% PS, 100 μg mL−1 of Aβ 1–42 monomer, oligomer, and ﬁlament were added to incubate for another 24 h. Then the medium containing materials was removed, and cell viability was measured using a CCK8 Kit. The cROS detection in PC-12 cells (CRL-1721, ATCC) (They were maintained in RPMI 1640 medium supplemented with 10% horse serum (HS), FBS 5% FBS, 1% PS, and pre-induced with 50 ng mL−1 nerve growth factor (NGF) for neuronal differentiation for 10 d) was detected in adding 10 µM DCFH-DA working solution for 30 min at 37 °C. After digestion with trypsin, 1 × 104 cells were collected and analyzed using ﬂow cytometry (BD FACSCalibur) (following the gating strategy in Supplementary Fig. 31) for measuring the ﬂuorescence intensity of these cROS levels. SAXS measurements were performed on the solution samples of a concentration of 3.75 mM loaded in a low noiseﬂowcellusing a Xenocs Xeuss 2.0 instrument equipped with a Dectris Pilatus 300k detector (pixel size 172 μm) and a Cu-Kα radiation source (wavelength λ = 1.54189 Å). 2D SAXS data were collected at an exposure time of 1200 s (room temperature). The SAXS data of the Silver Behenate standard were also collected and used to calibrate the sample-to- detector distance (2480 mm). Electron microscopy for peptide samples Samples were dropped on a silicon wafer at room temperature (~23 °C). The SEM images were recorded using a Hitachi SU8010 (Japan), and TEM images were recorded using a Tecnai Spirit (FEI) (USA) operating at 100 kV dropped on a double copper net. For AFM, liquid Fmoc–F–F (His) (1 mL) was dropped on a mica sheet and observed by Bruker Dension Icon (Germany). For negative staining TEM, the co-assembled Fmoc–F–F (His) was stained with uranyl acet- ate. Cryo-EM and two-dimensional classiﬁcation data were collected by Talos Arctica 200 kV FEG (USA), and the sample was pre-frozen at −184 °C. Computational methods All quantum chemical calculations were performed using DFT meth- ods with an empirical dispersion correction (D3) implemented using the Gaussian09 package. Typical Fmoc–F–F/His dimers and trimers predominantly stabilized by the intermolecular hydrogen bonds were constructed and optimized at the level of B3LYP/6-31 g (d) theory. Harmonic vibrational frequency calculations of the optimized geo- metries were also performed to ensure the structures at local minima. The Fmoc–F–F/His binding energies (ΔE) were calculated as the energy difference between their molecular clusters and the sum of the ener- gies of Fmoc–F–F and His (Eq. 4). Cellular and in vivo ROS assessment The 2D SAXS data of the samples were reduced to obtain 1D scattering intensity proﬁle I(q), where q is the scattering vector (q = 4πsinθ/λ (4), 2θ is the scattering angle). The raw data were corrected for the sample background (ﬂow cell containing water) in the data reduction process using the SasView5.0.6 software package37,38 (the instructions could be obtained from https://www. sasview.org/). For ThT-binding analysis, 20 µL of assembled solution was added to 160 µL of DDH2O in a 96-well blackboard. Then, 20 µL of 200 µM ThT was immediately added. Excitation was set to 438 nm, and ﬂuores- cence intensity was recorded at an emission wavelength of 485 nm. Zeta potentials of samples were determined using a Zetasizer Nano ZS90 (England) via three tests in a 1-mL sample cell. The UV spectrum at 200–800 nm was collected using a Tanon-3500R Gel Imaging System (Shanghai, China). The ﬂuorescence emission spec- trum at 300–550 nm and 280 nm excitation was collected using a F-7000 FL spectrophotometer (Japan) with a scan speed of 1200 nm/ min. The PMT voltage and response were set to 500 V and 0.1 s, respectively. Both EX Slit and EM Slit were set to 2.5 nm. To evaluate the ability of Aβ 1–42 ﬁlaments to generate cROS in vivo, 10 µl Aβ ﬁlaments (1 mg mL−1) and PBS were injected into the bilateral hippocampus of the male SD rats39 (6–8 weeks, all animals were housed in a temperature controlled (25 °C) condition, and were given free access to water and diet.), respectively. After the SD rats were observed for 5 days, the right hippocampus tissue was taken and prepared into a cell suspension following the below procedures. Firstly, the rats were dislocated and sacriﬁced after anesthesia, and the brain was collected (about 3–5 min). Next, after washing with normal saline, the right hippocampus was peeled off on the ice bag and quickly Preparation of dipeptide and amino acid assemblies Using liquid chromatography-mass spectrometry (LC-MS), peptides with purities >97% were veriﬁed. The F–F was stored at −20 °C, and Fmoc–F–F was stored at −20 °C. For assembly, amino acids (con- centration 20 mg mL−1) were weighed and dissolved in deionized water after 8 min treatment of sonication. Peptides (concentration 2 mg mL−1) were weighed and dispersed in amino acid aqueous Consequently, we speculated that Aβ1–42 ﬁlaments as a natural nanozyme with peroxidase-like activity may help understand the role of Aβ deposition in AD pathogenesis, as their detailed pathogenesis in AD is not yet fully elucidated. The Aβ cascade hypothesis is one of the Nature Communications\| (2023) 14:5808 9 https://doi.org/10.1038/s41467-023-41591-1 Article of buffer, 20 µL of TMB, 20 µL of Aβ, and 20 µL of H2O2 (625 mM) were added in order and reacted for 2 h at 37 °C. Absorbance at 652 nm was monitored using a microplate reader. For CAT activity, 500 mM H2O2 solution was prepared, with 3.6 mL of H2O2 and 0.4 mL of Fmoc–F–F (His) added. Immediately, the COD analyzer probe was inserted into the reaction solution to record the number of different reaction times. Evaluation of •OH was performed by the interaction between Aβ and H2O2. Brieﬂy, DMPO (50 mM) and H2O2 (62.5 mM) were dissolved in sodium acetate buffer (0.1 M, pH 3.5) containing Aβ (100 μg mL−1). The product DMPO-OH was detected using an A300-10/12 ESR instrument (Germany). solution. After sonication treatment and standing a while (30 min), clariﬁed Fmoc–F–F (His) instead of cloudy Fmoc–F–F solution was prepared. Characterizations based on spectroscopy, XRD, NMR, SAXS, ThT binding, and zeta potential The samples were characterized using FTIR spectroscopy. For FTIR, 3 mg of powdered sample and 50 mg of potassium bromide (KBr) were mixed for tablet compression analysis with transmittance at 400–4000 cm−1, collected using an iS10 FTIR spectrometer (USA). Measurements were performed by averaging 32 scans at 4-cm−1 reso- lution (signal-to-noise ratio (S/N) = 50000). The samples were char- acterized by XRD using a D8 ADVANCE (Germany). Measurements were performed at 1.5406 angstroms of the copper target wavelength, 40-kV tube voltage, and 40-mA tube current. As for NMR, solid sam- ples were characterized by JNM-ECZ600R at a frequency of 600 MHz at room temperature. The tube diameter, mass frequency, and relaxation delay were set as 3.2 mm, 15 kHz, and 5 s by averaging 16 scans. 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Reprints and permissions information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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Acknowledgements We thank Dr. Jianping Jia from the Clinical Center for Neurodegenerative Disease and Memory Impairment, Capital Medical University, for the discussion of the relationship between Aβ ﬁlament catalysis and AD pathogenesis. We thank the Testing and Analysis Center at the Institute of Biophysics, Chinese Academy of Sciences (CAS), for the character- ization of nanomaterials. We thank Can Peng for help with SEM samples. We thank Shuoguo Li and Yun Feng from the Center for Biological Imaging (CBI), Institute of Biophysics, CAS, for help with taking and analyzing TEM, Prof. Ningdong Huang from the University of Science and Technology of China for help with SAXS analysis. This work was sup- ported by the National Key R&D Program of China (2019YFA0709200), the National Natural Science Foundation of China (32201162), the 70th general grant of China Postdoctoral Science Foundation (2021M702947) N ti l N t l S i F d ti f Chi F d Author contributions L.G. conceived and organized the project. Y.Y. participated in all experiments. L.C. performed theoretical calculations and participated in structural and logical analyses. L.K. performed and collected cryo-EM, negative staining TEM, and two-dimensional classiﬁcation data. L.Q., M.C., Z.F., S.M., M.S., C.Z., and X.W. performed the cell tests and data analysis. J.J. guided the experiments. Y.L. performed TEM. L.G., X.Z., and © The Author(s) 2023 Nature Communications\| (2023) 14:5808 Nature Communications\| (2023) 14:5808 Nature Communications\| (2023) 14:5808 12
https://openalex.org/W4205293802	https://www.frontiersin.org/articles/10.3389/fchem.2021.785848/pdf	English	null	MALDI-TOF MS Based Bacterial Antibiotics Resistance Finger Print for Diabetic Pedopathy	Frontiers in chemistry	2,022	cc-by	5,889	ORIGINAL RESEARCH published: 14 January 2022 doi: 10.3389/fchem.2021.785848 MALDI-TOF MS Based Bacterial Antibiotics Resistance Finger Print for Diabetic Pedopathy Haojie Sun 1,2, Peng Lai 2, Wei Wu 3, Hao Heng 2, Shanwen Si 2, Yan Ye 2, Jiayi Li 2, Hehe Lyu 2, Caiyan Zou 2, Mengzhe Guo 4, Yu Wang 2, Houfa Geng 2 and Jun Liang 1,2* 1Medical College, Soochow University, Suzhou, China, 2Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical University, Xuzhou, China, 3Afﬁliated Hospital of Xuzhou Medical University, Xuzhou, China, 4Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, China Diabetes mellitus has become a major global health issue. Currently, the use of antibiotics remains the best foundational strategy in the control of diabetic foot infections. However, the lack of accurate identiﬁcation of pathogens and the empirical use of antibiotics at early stages of infection represents a non-targeted treatment approach with a poor curative effect that may increase the of bacterial drug resistance. Therefore, the timely identiﬁcation of drug resistant bacteria is the key to increasing the efﬁcacy of treatments for diabetic foot infections. The traditional identiﬁcation method is based on bacterial morphology, cell physiology, and biochemistry. Despite the simplicity and low costs associated with this method, it is time-consuming and has limited clinical value, which delays early diagnosis and treatment. In the recent years, MALDI-TOF MS has emerged as a promising new technology in the ﬁeld of clinical microbial identiﬁcation. In this study, we developed a strategy for the identiﬁcation of drug resistance in the diagnosis of diabetic foot infections using a combination of macro-proteomics and MALDI MS analysis. The macro-proteomics result was utilized to determine the differential proteins in the resistance group and the corresponding peptide fragments were used as the ﬁnger print in a MALDI MS analysis. This strategy was successfully used in the research of drug resistance in patients with diabetic foot infections and achieved several biomarkers that could be used as a ﬁnger print for 4 different drugs, including ceftazidime, piperacillin, levoﬂoxacin, and tetracycline. This method can quickly conﬁrm the drug resistance of clinical diabetic foot infections, which can help aid in the early treatment of patients. Edited by: Kezhi Jiang, Hangzhou Normal University, China Edited by: Kezhi Jiang, Hangzhou Normal University, China Edited by: Kezhi Jiang, Hangzhou Normal University, China Reviewed by: Haoyang Wang, Shanghai Institute of Organic Chemistry, China Guodong Cao, Anhui Medical University, China Correspondence: Jun Liang mwlj521@163.com Houfa Geng genghoufa@njmu.edu.cn Yu Wang wangyubh@163.com Reviewed by: Haoyang Wang, Shanghai Institute of Organic Chemistry, China Guodong Cao, Anhui Medical University, China Correspondence: Jun Liang mwlj521@163.com Houfa Geng genghoufa@njmu.edu.cn Yu Wang wangyubh@163.com Specialty section: This article was submitted to Analytical Chemistry, a section of the journal Frontiers in Chemistry Received: 29 September 2021 Accepted: 27 December 2021 Published: 14 January 2022 Specialty section: This article was submitted to Analytical Chemistry, a section of the journal Frontiers in Chemistry Received: 29 September 2021 Accepted: 27 December 2021 Published: 14 January 2022 INTRODUCTION Diabetes mellitus has become a major global health issue affecting approximately 9.3% of the population worldwide, and is expected to increase by 25% by 2030 (Sinclair et al., 2020). Approximately 30% of diabetic patients will develop diabetic pedopathy during their lifetime (Armstrong et al., 2017). Of all the complications of diabetes mellitus, diabetic pedopathy poses the most severe risks, and may result in a shortened life expectancy and a dramatic decline in the quality of life (Lavery et al., 2016). In the past few decades, diabetic pedopathy has ranked 10th among all diseases and is often a huge ﬁnancial burden to the patients family as well as society as a whole (Lazzarini et al., 2018). Keywords: MALDI TOF, macro-proteomics, drug resistance, diabetic foot, ﬁnger print Citation: Sun H, Lai P, Wu W, Heng H, Si S, Ye Y, Li J, Lyu H, Zou C, Guo M, Wang Y, Geng H and Liang J (2022) MALDI-TOF MS Based Bacterial Antibiotics Resistance Finger Print for Diabetic Pedopathy. Front. Chem. 9:785848. doi: 10.3389/fchem.2021.785848 January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 1 Sun et al. MALDI Drug Resistance Analysis We have developed a MALDI mass spectrometry (MS) ﬁnger- print analysis to be used in the diagnosis of antibiotic resistance in patients with diabetic foot infections (Figure 1). First, the bacteria from the affected tissue of patients were cultured and the species were identiﬁed. The bacterial cultures were then tested for drug resistance and divided into either a drug-resistant or drug- sensitive group. Next, the bacterial macro-proteomics analysis was introduced to identify the differential proteins between these two groups. Finally, the fragments of the identiﬁed differential proteins were compared using the MALDI analysis to identify the drug-resistance ﬁnger print. This study could aid in the establishment of a rapid drug resistance MALDI identiﬁcation method to be used for the clinical the determination of treatments for diabetic foot infections. At present, antibiotics have been the foundational strategy used to control diabetic foot infections. Due to the lack of accurate identiﬁcation of the pathogens, the empirical use of antibiotics at the early stages of infection represents a non-targeted treatment approach with a poor curative effect, and may result in bacterial drug resistance (Munita and Arias, 2016). The World Health Organization has recognized antibiotic resistance as one of the most important public health threats in the 21st century (World Health Organization, 2014). The ineffective use of antibiotics will hinder wound healing in patients with diabetic foot infections, and increase the length of stay in hospitals as well as hospital costs. Therefore, timely identiﬁcation of bacteria and drug resistance is the key in the appropriate treatment of diabetic foot infections (Caruso et al., 2021). Enterobacterales and Staphylococcus aureus are the most common pathogens identiﬁed in diabetic pedopathy (Ramirez et al., 2018; Alexi et al., 2021). They play a crucial role in MDR organisms, and their broad antibiotics resistance has increasingly attracted more attention from healthcare associated workers worldwide. Especially concerning is the resistance to cephalosporin and penicillin antibiotics, which may lead to life-threatening issues in the treatment of diabetes-related infections (Weinstein et al., 2019). Specimen Collection After ﬂushing the wound with sterile saline, a well-trained diabetic podiatrist took the deep necrotic tissue of the wound to avoid superﬁcial tissue contamination, which could cause inaccurate results. A total of 16 necrotic tissue specimens were collected and divided into two parts under sterile conditions: one part was used for conventional microbial culture and the other was used for MALDI TOF MS analysis. In recent years, MALDI-TOF MS has emerged as a promising new technology in the ﬁeld of clinical microbial identiﬁcation. It is fast, stable, accurate, sensitive, and has a high resolution (Tan et al., 2012; Scott et al., 2016). Its expanded research application in clinical microorganisms has involved many ﬁelds, such as bacterial identiﬁcation, drug resistance analysis, virulence, and epidemiology (Kostrzewa, 2018). Its potential for the rapid detection of bacterial drug resistance has made it especially attractive to many scholars. It falls within the mass range by giving the protein that is resistant to bacteria, and then analyzes the drug resistance of bacteria by looking for characteristic spectral peaks. This study explores its use in detecting bacterial drug resistance in diabetic foot infections and provides a basis for rapid clinical antibiotic selection. Patient Characteristics The subjects of the study included type 2 diabetic foot patients hospitalized in the Department of Endocrine and Metabolic Diseases of the Xuzhou Central Hospital from June 2021 to August 2021. The main inclusion criteria were: age was within the range of 18–80 years old, and Wagner 3–4. Exclusion criteria: no standing debridement, immunosuppressants had been used within 3 months prior to admission, and the use of antibiotics in the 4 weeks prior to admission. The diagnosis of diabetes mellitus is based on the 1999 WHO diabetes mellitus diagnostic criteria. The diagnosis of diabetic foot is based on the International Diabetes Foot working group (IWGDF) guide (Lipsky et al., 2020). This study was designed and implemented according to the declaration of Helsinki. All patients and their families were informed and agreed to participate in the study, which was approved by the hospital ethics committee of Xuzhou central hospital. Bacterial identiﬁcation is a crucial component of clinical practice. The traditional method is based on the assessment of bacterial morphology, cell physiology, and biochemistry (Sun et al., 2021). While the simplicity of this method and low costs make it an attractive option, it is time-consuming and has limited clinical value due to its low positive rate (Jasson et al., 2010; Oberhettinger et al., 2020). This results in diagnostic and treatment delays. Citation: Over the last decade, the resistance of Enterobacterales to cephalosporin has dramatically increased worldwide. The underlying resistance mechanisms are complex and difﬁcult to be identify. Additionally, carbapenem-hydrolyzing enzymes produced by bacterium are also of great concern (Mao et al., 2017). For example, Klebsiella pneumonia, which has the characteristics of easy-spreading and extensive-contaminating, could have a detrimental effect in its surroundings, such as healthcare settings (Tascini et al., 2015). S aureus has primarily exhibits penicillin-resistance. Due to the horizontal transfer and natural selection of genes that encode for a mutant penicillin- binding protein (Masters et al., 2020), this bacteria has a low afﬁnity to penicillin molecules, which underlies the penicillin- resistance. Frontiers in Chemistry \| www.frontiersin.org Label Free Macro-Proteomics Analysis y 100 µg of protein was reduced with 5 m Mdithiothreitol (DTT) for 1 h at 37°C and subsequently alkylated with 10 mM iodoacetamide for 45 min at RT (room temperature) in the dark. Samples were diluted 1:3 with 50 mM Tris-HCl (pH 8.0) and subjected to proteolytic digestion with trypsin (Promega) at a 1:50 enzyme-to- substrate ratio and incubated overnight at RT. The digested samples were then acidiﬁed with 50% triﬂuoroacetic acid (TFA, Sigma) to a pH value of approximately 2.0. Tryptic peptides were desalted on reversed-phase C18 SPE columns and dried using a Speed-Vac. A total of 16 samples from patients with diabetic foot infections were collected and used to create bacterial cultures. 5 Gram- positive strains and 11 Gram-negative strains were successfully cultivated. Under the authentication of MALDI MS, the Gram- positive strains consisted of staphylococcus aureus and the Gram- negative strains consisted of bacillus (Figure 2). The bacterial cultures were then processed to determine drug-resistance. Both the staphylococcus aureus and bacillus were tested using 2 types of drug resistance experiments. In the case for bacillus, ceftazidime and piperacillin were chosen as the test drugs, and levoﬂoxacin and tetracycline were chosen for staphylococcus aureus. The proteins in these bacterial cultures were then extracted and passed through the macro-proteomics and MALDI analysis. Peptides (0.8 μg) were separated on an Easy nLC 1200 UHPLC system (Thermo Scientiﬁc) on an in-house packed 20 cm × 75 mm diameter C18 column. The column the ﬂow rate was 0.200 μL/min with 0.1% formic acid and 2% acetonitrilein water (A) and 0.1% formic acid, 90% acetonitrile (B). The peptides were separated with a 6–30% B gradient in 84 min and analyzed using the Thermo Velos mass spectrometer (Thermo Scientiﬁc). Parameters were as follows: MS1: resolution—60,000, mass range 350–1800 m/z, MS2: resolution −50,000, high-energy collision dissociation activation energy (HCD) was 37 eV, isolation width (m/z) was 0.7, AGC Target was 2.0 e5, Max IT was 105 ms. For the bacillus, a total 1,500 proteins were detected by the macro-proteomics analysis. Additionally, there were over 500 identical proteins among them. The samples were then divided into two groups according to the drug resistance test results. As shown in Supplementary Table S1, there are ﬁve bacillus samples in the ceftazidime resistance group and six bacillus samples in the other group. Moreover, PCA analyses were carried out for these two groups according to the identical proteins. Data Statistics Experiments were repeated at least three times with consistent results. Data are presented as the Mean ± SEM (standard error of the mean) or Mean ± SD (standard deviation). Differences between groups were determined using a two-tailed Mann- Whitney U test or two-tailed Student’s t-test. Pearson correlation coefﬁcients (r) were calculated to evaluate correlation and statistical signiﬁcance was assessed by a two- tailed t-test. The results of western blot analysis are the representative images of at least three independent experiments. For boxplots, the center line represents the median, the box limits show the upper and lower quartiles, and the outliers are represented as individual data points. MALDI TOF MS Method MALDI TOF MS analysis was performed by a Bruker ultraﬂe Xtreme MALDI TOF/TOF mass spectrometry (Bruker Daltonics, France). α-cyano-4- hydroxycinnamic acid (HCCA) was used as the matrix and was dissolved into ACN. The extracted proteins were dissolved into water and 1 µl of protein solution was mixed with 1 µl of matrix. The mixture was dripped on a reusable polished steel target and left to dry. The ion mode was positive ion mode with delay: 150 ns; ion source 1 voltage: 20 kV; ion source 2 voltage: 18 kV; lens voltage: 6 kV. All spectra were shown baseline-subtracted, smoothed, and auto-scaled in the Y-direction, covering a range of 300–3,000 Da. Bacterial Inactivation and Protein Extraction Bacterial inactivation was performed before MS analysis. The bacterial colonies after 72 h incubation were resuspended in 200 μl of pure water. The concentration of bacteria was chosen to be 5 × 106 CFU/ml. Then 600 μl of methanol (MeOH) was added to obtain a ﬁnal 75% MeOH. This mixture was incubated for 15 min and then centrifuged at 12,000 rom for 10 min to obtain a bacterial pellet. The supernatant was removed and the bacterial pellet was resuspended in 100% acetonitrile (ACN) with 0.1% formic acid. The mixture was passed through the secondary January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 2 Sun et al. MALDI Drug Resistance Analysis FIGURE 1 \| The strategy of the drug resistance ﬁnger print research for diabetic foot. FIGURE 1 \| The strategy of the drug resistance ﬁnger print research for diabetic foot. centrifugation (also 12,000 rpm for 10 min) and the bacterial proteins were extracted. Label Free Macro-Proteomics Analysis As shown in January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 3 Sun et al. MALDI Drug Resistance Analysis FIGURE 2 \| The identiﬁcation of Klebsiella pneumonia (A) and Staphylococcus aureus (B) based on MALDI TOF analysis after bacterial cultured. RE 2 \| The identiﬁcation of Klebsiella pneumonia (A) and Staphylococcus aureus (B) based on MALDI TOF analysis after bacterial cultured. \| The identiﬁcation of Klebsiella pneumonia (A) and Staphylococcus aureus (B) based on MALDI TOF analysis after bacterial cultu Figure 3, these two groups can be well distinguished, with the Q2 over 0.8. A total of 10 differential proteins (p < 0.05 in t-test) were obtained, including fumC, fmt, rpsT, proX, ﬁsH, nuoC, proC, tyrS, ribC, and bfr. Additionally, the digested peptides fragments were found in the database, and were compared with the MALDI TOF analysis. Finally, three peptides were consistently obtained in both results (Figures 3C,D, Supplementary Figure S1), including the peptides from nuoC with a sequence of EALEWGTTGAGLR (m/z 1,360.7), from ﬁsH with a sequence of ESTAYHEAGHAIIGR (m/z 1,611.8), and from tyrS with a sequence of LAEEIIYGPEHVSTGASNDIK (m/z 2243.1). These three peaks of peptides can be considered the ﬁnger print for the resistance of bacillus to ceftazidime. Figure 3, these two groups can be well distinguished, with the Q2 over 0.8. A total of 10 differential proteins (p < 0.05 in t-test) were obtained, including fumC, fmt, rpsT, proX, ﬁsH, nuoC, proC, tyrS, ribC, and bfr. Additionally, the digested peptides fragments were found in the database, and were compared with the MALDI TOF analysis. Finally, three peptides were consistently obtained in both results (Figures 3C,D, Supplementary Figure S1), including the peptides from nuoC with a sequence of EALEWGTTGAGLR (m/z 1,360.7), from ﬁsH with a sequence of ESTAYHEAGHAIIGR (m/z 1,611.8), and from tyrS with a sequence of LAEEIIYGPEHVSTGASNDIK (m/z 2243.1). These three peaks of peptides can be considered the ﬁnger print for the resistance of bacillus to ceftazidime. including the peptides from acnA with a sequence of SDTYGWQEDSTYIR (m/z 1720.8), from ompA with a sequence of ATLKPEGQAALDQLYSQLSNLDPK (m/z 2600.4), and from rpmI with a sequence of GDLGLVIACLPYA (m/z 1,361.7). These three peaks of peptides can be considered as the ﬁnger print for the resistance of bacillus to piperacillin.. Label Free Macro-Proteomics Analysis including the peptides from acnA with a sequence of SDTYGWQEDSTYIR (m/z 1720.8), from ompA with a sequence of ATLKPEGQAALDQLYSQLSNLDPK (m/z 2600.4), and from rpmI with a sequence of GDLGLVIACLPYA (m/z 1,361.7). These three peaks of peptides can be considered as the ﬁnger print for the resistance of bacillus to piperacillin.. For the staphylococcus aureus, a total 1,000 proteins were detected by macro-proteomics analysis. In addition, there are over 200 identical proteins among them. The samples were divided into two groups according to the two other drug resistance test results. As shown in Supplementary Table S2, there are two staphylococcus aureus samples in levoﬂoxacin resistance group and three staphylococcus aureus samples in the sensitive group. Moreover, the PCA analysis were carried out for these two groups according to the identical proteins. As shown in Figure 5, these two groups can be well distinguished with the Q2 over 0.8. But only 3 differential proteins (p < 0.05 in t-test) were obtained, including rpsS, rplV, and rpsQ. Additionally, the digested peptides fragments were found in the database, which were compared with the MALDI TOF analysis. Finally, three peptides were obtained consistently in both results (Figures 5C,D, Supplementary Figure S3), including the peptides from rpsS with the sequence QHVPVFVTDRMVGHK (m/z 1722.9), from rplV with the sequence VLESAIANARHNDGADIDDLKVTK (m/z 2538.3), The samples were also divided into two groups according to piperacillin resistance. As shown in Supplementary Table S1, there are six bacillus samples in the piperacillin resistance group and ﬁve bacillus samples in the other group. The PCA analyses were carried out for these two groups according to the identical proteins. As shown in Figure 4, these two groups can be well distinguished, with the Q2 over 0.8. A total of 14 differential proteins (p < 0.05 in t-test) were obtained, including ybjP, rpmI, rof, rim, acnA, lolA, engB, hisS, bglX, cpxR, phoP, trxA, ompA, and bfr. Additionally, the digested peptides fragments were found in the database, which were compared with the MALDI TOF analysis. Finally, three peptides were obtained consistently in both results (Figures 4C,D, Supplementary Figure S2), January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 4 MALDI Drug Resistance Analysis Sun et al. Label Free Macro-Proteomics Analysis GURE 3 \| (A) The PCA analysis of drug resistance of ceftazidime for Klebsiella pneumonia; (B) The differential proteins between drug resistance group and drug ensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. FIGURE 3 \| (A) The PCA analysis of drug resistance of ceftazidime for Klebsiella pneumonia; (B) The differential proteins between drug resistance group and drug sensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. Frontiers in Chemistry \| www.frontiersin.org January 2022 \| Volume 9 \| Article 785848 GURE 4 \| (A) The PCA analysis of drug resistance of piperacillin for Klebsiella pneumonia; (B) The differential proteins between drug resistance group and drug nsitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. et al. MALDI Drug Resistance Analysi MALDI Drug Resistance Analysis Sun et al. FIGURE 4 \| (A) The PCA analysis of drug resistance of piperacillin for Klebsiella pneumonia; (B) The differential proteins between drug resistance group and drug sensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. FIGURE 4 \| (A) The PCA analysis of drug resistance of piperacillin for Klebsiella pneumonia; (B) The differential proteins between drug resistance group and drug sensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. Frontiers in Chemistry \| www.frontiersin.org January 2022 \| Volume 9 \| Article 785848 6 MALDI Drug Resistance Analysis Sun et al. GURE 5 \| (A) The PCA analysis of drug resistance of levoﬂoxacin for Staphylococcus aureus; (B) The differential proteins between drug resistance group and ug sensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. FIGURE 5 \| (A) The PCA analysis of drug resistance of levoﬂoxacin for Staphylococcus aureus; (B) The differential proteins between drug resistance group and FIGURE 5 \| (A) The PCA analysis of drug resistance of levoﬂoxacin for Staphylococcus aureus; (B) The differential proteins between drug resistance group and drug sensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 7 MALDI Drug Resistance Analysis Sun et al. Label Free Macro-Proteomics Analysis GURE 6 \| (A) The PCA analysis of drug resistance of tetracycline for Staphylococcus aureus; (B) The differential proteins between drug resistance group and ug sensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. FIGURE 6 \| (A) The PCA analysis of drug resistance of tetracycline for Staphylococcus aureus; (B) The differential proteins between drug resistance group and drug sensitive group; (C) The corresponding differential peptides and (D) the ﬁnger print in MALDI TOF. January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 8 MALDI Drug Resistance Analysis Sun et al. strains consisted of bacillus. Subsequently, a macro-proteomics analysis was carried out to detect the proteins associated with antibiotic resistance. As a result, a total of 1,500 proteins and 1,000 proteins were detected in the bacillus and staphylococcus aureus by macro-proteomics analysis, respectively. Combined with the results of bacterial culture, MALDI TOF, and proteomics, we found a series of differentially expressed proteins. According to the antibiotics resistance, subgroup analyses have conﬁrmed the presence of a ﬁnger print of the different bacterium. The peaks of the nuoC, ﬁsH, and tyrS peptides can be considered as the ﬁnger print for the resistance of bacillus to ceftazidime resistance. The peaks of the acnA, ompA, and rpmI peptides can be considered as the ﬁnger print for the resistance of bacillus to ceftazidime. The peaks of the rpsS, rplV, and rpsQ peptides can be considered as the ﬁnger print for the resistance of staphylococcus aureus to levoﬂoxacin resistance. The peaks of the rplE, guaA, and proS peptides can be considered as the ﬁnger print for the resistance of staphylococcus aureus to tetracycline. and from rpsQ with the sequence LHVHDENNECGIGDVVEIR (m/z 2205.1). These three peaks of peptides can be considered as the ﬁnger print for the levoﬂoxacin resistance of staphylococcus aureus. The staphylococcus aureus samples were also divided into two groups according to the tetracycline resistance results. As shown in Supplementary Table S2, there are three staphylococcus aureus samples in levoﬂoxacin resistance group and two staphylococcus aureus samples in the sensitive group. As shown in Figure 6, these two groups can be well distinguished with the Q2 over 0.8. And 10 differential proteins (p < 0.05 in t-test) were obtained, including rpmA, rpsT, dnaN, guaA, proS, alaS, frr, adk, groS, and rplE. DISCUSSION Recent studies have suggested that the microbiota in diabetic foot infections are diverse, which is typically beyond the identiﬁcation capabilities of the traditional culture method. Although molecular-based methods, such as qPCR and mNGS, can effectively identify the bacterial species, they are difﬁcult to routinely carry out in clinical practice and also have several inherent biases (Malone et al., 2017; Sadeghpour Heravi et al., 2019). Overall, there is still no simple and speciﬁc method to identify the antibiotics resistance of bacteria. Using bacterial cultures made from diabetic pedopathy patients, we further conducted the MALDI TOF analysis. Consistent with previous studies (Złoch et al., 2021), our study found both Gram-positive and Gram-negative bacterium. A total of 5 strains of Gram-positive bacteria and 11 strains of Gram- negative bacteria were used for further MALDI detection and analysis, which authenticated that the Gram-positive strains consisted of staphylococcus aureus and the Gram-negative CONCLUSION In this work, we have developed a strategy for the identiﬁcation of drug resistance in the diagnosis of diabetic foot. Macro-proteomics and MALDI MS analysis were combined in this strategy. From the macro-proteomics result, the differential proteins which in the resistance group were obtained and the correspondence peptide fragments were used as the ﬁnger print in MALDI MS analysis. Then this strategy was successfully used in the drug resistance research in diabetic foot patients and achieved several biomarkers as ﬁnger print for 4 drugs, including ceftazidime, piperacillin, levoﬂoxacin, and tetracycline. This method can quickly conﬁrm the drug resistance of diabetic foot in clinical, which can help the treatment of patients as early as possible. Label Free Macro-Proteomics Analysis Additionally, the digested peptides fragments were found in the database, which were compared with the MALDI TOF analysis. Finally, three peptides were obtained consistently in both results (Figures 6C,D, Supplementary Figure S4), including the peptides from rplE with the sequence AKLHDYYKDEVVKK (m/z 1735.9), from guaA with the sequence DFNPSGIILSGGPESTTEENSPR (m/z 2404.2), and from proS with the sequence DAYSFHTSQESLQETYDAMYAAYSK (m/z 2906.3). These three peaks of peptides can be considered as the ﬁnger print for the tetracycline resistance of staphylococcus aureus. An appropriate antibiotic strategy is well known to be essential in the management of diabetic foot infections. However, the prompt identiﬁcation of antibiotic resistance is the cornerstone of anti-infection therapy. The bacterial resistance ﬁngerprint identiﬁed by this study can guide modiﬁcations to clinical antibiotic regimens in the earliest period, which may enhance infection control and wound healing of diabetic pedopathy, shorten the length of hospital stays, and reduce overall costs. Based on the convenience and accuracy of the new method in the identiﬁcation of a bacteria ﬁnger print, as well as results from a previous study (Asghari et al., 2021), has conﬁrmed that MALDI TOF could be widely used in clinic and guide decision-making regarding the use of antibiotics. In addition, we added 12 bacterial samples from diabetic podiatry to validate our method, including 7 samples of ceftazidime resistance of bacillus, and 5 samples of levoﬂoxacin resistance of staphylococcus aureus. We examined whether there were characteristic peaks of resistance in these samples. 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Microbiol. 50 (10), 3301–3308. doi:10.1128/ JCM.01405-12 Lipsky, B. A., Senneville, É., Abbas, Z. G., Aragón-Sánchez, J., Diggle, M., Embil, J. M., et al. (2020). Guidelines on the Diagnosis and Treatment of Foot Infection in Persons with Diabetes (IWGDF 2019 Update). Diabetes Metab. Res. Rev. 36, e3280. doi:10.1002/dmrr.3280 Malone, M., Johani, K., Jensen, S. O., Gosbell, I. B., Dickson, H. G., Hu, H., et al. (2017). Next Generation DNA Sequencing of Tissues from Infected Diabetic Foot Ulcers. EBioMedicine 21, 142–149. doi:10.1016/ j.ebiom.2017.06.026 Tascini, C., Lipsky, B. A., Iacopi, E., Ripoli, A., Sbrana, F., Coppelli, A., et al. (2015). KPC-producing Klebsiella pneumoniae Rectal Colonization Is a Risk Factor for Mortality in Patients with Diabetic Foot Infections. Clin. Microbiol. Infect. 21 (8), e1. doi:10.1016/j.cmi.2015.04.010 Mao, W., Xia, L., and Xie, H. (2017). Detection of Carbapenemase-Producing Organisms with a Carbapenem-Based Fluorogenic Probe. Angew. Chem. Int. Ed. 56 (16), 4468–4472. doi:10.1002/anie.201612495 Masters, E. A., de Mesy Bentley, K. L., Gill, A. L., Hao, S. P., Galloway, C. A., Salminen, A. T., et al. (2020). Identiﬁcation of Penicillin Binding Protein 4 (PBP4) as a Critical Factor for Staphylococcus aureus Bone Invasion during Weinstein, E. J., Han, J. H., Lautenbach, E., Nachamkin, I., Garrigan, C., Bilker, W. B., et al. (2019). AUTHOR CONTRIBUTIONS HS, PL, and WW were contributed in sample collection, experimental operations, and data analysis, which can be considered as the co-ﬁrst authors. HH, SS, YW, and CZ, YY, JL, HL were contributed into data analysis and improving paragraphs. MG, HG, and JuL were contributed in experimental design. HS, PL, and WW were contributed in sample collection, experimental operations, and data analysis, which can be considered as the co-ﬁrst authors. HH, SS, YW, and CZ, YY, JL, HL were contributed into data analysis and improving paragraphs. MG, HG, and JuL were contributed in experimental design. 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 authors. January 2022 \| Volume 9 \| Article 785848 9 MALDI Drug Resistance Analysis Sun et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2021.785848/ full#supplementary-material FUNDING The studies involving human participants were reviewed and approved by Xuzhou Central Hospital. The patients/participants provided their written informed consent to participate in this study. This work was supported by National Natural Science Foundation of China, 81870540; Jiangsu provincial primary talents program, ZBRCC2016022; Postdoctoral study of Department of human resources and social security, Jiangsu Province, China, (2017) No. 279; Science and Education Project for Young medical talents, Jiangsu Province, China, No. QNRC2016388; Xuzhou Primary Research & Development projects, KC21231; Xuzhou Primary Research & Development projects, KC21274. World Health Organization (2014). Antimicrobial Resistance: Global Report on Surveillance 2014. Available at: https://apps.who.int/mediacentre/news/ releases/2014/amr-report/en/index.html (Accessed September 25, 2021). 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. Enterobacterales Urinary Tract Infection. Open Forum Infect. Dis. 6 (4), ofz164. doi:10.1093/oﬁd/ofz164 Złoch, M., Maślak, E., Kupczyk, W., Jackowski, M., Pomastowski, P., and Buszewski, B. (2021). Culturomics Approach to Identify Diabetic Foot Infection Bacteria. Int. J. Mol. Sci. 22 (17), 9574. doi:10.3390/ ijms22179574 Frontiers in Chemistry \| www.frontiersin.org January 2022 \| Volume 9 \| Article 785848 Enterobacterales Urinary Tract Infection. Open Forum Infect. Dis. 6 (4), ofz164. doi:10.1093/oﬁd/ofz164 World Health Organization (2014). Antimicrobial Resistance: Global Report on Surveillance 2014. Available at: https://apps.who.int/mediacentre/news/ releases/2014/amr-report/en/index.html (Accessed September 25, 2021). Złoch, M., Maślak, E., Kupczyk, W., Jackowski, M., Pomastowski, P., and Buszewski, B. (2021). Culturomics Approach to Identify Diabetic Foot Infection Bacteria. Int. J. Mol. Sci. 22 (17), 9574. doi:10.3390/ ijms22179574 Enterobacterales Urinary Tract Infection. Open Forum Infect. Dis. 6 (4), ofz164. doi:10.1093/oﬁd/ofz164 REFERENCES A Clinical Prediction Tool for Extended- Spectrum Cephalosporin Resistance in Community-Onset January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 10 Sun et al. MALDI Drug Resistance Analysis 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. Złoch, M., Maślak, E., Kupczyk, W., Jackowski, M., Pomastowski, P., and Buszewski, B. (2021). Culturomics Approach to Identify Diabetic Foot Infection Bacteria. Int. J. Mol. Sci. 22 (17), 9574. doi:10.3390/ ijms22179574 Copyright © 2022 Sun, Lai, Wu, Heng, Si, Ye, Li, Lyu, Zou, Guo, Wang, Geng and Liang. 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: 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. January 2022 \| Volume 9 \| Article 785848 Frontiers in Chemistry \| www.frontiersin.org 11
https://openalex.org/W2169617361	https://eprints.qut.edu.au/72550/54/72550.pdf	English	null	Heat Transfer Analysis of Viscous Incompressible Fluid by Combined Natural Convection and Radiation in an Open Cavity	Mathematical problems in engineering	2,014	cc-by	10,000	This may be the author’s version of a work that was submitted/accepted for publication in the following source: ( ) Heat transfer analysis of viscous incompressible ﬂuid by combined natural convection and radiation in an open cavity. This ﬁle was downloaded from: https://eprints.qut.edu.au/72550/ © Copyright 2014 M. Seleem et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and re- production in any medium, provided the original work is properly cited. Notice: Please note that this document may not be the Version of Record (i.e. published version) of the work. Author manuscript versions (as Sub- mitted for peer review or as Accepted for publication after peer review) can be identiﬁed by an absence of publisher branding and/or typeset appear- ance. If there is any doubt, please refer to the published source. https://doi.org/10.1155/2014/412480 https://doi.org/10.1155/2014/412480 Hindawi Publishing Corporation Mathematical Problems in Engineering Volume 2014, Article ID 412480, 14 pages http://dx.doi.org/10.1155/2014/412480 Hindawi Publishing Corporation Mathematical Problems in Engineering Volume 2014, Article ID 412480, 14 pages http://dx.doi.org/10.1155/2014/412480 Correspondence should be addressed to Suvash C. Saha; s c saha@yahoo.com Correspondence should be addressed to Suvash C. Saha; s c saha@yahoo.com Received 10 March 2014; Revised 24 May 2014; Accepted 3 June 2014; Published 25 June 2014 Academic Editor: Hang Xu Copyright © 2014 M. Saleem 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. The effect of radiation on natural convection of Newtonian fluid contained in an open cavity is investigated in this study. The governing partial differential equations are solved numerically using the Alternate Direct Implicit method together with the Successive Overrelaxation method. The study is focused on studying the flow pattern and the convective and radiative heat transfer rates are studied for different values of radiation parameters, namely, the optical thickness of the fluid, scattering albedo, and the Planck number. It was found that, in the optically thin limit, an increase in the optical thickness of the fluid raises the temperature and radiation heat transfer of the fluid. However, a further increase in the optical thickness decreases the radiative heat transfer rate due to increase in the energy level of the fluid, which ultimately reduces the total heat transfer rate within the fluid. M. Saleem,1 M. A. Hossain,2 Suvash C. Saha,3 and Y. T. Gu3 M. Saleem, M. A. Hossain, Suvash C. Saha, and Y. T. Gu 1 Department of Electrical and Computer Engineering, Center for Advanced Studies in Engineering, 19 Atta Turk Avenue, G-5/1, Islamabad 44000, Pakistan 2 University of Dhaka, Dhaka 1000, Bangladesh 3 School of Chemistry, Physics & Mechanical Engineering, Queensland University of Technology, G.P.O. Box 2434, Brisbane, QLD 4001, Australia trical and Computer Engineering, Center for Advanced Studies in Engineering, 19 Atta Turk Avenue, G-5/1, akistan 3 School of Chemistry, Physics & Mechanical Engineering, Queensland University of Technology, G.P.O. Box 2434, Brisbane, QLD 4001, Australia 1. Introduction The density of fluid with temperature is considered to vary under Boussinesq approximation. Boussinesq approximation is a better choice for laminar case of such fluids. (See also [26].) Further, it is also worth mentioning here that modeling such flows requires that the temperature of the gas does not rise enough to a level where it causes generation of large number of ions in the gas. So high energy photons due to ionic exchange do not come into account and the frequency/energy level of radiation does not vary over a wide range (Modest [2]), and gray approximation remains valid therein. Air (Pr = 0.71) can be chosen for modeling such phenomenon (see also [3, 4, 6]) so that the ionic exchange does not take place even at high temperatures. Thus we consider a Boussinesq type fluid with gray radiant properties and consider the Rayleigh number only in the range of 2 × 105 ≤Ra ≤7 × 105. Attention is focused on investigating the effect of radiation parameters, namely, the optical thickness, scattering albedo, and the Planck number. Most of the study of natural convection in enclosures has been devoted to the study of streamlines and isotherms. However, the investigation of heat function is also useful in studying the heat transfer characteristics of the phenomenon of natural convection. Kimura and Bejan [16] introduced heat function as the energy analog of stream function. Also, isotherms provide credible information about heat flow only in conduction dominated regime, whereas heatlines are locally parallel to the actual direction of energy flow in a domain. Thus heatlines study helps to comprehend the flow and temperature profile as the heat function formu- lation is based on identically satisfying the thermal energy equation (see also Bello-Ochende [17], Costa [18, 19], and Deng and Tang [20]). The study of heat function profile becomes more important when the natural convection flow is combined with radiation, as the inclusion of radiation raises the energy level of the fluid in domain considerably (see Larson and Viskanta [3]). A very recent study of heatlines formulation for surface radiation case in enclosures has been made by Hossain et al. [21]. Further, the problem of natural convection in open ended cavity is recently addressed by Saleem et al. [22, 23]. As far as the study of the effect of surface radiation in open cavity is concerned, the model was considered by Hinojosa et al. [24]. 1. Introduction [7] studied the effects of optical thickness and thermal gradients on the stability and structure of flows in a cylindrical container heated from below. Modest [2, page 466] indicated that there is a general misconception about lowest order differential approximation, that it may fail in optically thin limit. Rather there is some loss of accuracy only when small optically thin medium is sandwiched between hot and cold surfaces in the presence of Collimated irradiation. Derby et al. [8] examined the performance of the approxima- tions for modeling a representative problem of heat transfer and buoyant flow in optically thick fluids. They remarked that although Rosseland diffusion had an advantage of less computational cost, an inaccuracy in the thermal boundary layer appears in it. However despite the fact that differential approximation is more costly in terms of computation due to an additional elliptic type differential equation, it gives more accurate results. Ridouane et al. [9] studied both transient and steady conditions for the effects of surface radiation on natural convection in a square enclosure heated from below and cooled from above. They showed that heat transfer across the cavity rises quickly due to active walls emissivity. Recent boundary layer study of combined mechanism of convection and radiation under different conditions can be seen in [10–14]. Liu et al. [15] used discrete ordinate method to investigate the combined mechanism of natural convection and radiation in a cavity and showed that radiative heat transfer was significantly reduced as the optical thickness was increased. angle on the Nusselt number distributions. Also the case of conjugate natural convection with conduction and surface radiation in open cavity was studied by Nouanegue et al. [25]. The present study aims to investigate the basic flow pat- tern and heat transfer characteristics in open ended domain. Since, to author’s knowledge, the study of participating fluids in open ended cavities is not found in literature, such kind of models may have their applications in the situations where heat is rejected via spaces between reflecting surfaces, energy transfer in vacuum tubes, and flow of air and water as coolant in power plants, where heat is mainly rejected via radiation. Thus here we consider the case of combined natural con- vection and radiation of Newtonian fluid in an open ended cavity, whose left wall is maintained at a higher temperature. 1. Introduction In general, the combined mechanism of radiant and convective heat transfer in finite enclosures has received considerable attention. Larson and Viskanta [3] investigated the effects of free burning by taking a model of transient natural convection and radiation for gray diffuse walls with an arbitrary temperature in an enclosure. They showed that the difference between the temperatures of the hot and the cold walls reduces to about 1% of that of the hot wall, due to radiation effect. More interestingly, the air in the core region generally reached 33% of the hot wall temperature, whereas it obtains 13% of the hot wall temperature in the absence of radiation effect at the same time level. Lauriat [4] analyzed the effect of combined radiation and radiation phenomena for gray fluids. Bouallou and Sacadura [5] considered the porous media case of participating medium. Draoui et al. [6] further made an investigation of natural convection of participating fluids in a square enclosure. The study of flow properties for such kind of flows showed that there is a tendency of flow reduction and an increase in heat transfer for the process of combined conduction-radiation of fluids in There are many physical phenomena in which energy exchange due to radiation plays an important role, for instance, heat transfer in furnaces and combustion chambers, solar simulators and the utilization of solar energy, flow of the earth’s mantle, the flow of oxide melts during crystal growth, processing of molten glass, and the solar air receivers. Heat leakage in evacuated spaces, energy dissipation in vacuum tubes, role of air and water as coolant in power plants, and cooling of electronic and optoelectronic devices also involve the energy exchange via radiation. One of the important features of radiation heat transfer is the nature of its dependency on temperature. Review of the radiation models that exist in the literature is given by Siegel and Howell [1]. It should also be noted that the study of heat transfer in fluids which absorb and emit radiant energy becomes a complicated task due to the coupled, non-linear physical phenomena of internal radiation and natural convection. A detailed review of this coupling phenomenon of radiative and fluid transport can also be found in Modest [2]. Mathematical Problems in Engineering 2 2 enclosures. Moreover the centrosymmetric property of flow is perturbed due to the effect of radiation (see also Lauriat [4]). Salinger et al. 1. Introduction They investigated the Nusselt number distribution for the natural convection and surface thermal radiation in a square tilted open cavity. However, more emphasis was laid on the effect of tilt 2. Mathematical Formulation Consider two-dimensional flow of a viscous incompressible absorbing/emitting and scattering Boussinesq type fluid con- fined in an open rectangular cavity formed by the regions between two horizontal planes at 𝑦= 0 and 𝑦= 𝑌, and the two vertical planes at 𝑥= 0 and the open end along 𝑥= 𝑋, where 𝑋is the length and 𝑌is the height of the cavity. The left wall is assumed to be emissive, whereas its temperature is isothermally maintained at 𝑇𝐻. The temperature of the fluid that enters the cavity region is supposed to be at 𝑇𝐶 at 𝑡 = 0 (where 𝑇𝐻 > 𝑇𝐶). In order to represent the temperature at the open end, we use the subscripts “in” for incoming and “out” for outgoing fluid, respectively. Flow con- figuration and dynamical boundary conditions are shown in Figure 1. Here, 𝑢and V are the components of velocity along the 𝑥and 𝑦axes, respectively, 𝑇is the fluid temperature, and 𝑔 is the magnitude of acceleration due to gravity whereas 𝜖𝑖, (𝑖= 𝑖, 2, 3) is the emissivity of left, top, and bottom walls, respectively. The unsteady motion of incompressible fluid and the equations for conservation of mass, momentum, and Mathematical Problems in Engineering 3 𝑡≥0. First we describe the boundary conditions at the solid walls. The velocity components at the solid walls are given by no slip condition y 𝜀2 = 𝜕T 𝜕y = u = = 0 𝜀3 = 𝜕T 𝜕y = u = = 0 T = TH u = 0 = 0 𝜀1 = 1 u g Tin = TC x 𝜕T 𝜕x out = 0 𝜕u 𝜕x = 𝜕 𝜕y 𝜕 𝜕x = 0 − Figure 1: Flow configuration in coordinate system. 𝜀2 = 𝜕T 𝜕y = u = = 0 𝑢= V = 0. (5) (5) The temperature of the left wall is given by The temperature of the left wall is given by 𝑇= 𝑇𝐻, 𝑥= 0, 0 ≤𝑦≤𝑌, (6) (6) whereas the temperature at the nonisothermal walls is given by 𝑘𝜕𝑇 𝜕𝑦+ 1 3𝛼𝑚 𝜕𝐺 𝜕𝑦= 0, 𝑦= 0, 𝑌, 0 ≤𝑥≤𝑋. (7) (7) Finally the boundary conditions at the solid walls for thermal radiation are 𝜕𝐺 𝜕𝑛𝑖 = 3𝛼𝑚𝜖𝑖 4 −2𝜖𝑖 {𝐺−4𝜎(𝑇𝑖−𝑇𝐶) 4} . (8) (8) Figure 1: Flow configuration in coordinate system. 2. Mathematical Formulation Here, 𝑛is the direction normal to the wall and 𝑖= 1, 2, 3, respectively, represent the left, top, and bottom wall as labeled in Figure 1. (See [6].) energy in two-dimensional Cartesian coordinate system are given by To set up the open end boundary conditions, notice that the velocity component 𝑢, V should satisfy the continuity equation at the open end. Moreover, 𝜕V/𝜕𝑥= 0 is a physically more realistic condition for vertical velocity at the open end as compared to V = 0, as the fluid may not necessarily leave the domain horizontally. Further, (𝜕𝑇/𝜕𝑥)out = 0 is found to be a suitable condition at the open end for temperature. Most recently, these conditions are also addressed by the same authors (Saleem et al. [22, 23] and see the references therein). Further, since it is required to confine the effects of wall radiation within the cavity, this suggests that the divergence of radiation intensity at the open end for thermal radiation should be zero. Thus the boundary conditions at the open end of the cavity are summarized by the following equations 𝜕𝑢 𝜕𝑥+ 𝜕V 𝜕𝑦= 0, 𝜕𝑢 𝜕𝑡+ 𝑢𝜕𝑢 𝜕𝑥+ V𝜕𝑢 𝜕𝑦= −1 𝜌 𝜕𝑝 𝜕𝑥+ ] (𝜕2𝑢 𝜕𝑥2 + 𝜕2𝑢 𝜕𝑦2 ) , 𝜕V 𝜕𝑡+ 𝑢𝜕V 𝜕𝑥+ V 𝜕V 𝜕𝑦= −1 𝜌 𝜕𝑝 𝜕𝑦+ ] (𝜕2V 𝜕𝑥2 + 𝜕2V 𝜕𝑦2) + 𝑔𝛽(𝑇−𝑇𝐶), (1) 𝜌𝐶𝑝(𝜕𝑇 𝜕𝑡+ 𝑢𝜕𝑇 𝜕𝑥+ V𝜕𝑇 𝜕𝑦) = 𝑘(𝜕2𝑇 𝜕𝑥2 + 𝜕2𝑇 𝜕𝑦2 ) −∇⋅𝑞𝑟, (2) (1) where ] is the kinematic viscosity, 𝛽is the coefficient of volume expansion, 𝜌is the density, 𝐶𝑝is the molar specific heat at constant pressure, 𝑘is the coefficient of thermal conductivity, 𝑡is the time, and 𝑞𝑟is the radiation flux vector in two dimensions. We have considered semitransparent fluid with gray radiant properties. The equation of radiant energy for optically thick absorbing/emitting and scattering fluid is thus given by (see [4–6]) 𝜕𝑢 𝜕𝑥= −𝜕V 𝜕𝑦, 𝜕V 𝜕𝑥= 𝜕𝐺 𝜕𝑥= 0, (𝜕𝑇 𝜕𝑥) out = 0, 𝑇in = 𝑇𝐶, 𝑥= 𝑋, 0 ≤𝑦≤𝑌. (9) 𝜕𝑢 𝜕𝑥= −𝜕V 𝜕𝑦, 𝜕V 𝜕𝑥= 𝜕𝐺 𝜕𝑥= 0, (𝜕𝑇 𝜕𝑥) out = 0, 𝑇in = 𝑇𝐶, 𝑥= 𝑋, 0 ≤𝑦≤𝑌. (9) (𝜕𝑇 𝜕𝑥) ou (𝜕𝑇 𝜕𝑥) out = 0, 𝑇in = 𝑇𝐶, 𝑥= 𝑋, 0 ≤𝑦≤𝑌. 2. Mathematical Formulation (9) (9) 𝜕2𝐺 𝜕𝑥2 + 𝜕2𝐺 𝜕𝑦2 = 3𝛼2 𝑚(1 −Ω0) {𝐺−𝜎(𝑇−𝑇𝐶) 4} , (3) In order to transform the problem into stream vorticity form, we define the stream function and vorticity by the following relations: In order to transform the problem into stream vorticity form, we define the stream function and vorticity by the following relations: (3) where 𝛼𝑚is mean extinction coefficient, Ω0 is scattering albedo, and 𝜎is Stefan-Boltzmann constant. The boundary conditions are expressed as follows: 𝜎is Stefan-Boltzmann constant. The boundary e expressed as follows: = 𝐺= 0, 0 ≤𝑦≤𝑌, 0 ≤𝑥≤𝑋, (4) 𝜔= 𝜕V 𝜕𝑥−𝜕𝑢 𝜕𝑦, 𝑢= 𝜕𝜓 𝜕𝑦, V = −𝜕𝜓 𝜕𝑥. (10) 𝜔= 𝜕V 𝜕𝑥−𝜕𝑢 𝜕𝑦, 𝑢= 𝜕𝜓 𝜕𝑦, V = −𝜕𝜓 𝜕𝑥. 2. Mathematical Formulation (10) (10) 𝑡< 0 𝑢= V = 𝑇= 𝐺= 0, 0 ≤𝑦≤𝑌, 0 ≤𝑥≤𝑋, ( 𝑡< 0 𝑢= V = 𝑇= 𝐺= 0, 0 ≤𝑦≤𝑌, 0 ≤𝑥≤𝑋, (4) (4) 𝑢= V = 𝑇= 𝐺= 0, 0 ≤𝑦≤𝑌, 0 ≤𝑥≤𝑋, (4) Mathematical Problems in Engineering Mathematical Problems in Engineering 4 𝑢= V = 𝜓= 0, 𝜔= 𝜕V 𝜕𝑥, 𝜃= 1, 𝑥= 0, 0 ≤𝑦≤1, 𝜕𝑢 𝜕𝑥= −𝜕V 𝜕𝑦, 𝜔in = 𝜃in = 𝐺in = 0, 𝑥= 1 𝐴, 0 ≤𝑦≤1, 𝜕V 𝜕𝑥= (𝜕𝜔 𝜕𝑥) out = (𝜕𝜃 𝜕𝑥) out = (𝜕𝐺 𝜕𝑥) out = 0, 𝑥= 1 𝐴, 0 ≤𝑦≤1, (17) 𝑢= V = 𝜓= 0, 𝜔= 𝜕V 𝜕𝑥, 𝜃= 1, 𝑥= 0, 0 ≤𝑦≤1, 𝜕𝑢 𝜕𝑥= −𝜕V 𝜕𝑦, 𝜔in = 𝜃in = 𝐺in = 0, 𝑥= 1 𝐴, 0 ≤𝑦≤1, Further introduce the nondimensional variables by using the following transformations: 𝑥= 𝑥 𝑌, 𝑦= 𝑦 𝑌, 𝑡= 𝑡𝛼 𝑌2 , 𝑢= 𝑢𝑌 𝛼, V = V𝑌 𝛼, 𝜓= 𝜓 𝛼, 𝜔= 𝜔𝑌2 𝛼, Δ𝑇= 𝑇𝐻−𝑇𝐶, 𝑇0 = 𝑇𝐻+ 𝑇𝐶 2 , 𝜃= 𝑇−𝑇𝐶 Δ𝑇 , 𝐴= 𝑌 𝑋, 𝜃0 = 𝑇0 Δ𝑇 , 𝑞𝑟= 𝑞𝑟𝐴 𝜎𝑇 4 0 , 𝐺= 𝐺 𝜎𝑇 4 0 , 𝜏0 = 𝛼𝑚𝑋, (11) 𝜕V 𝜕𝑥= (𝜕𝜔 𝜕𝑥) out = (𝜕𝜃 𝜕𝑥) out = (𝜕𝐺 𝜕𝑥) out = 0, 𝜕V 𝜕𝑥= (𝜕𝜔 𝜕𝑥) out = (𝜕𝜃 𝜕𝑥) out = (𝜕𝐺 𝜕𝑥) out = 0, 𝑥= 1 𝐴, 0 ≤𝑦≤1, 𝑥= 1 𝐴, 0 ≤𝑦≤1, (17) where Pr = ] 𝛼, Ra = 𝑔𝛽Δ𝑇𝐻3 𝛼] , 𝑁CR = 𝑘𝛼𝑚Δ𝑇 𝜎𝑇 4 0 (18) (11) (18) where 𝑥, 𝑦are the nondimensional coordinate axis, 𝑢, V are the nondimensional velocity components, 𝜓and 𝜔are the nondimensional stream and vorticity functions, 𝑡is the time, 𝑇0 is the reference temperature, 𝜃is the nondimensional temperature, 𝜃0 is the mean temperature in nondimensional form, 𝐺is the dimensionless radiant energy, and 𝜏0 is the opti- cal thickness. The treatment of radiative flux divergence in (2) is based on two-dimensional differential approximation. The nondimensional form of radiative flux and its divergence are given by the following relations (see also [1, 2, 4–6]): are, respectively, the Prandtl number, the Rayleigh number, and the Planck number (also known as conduction-radiation parameter). 2. Mathematical Formulation The temperature boundary conditions for hori- zontal walls and radiation energy conditions at the three solid walls in nondimensional form are now given by ([1, 2, 4–6]) are, respectively, the Prandtl number, the Rayleigh number, and the Planck number (also known as conduction-radiation parameter). The temperature boundary conditions for hori- zontal walls and radiation energy conditions at the three solid walls in nondimensional form are now given by ([1, 2, 4–6]) 𝜕𝜃 𝜕𝑦+ 1 3𝑁CR 𝜕𝐺 𝜕𝑦= 0, 𝑦= 0, 1, 0 ≤𝑥≤1 𝐴, 𝜕𝐺 𝜕𝑛𝑖 = 3𝜏0𝐴𝜖𝑖 4 −2𝜖𝑖 (𝐺−4𝜃4 𝑖 𝜃4 0 ) . (19) (19) 𝑞𝑟,𝑥= −1 3𝜏0 𝜕𝐺 𝜕𝑥, 𝑞𝑟,𝑦= −1 3𝜏0 𝜕𝐺 𝜕𝑦, (12) (12) Heatlines are a measure of the path followed by the heat function across the flow region. Following the formulation of heat function defined by Kimura and Bejan [16], we construct a Poisson-type heat function equation that identically satisfies the energy transport equation (2) given by (see also [15–19]) where 𝑞𝑟,𝑥and 𝑞𝑟,𝑦are the radiative fluxes along the 𝑥and 𝑦components, respectively. Thus the governing equations in nondimensional form finally become 𝜕2𝜓 𝜕𝑥2 + 𝜕2𝜓 𝜕𝑦2 = −𝜔, (13) 𝜕𝜔 𝜕𝑡+ 𝑢𝜕𝜔 𝜕𝑥+ V𝜕𝜔 𝜕𝑦= Pr (𝜕2𝜔 𝜕𝑥2 + 𝜕2𝜔 𝜕𝑦2 ) + RaPr𝜕𝜃 𝜕𝑥, (14) 𝜕𝜃 𝜕𝑡+ 𝑢𝜕𝜃 𝜕𝑥+ V𝜕𝜃 𝜕𝑦= (𝜕2𝜃 𝜕𝑥2 + 𝜕2𝜃 𝜕𝑦2 ) + 1 3𝑁CR (𝜕2𝐺 𝜕𝑥2 + 𝜕2𝐺 𝜕𝑦2 ) , (15) 𝜕2𝐺 𝜕𝑥2 + 𝜕2𝐺 𝜕𝑦2 = 3𝜏2 0𝐴2 (1 −Ω0) (𝐺−4𝜃4 𝜃4 0 ) , (16) 𝜕2𝜓 𝜕𝑥2 + 𝜕2𝜓 𝜕𝑦2 = −𝜔, (13) (13) 𝜕𝐻 𝜕𝑦= 𝜌𝐶𝑝𝑢(𝑇−𝑇𝐶) −𝑘𝜕𝑇 𝜕𝑥+ 𝑞𝑟,𝑥, −𝜕𝐻 𝜕𝑥= 𝜌𝐶𝑝V (𝑇−𝑇𝐶) −𝑘𝜕𝑇 𝜕𝑦+ 𝑞𝑟,𝑦. (20) 𝜕𝜔 𝜕𝑡+ 𝑢𝜕𝜔 𝜕𝑥+ V𝜕𝜔 𝜕𝑦= Pr (𝜕2𝜔 𝜕𝑥2 + 𝜕2𝜔 𝜕𝑦2 ) + RaPr𝜕𝜃 𝜕𝑥, (14) (20) 𝜕𝜃 𝜕𝑡+ 𝑢𝜕𝜃 𝜕𝑥+ V𝜕𝜃 𝜕𝑦= (𝜕2𝜃 𝜕𝑥2 + 𝜕2𝜃 𝜕𝑦2 ) + 1 3𝑁CR (𝜕2𝐺 𝜕𝑥2 + 𝜕2𝐺 𝜕𝑦2 ) , (15) Now defining 𝐻= 𝐻/𝑘Δ𝑇as the nondimensional heat function and making use of (12), we get the following definition of heat function: Now defining 𝐻= 𝐻/𝑘Δ𝑇as the nondimensional heat function and making use of (12), we get the following definition of heat function: 𝜕2𝐺 𝜕𝑥2 + 𝜕2𝐺 𝜕𝑦2 = 3𝜏2 0𝐴2 (1 −Ω0) (𝐺−4𝜃4 𝜃4 0 ) , (16) (16) 𝜕2𝐻 𝜕𝑥2 + 𝜕2𝐻 𝜕𝑦2 = 𝑢𝜕𝜃 𝜕𝑦+ V𝜕𝜃 𝜕𝑥−𝜃𝜔. 2. Mathematical Formulation (21) (21) with the following boundary conditions: The wall boundary conditions for the heatlines are followed by the definition of heat function given by (17) (see also [15– 19]): The wall boundary conditions for the heatlines are followed by the definition of heat function given by (17) (see also [15– 19]): 𝑡< 0 𝑢= V = 𝜓= 𝜔= 𝜃= 𝐺= 0, 0 ≤𝑦≤1, 0 ≤𝑥≤1 𝐴 𝑡≥0 𝑡< 0 𝑢= V = 𝜓= 𝜔= 𝜃= 𝐺= 0, 0 ≤𝑦≤1, 0 ≤𝑥≤1 𝐴 𝜕𝐻 𝜕𝑦= −𝜕𝑇 𝜕𝑥, 𝑦= 0, 1, 0 ≤𝑥≤1 𝐴, 𝜕𝐻 𝜕𝑥= 𝜕𝑇 𝜕𝑦, 𝑥= 0, 0 ≤𝑦≤1, 𝜕𝐻 𝜕𝑥= 0, 𝑥= 1 𝐴, 0 ≤𝑦≤1. (22) 𝑡≥0 𝑢= V = 𝜓= 0, 𝜔= −𝜕𝑢 𝜕𝑦, 𝑦= 0, 0 ≤𝑥≤1 𝐴, 𝑢= V = 𝜓= 0, 𝜔= −𝜕𝑢 𝜕𝑦, 𝑦= 1, 0 ≤𝑥≤1 𝐴, (22) 5 Mathematical Problems in Engineering 5 t 0 0.01 0.02 0.03 0.04 15 20 25 30 35 40 45 Nu Grids 31 × 31 41 × 41 51 × 51 61 × 61 71 × 71 Error (%) 5.41 3.66 1.98 0.86 Figure 2: Average heat transfer rate of the heated wall against time for various grid choices at Ra = 5 × 105, Pr = 0.7, and 𝜏0 = Ω0 = 𝑁CR = 0.5. Finally we define the heat transfer from the left wall. When energy transfer takes place in combined mode, the net heat transfer rate is also defined as the combined mechanism of these modes (Lauriat [4]). Thus for combined mechanism of convection and radiation the net heat transfer for solid wall boundaries can be defined by the following relations (see [4– 6, 21]): Nu𝐶= −(𝜕𝜃 𝜕𝑥) 𝑥=0 , Nu𝑅= − 1 3𝑁CR (𝜕𝐺 𝜕𝑥) 𝑥=0 , Nu = Nu𝐶+ Nu𝑅. (23) (23) Nu = Nu𝐶+ Nu𝑅. From here it follows that the average Nusselt number is defined as From here it follows that the average Nusselt number is defined as Nu = Nu𝐶+ Nu𝑅= ∫ 1 0 Nu 𝑑𝑦 Nu = Nu𝐶+ Nu𝑅= ∫ 1 0 Nu 𝑑𝑦 = ∫ 1 0 [(−𝜕𝜃 𝜕𝑥) 𝑥=0 + (− 1 3𝑁CR 𝜕𝐺 𝜕𝑥) 𝑥=0 ] 𝑑𝑦, (24) (24) where 𝑑𝑦is the element of length 𝑌along the wall. Equations (13) to (16) are solved with the boundary conditions given by (17) and (19) to solve the system. 3. Method of Solution The flow is developed by coupling of buoyancy term in (14), which is updated by (15) and (16), stream function is obtained by coupling of (13) and (14), and finally velocity profile is updated using the nondimensional form of (10). The solution is complemented with the implementation of boundary con- ditions given in (17) and (19). The stream function equation (13) is solved using Successive Over Relaxation (SOR) method with residual tolerance of order 10−5. With 𝑌as the reference height of the cavity, we have considered a uniform grid of size ℎ= 𝑌/(𝐽−1), where 𝐽is the maximum number of grids along coordinate axes. Throughout the computation we take 𝑌= 1. The time dependent vorticity transport equation (14) and thermal conduction-convection equation (15) are solved using the Alternate Direct Implicit method. The details of the proposed method are also given in [21–23, 27]. A simple discretization procedure given by Bello-Ochende [17] is adopted for the solution of radiation equation (16) and heat function equation given by (21). As a simple case, all computations are performed for square cavity. That is, 𝐴= 1. Now in order to meet the criteria of convergence to reach the steady state, we define the error bound in the computed value of a variable 𝐹, by the relation Figure 2 shows the result of grid dependence study at Ra = 5 × 105, 𝜏0 = Ω0 = 𝑁CR = 0.5 for the choice of appropriate mesh size ℎ. For any variable 𝐹, we define the relative error % between the computed values taking different grid points as given by Error% = 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝐹(𝜅+10,𝜅+10) −𝐹𝜅,𝜅 𝐹𝜅,𝜅 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 , (26) (26) where 𝐹𝜅,𝜅is previously calculated value of a variable for (𝜅× 𝜅) grid points. As a demonstration, Figure 2 shows the percentage error in the values obtained from the difference between the computed values of the Average Nusselt number, for different choices of mesh points. It can be seen that the maximum error between mesh sizes 61 × 61 and 71 × 71, in terms of Nu, drops to less than 1%. Thus a mesh of 61 × 61 is considered sufficient for the entire computation. The reduction in relative error justifies the grid independence of the solution. In order to further check the validity for the solution method of the proposed model, we have also revisited the work of Draoui et al. 2. Mathematical Formulation The heat lines are given by (21) whereas the average heat transfer is calculated from (24). Figure 2: Average heat transfer rate of the heated wall against time for various grid choices at Ra = 5 × 105, Pr = 0.7, and 𝜏0 = Ω0 = 𝑁CR = 0.5. 3. Method of Solution [6] using the solid wall conditions and the temperature and radiation condition given by [6] for the right wall. The comparison of streamlines, isotherms, and heatlines, for Ra = 105, Pr = 0.7, 𝜏0 = 1, Ω0 = 0.2, and 𝑁CR = 0.1, is shown in Figure 3. From this figure we assert that the numerical solution we obtained agrees where 𝐹𝜅,𝜅is previously calculated value of a variable for (𝜅× 𝜅) grid points. As a demonstration, Figure 2 shows the percentage error in the values obtained from the difference between the computed values of the Average Nusselt number, for different choices of mesh points. It can be seen that the maximum error between mesh sizes 61 × 61 and 71 × 71, in terms of Nu, drops to less than 1%. Thus a mesh of 61 × 61 is considered sufficient for the entire computation. The reduction in relative error justifies the grid independence of the solution. In order to further check the validity for the solution method of the proposed model, we have also revisited the work of Draoui et al. [6] using the solid wall conditions and the temperature and radiation condition given by [6] for the right wall. The comparison of streamlines, isotherms, and heatlines, for Ra = 105, Pr = 0.7, 𝜏0 = 1, Ω0 = 0.2, and 𝑁CR = 0.1, is shown in Figure 3. From this figure we assert that the numerical solution we obtained agrees 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝐹𝑚+1 (𝑖,𝑗) −𝐹𝑚 (𝑖,𝑗) 𝐹𝑚 (𝑖,𝑗) 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 < 10−8, (25) (25) where the superscript 𝑚refers to the number of time steps and (𝑖, 𝑗) is a grid location on coordinate axes. A grid dependence study has been carried out for the choice of suitable number of grid points whose results are shown in Figure 2. Mathematical Problems in Engineering 6 6 Mathematical Problems in Engineering 0 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 0.2 1 −12.3 −10.0 −2.9 (a) 0 0.2 0.4 0.6 0.8 1 0.4 0.6 0.8 0 0.2 1 0.5 0.7 0.4 0 0.2 0.4 0.6 0.8 1 0.4 0.6 0.8 0 0.2 1 0.5 0.7 0.4 (b) 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 3.8 0.8 0 1 0 1 −0.7 −0.2 (c) gure 3: Comparison of (a) streamlines and (b) isotherms. (c) Heat lines with the results of Draoui et al. 3. Method of Solution By further increasing the optical thickness, the flow develops into a multicell pattern due to the dominance of radiation effects into the core region. It may be due to the reason that the fluid in the core region experiences 3. Method of Solution [6] for Ra = 105, Pr = 0.7, 𝜏0 = 1, = 0.2, and 𝑁CR = 0.1 with the conditions given by [6]. 0 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 0.2 1 −12.3 −10.0 −2.9 (a) 0 0.2 0.4 0.6 0.8 1 0.4 0.6 0.8 0 0.2 1 0.5 0.7 0.4 0 0.2 0.4 0.6 0.8 1 0.4 0.6 0.8 0 0.2 1 0.5 0.7 0.4 (b) (b) (a) (b) 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 3.8 0.8 0 1 0 1 −0.7 −0.2 (c) 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 3.8 0.8 0 1 0 1 −0.7 −0.2 (c) (c) Figure 3: Comparison of (a) streamlines and (b) isotherms. (c) Heat lines with the results of Draoui et al. [6] for Ra = 105, Pr = 0.7, 𝜏0 = 1, Ω0 = 0.2, and 𝑁CR = 0.1 with the conditions given by [6]. well with the existing literature of heatlines. Intel 1.83 GHz processing machine is used for the entire computation. streamlines, isotherms, heatlines, and heat transfer rate for different values of these governing physical parameters. 4.1. Effect of Optical Thickness. Figure 4 represents the selected results of steady state pattern of streamlines while Ra = 7×105, Ω0 = 0.2, and 𝑁CR = 0.1 for (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0, respectively. The result of Figure 4(a) shows the streamlines in the absence of radiation effects. However, with the increase in optical thickness, the flow in the core region decreases as shown in Figure 4(b); rather it is minimum at this value of 𝜏0. By further increasing the optical thickness, the flow develops into a multicell pattern due to the dominance of radiation effects into the core region. It may be due to the reason that the fluid in the core region experiences 4.1. Effect of Optical Thickness. Figure 4 represents the selected results of steady state pattern of streamlines while Ra = 7×105, Ω0 = 0.2, and 𝑁CR = 0.1 for (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0, respectively. The result of Figure 4(a) shows the streamlines in the absence of radiation effects. However, with the increase in optical thickness, the flow in the core region decreases as shown in Figure 4(b); rather it is minimum at this value of 𝜏0. 4. Results and Discussion We have considered combined natural convection and radia- tion phenomena for participating fluid confined in an open square cavity. The left wall is considered at a temperature higher than that of the fluid entering from the ambient region. The effect of relevant radiation parameters, namely, the optical thickness, scattering albedo, and Planck number, on the flow profile and heat transfer rate has been numerically studied. The result is presented graphically in terms of Mathematical Problems in Engineering 7 hematical Problems in Engineering 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 1 −32.0 −8.8 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −0.1 −152.4 (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 9.6 −143.7 −1.0 (c) Figure 4: Steady state pattern of streamlines for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0. 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 1 −32.0 −8.8 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −0.1 −152.4 (b) (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 9.6 −143.7 −1.0 (c) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 9.6 −143.7 −1.0 (c) Figure 4: Steady state pattern of streamlines for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0. Figure 4: Steady state pattern of streamlines for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0 ate pattern of streamlines for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0 Figure 6 shows the heatlines while Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 for (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0, respectively. Obviously, at 𝜏0 = 0.0, there is no contribution of radiation in heat transfer and fluid flow. 4. Results and Discussion All fluid entering the flow domain absorbs heat from the solid wall and heatlines are negative throughout the flow region with higher values close to the solid wall as shown in Figure 6(a). The flow region, in which the recirculation is weaker (as in Figure 4(b)), is the region which absorbs heat at 𝜏0 = 0.5, given in Figure 6(b). The positive part of the heatlines pattern is the region of fluid with higher energy level. Figure 6(c) shows the heatlines pattern at 𝜏0 = 1.0. This clearly indicates the high energy level of fluid due to dominance of radiation effect. The heatlines are positive throughout the region, which indicates that the fluid in the region is at high energy level and emitting heat. The effect of a force from the incoming fluid, whereas the recirculation is higher in the half open vortex close to the opening. This counteracting mechanism develops a multicellular pattern as shown in Figure 4(c). Figure 5 shows the isotherms at these values of optical thickness. Figure 5(a) shows that, at 𝜏0 = 0.0, the isotherms are clustered close to the heated wall. It is due to the dominance of convection in the boundary layer region. However with the increase in optical thickness, the boundary layer thickness increases. This effect is qualitatively in agreement with the case of completely confined enclosures. The radiation energy adds to the total energy of isotherms and thus the high energy isotherms shift towards the core region as shown in Figure 5(b). At 𝜏0 = 1.0, the whole flow domain has the isotherms nearly equal to unity due to the dominance of radiant energy, shown in Figure 5(c). This might well be understood also from the heatlines pattern of Figure 6. a force from the incoming fluid, whereas the recirculation is higher in the half open vortex close to the opening. This counteracting mechanism develops a multicellular pattern as shown in Figure 4(c). Figure 5 shows the isotherms at these values of optical thickness. Figure 5(a) shows that, at 𝜏0 = 0.0, the isotherms are clustered close to the heated wall. It is due to the dominance of convection in the boundary layer region. However with the increase in optical thickness, the boundary layer thickness increases. This effect is qualitatively in agreement with the case of completely confined enclosures. 4. Results and Discussion The radiation energy adds to the total energy of isotherms and thus the high energy isotherms shift towards the core region as shown in Figure 5(b). At 𝜏0 = 1.0, the whole flow domain has the isotherms nearly equal to unity due to the dominance of radiant energy, shown in Figure 5(c). This might well be understood also from the heatlines pattern of Figure 6. 8 Mathematical Problems in Engineering 8 Mathematical Problems in Engineeri 0.11 0.60 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 1 0 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0 0.70 0.79 0.95 (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0 0.9938 0.9917 0.9886 0.9519 (c) Figure 5: Isotherms for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0. 0.11 0.60 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 1 0 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0 0.70 0.79 0.95 (b) (a) (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0 0.9938 0.9917 0.9886 0.9519 (c) Figure 5: Isotherms for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0. 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0 0.9938 0.9917 0.9886 0.9519 (c) (c) Figure 5: Isotherms for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0. 4.2. Effect of Scattering Albedo. Scattering albedo is a measure of radiative participation of the fluid. 0 ≤Ω0 ≤1 shifts the fluid regime from perfectly participating to nonparticipating. Figure 8 shows the average total heat transfer against time at Ra = 2 × 105, 𝜏0 = 0.5, and 𝑁CR = 0.2 for different values of scattering albedo Ω0. The right hand side of (16) signifies that the fluid is perfectly transmitting at Ω0 = 0. 4. Results and Discussion This justifies that with the increase in the value of Ω0, the scattering increases, which enhances the effect of convective heat transfer; ultimately Nu𝐶increases whereas the radiative heat transfer decreases and the decrease seems more prominent because Ω0 = 1 decouples the radiation equation from the thermal energy. Thus, at Ω0 = 1, the radiative heat transfer is zero. Figure 8 signifies that Ω0 has no significant effect on the overall heat transfer 0 ≤Ω0 ≤0.75; optical thickness on heat transfer is described in Figure 7. It can be discerned that the overall heat transfer Nu pattern is dominated by the radiation heat transfer Nu𝑅. In particular, overall heat transfer is almost the same as radiative heat transfer beyond 𝜏0 > 0.5. Heat transfer is maximum at 𝜏0 = 0.5, when the flow in the core region is very weak. The contribution of convective heat transfer asymptotically vanishes with the increase in the contribution of optical thickness. At 𝜏0 = 1.25, both the radiative and convective heat transfer rates coincide with “0,” beyond which the negative heat transfer signifies the energy level of fluid due to the fact that contribution of radiant energy is so high that it serves to reject heat to the solid wall. However, the smooth curve, for 𝜏0 > 1.75, shows that the flow pattern and transfer rates are not much affected beyond 𝜏0 = 1.75. Mathematical Problems in Engineering 9 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 1 −2.1 −12.7 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 22.0 −7.3 (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 12.9 63.9 (c) Figure 6: Heat lines for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0. 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 1 −2.1 −12.7 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 22.0 −7.3 (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 12.9 63.9 (c) Figure 6: Heat lines for Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1 at (a) 𝜏0 = 0.0, (b) 𝜏0 = 0.5, and (c) 𝜏0 = 1.0. 4. Results and Discussion rather, there is a considerable decrease only between Ω0 = 0.75 and Ω0 = 1 due to the reason that at Ω0 = 1 the fluid surface is perfectly scattering, and the contribution of Nu𝑅 vanishes at this value of Ω0. are comparable, and the flow in the core region develops into a unicellular pattern with maximum value of the flow at the opening. At 𝑁CR = 4.0, the conduction effect is dominant and the flow further decreases. The flow in Figure 9(c) is weaker than the one noticed in Figure 9(b), because of the gradual exclusion of radiant energy from the flow. Figure 10 shows the heatlines at Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0 for (a) 𝑁CR = 0.2, (b) 𝑁CR = 2.0, and (c) 𝑁CR = 4.0. Obviously at 𝑁CR = 0.2, the radiant energy is a dominant factor in the flow, because the fluid in most of the domain is at higher energy level, shown by positive values of heatlines. However, the heatline labeled −6 at the opening signifies that there is a small portion of the incoming fluid that absorbs heat from the flow domain. Figures 10(b) and 10(c) show that as the value of Planck number increases, more and more fluid in the 4.3. Effect of Planck Number. Planck number represents the ratio of conduction to radiation effects on the fluid. The greater the value of 𝑁CR is, the lesser the effect of radiation on the fluid will be. Figure 9 shows the streamlines at Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0 for (a) 𝑁CR = 0.2, (b) 𝑁CR = 2.0, and (c) 𝑁CR = 4.0, respectively. At 𝑁CR = 0.2, the radiation effect is dominant and the flow in the core region is weaker. Rather, a multicellular pattern appears due to the reason that the lower energy fluid is trapped by the recirculating fluid close to the opening. However, at 𝑁CR = 2.0, the two effects Mathematical Problems in Engineering 10 0 0.5 1 1.5 2 2.5 3 0 10 20 30 𝜏0 Nuc NuR Nu Figure 7: Average heat transfer rates against various values of optical thickness 𝜏0 at Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1. Nomenclature English Letters 𝐴: Aspect ratio 𝐶𝑝: Molar specific heat at constant pressure (JK−1) 𝑔: Acceleration due to gravity (ms−2) 𝐺: Radiant energy Wm−2 English Letters 𝐴: Aspect ratio 𝐶𝑝: Molar specific heat at constant pressure (JK−1) 𝑔: Acceleration due to gravity (ms−2) 𝐺: Radiant energy Wm−2 4. Results and Discussion t 0 0.05 0.1 0 20 40 60 0.00 0.25 0 50 0.75 1.00 Ω0 Nu 0 0.5 1 1.5 2 2.5 3 0 10 20 30 𝜏0 Nuc NuR Nu Figure 7: Average heat transfer rates against various values of optical thickness 𝜏0 at Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1. 0 0.5 1 1.5 2 2.5 3 0 10 20 30 𝜏0 Nuc NuR Nu Figure 7: Average heat transfer rates against various values of optical thickness 𝜏0 at Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1. t 0 0.05 0.1 0 20 40 60 0.00 0.25 0.50 0.75 1.00 Ω0 Nu t 0 0.05 0.1 0 20 40 60 0.00 0.25 0.50 0.75 1.00 Ω0 Nu Figure 8: Average heat transfer rate against time while Ra = 2×105, 𝜏0 = 0.5, and 𝑁CR = 0.2 for different values of scattering albedo Ω0. Figure 7: Average heat transfer rates against various values of optical thickness 𝜏0 at Ra = 7 × 105, Ω0 = 0.2, and 𝑁CR = 0.1. core region absorbs heat by conduction due to decrease in the energy level of the fluid in the core region. Figure 8: Average heat transfer rate against time while Ra = 2×105, 𝜏0 = 0.5, and 𝑁CR = 0.2 for different values of scattering albedo Ω0. gyl g Finally Figure 11 shows the convective, radiative, and total heat transfer as a function of 𝑁CR while 𝜏0 = 1, Ω0 = 0.2, and Ra = 5 × 105. It can be observed that, for sufficiently low value of Planck number (𝑁CR ≤0.5), both the radiation and convective heat transfer increase. The reason might be that, for low value of Planck number, radiation is assisted by natural convection. Looking up at (15) and (24), it becomes obvious that the contribution of radiation in total energy and total heat transfer should fade away in the limit 𝑁CR → ∞, and for sufficiently large 𝑁CR, the energy equation (15) gets decoupled from the radiant energy, whereas natural convection becomes the main mechanism to drive the flow. 4. Results and Discussion This is also physically evident from the definition of Planck number that, for 𝑁CR ≫1, the conduction effects would become dominant. However, the convective and total heat transfer attain almost a steady value beyond 𝑁CR > 15, due to the fact that the contribution of radiation in the total thermal energy becomes less and less as we increase the value of 𝑁CR. 4. Results and Discussion This is now evident from Figure 11 that, beyond 𝑁CR0.5, there is a decrease in the radiation heat transfer with the increasing values of 𝑁CR, and it asymptotically reaches to zero in the limit 𝑁CR > 20 in this case, whereas the convective heat transfer rate increases and approaches total heat transfer curve for 𝑁CR > 20. Thus in this case for 𝑁CR > 20, the overall heat transfer rate is represented by convective heat transfer rate and the contribution of radiative heat transfer vanishes. Therefore we can say that there is a smooth transition of flow from radiation dominated regime to convection dominated regime. This is also physically evident from the definition of Planck number that, for 𝑁CR ≫1, the conduction effects would become dominant. However, the convective and total heat transfer attain almost a steady value beyond 𝑁CR > 15, due to the fact that the contribution of radiation in the total thermal energy becomes less and less as we increase the value of 𝑁CR. has been carried out. The main focus was the study of flow pattern and heat transfer rates for different values of radiation parameters 0 ≤𝜏0 ≤2.5, 0 ≤Ω0 ≤1, and 0.05 ≤𝑁CR ≤20. The following conclusions are obtained. (1) It was seen that with the increase in the optical thick- ness the strength of flow and energy level of the fluid increases, which ultimately results in the negation of heat transfer rate. Convective heat transfer Nu𝐶 decreases asymptotically to zero, whereas radiative heat transfer Nu𝑅first increases in the range 0 ≤𝜏0 ≤ 0.5 and then decreases due to rapid attenuation along propagation in the optically thick media. (2) The total heat transfer is not significantly affected in the range 0 ≤ Ω0 ≤ 0.75, whereas due to rapid decrease in radiative heat transfer, overall heat transfer decreases near Ω0 = 1. (3) Both the strength of flow and energy level of the fluid decrease with the increase in Planck number 𝑁CR. The contribution of radiative heat transfer asymptotically fades to zero with the increase in the value of 𝑁CR, whereas convective heat transfer remains the main mechanism of heat transfer for large values of 𝑁CR. Therefore we can say that there is a smooth transition of flow from radiation dominated regime to convection dominated regime. 5. Conclusion An investigation of the effect of radiation and natural convec- tion of viscous incompressible fluid in a square open cavity Mathematical Problems in Engineering 11 2 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 1 −2 −173 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −78 −27 −10 (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −6 −69 (c) Figure 9: Streamlines for Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0 at (a) 𝑁CR = 0.2, (b) 𝑁CR = 2.0, and (c) 𝑁CR = 4.0. 2 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 1 −2 −173 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −78 −27 −10 (b) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −78 −27 −10 (b) 2 0 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1 0 1 −2 −173 (a) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −6 −69 ( ) 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −6 −69 (c) Figure 9: Streamlines for Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0 at (a) 𝑁CR = 0.2, (b) 𝑁CR = 2.0, and (c) 𝑁CR = 4.0. 𝐺: Dimensionless radiant energy 𝐻: Heat function Wm−1 𝐻: Dimensionless heat function ℎ: Mesh spacing (𝑖, 𝑗): Nodal locations of (𝑥, 𝑦) on grid 𝐼, 𝐽: The number of grid points in each direction 𝑘: Coefficient of thermal conductivity (Wm−1K−1) 𝑁CR: Planck number Nu𝐶: Convective Nusselt number Nu𝐶: Average convection Nusselt number Nu𝑅: Radiation heat transfer Nu𝑅: Average radiation heat transfer Nu: Total Nusselt number Nu: Average Nusselt numbers 𝑝: Fluid pressure (Pa) 𝑞𝑟: Radiant flux Wm−2 𝑞𝑟: Dimensionless radiant flux Pr: Prandtl number Ra: Raleigh number 𝑇: Dimensional temperature (K) 𝑇𝐻, 𝑇𝐶: Maximum and minimum temperature (K) 𝑇0: Average/reference temperature (K) 𝑡: Dimensional time (s) 𝑡: Nondimensional time 𝑢,V: Velocity components (ms−1) 𝑢, V: Nondimensional velocity components 𝑋, 𝑌: Length and height of the cavity (m) 𝑥, 𝑦: Dimensional coordinate axis (m) 𝑥, 𝑦: Nondimensional coordinate axis. References [17] F. L. Bello-Ochende, “A heat function formulation for thermal convection in a square cavity,” International Communications in Heat and Mass Transfer, vol. 15, no. 2, pp. 193–202, 1988. [1] R. Siegel and J. R. Howell, Thermal Radiation Heat Transfer, Hemisphere, Washington, Wash, USA, 3rd edition, 1992. [18] V. A. F. Costa, “Unification of the streamline, heatline and massline methods for the visualization of two-dimensional transport phenomena,” International Journal of Heat and Mass Transfer, vol. 42, no. 1, pp. 27–33, 1999. [2] M. F. Modest, Radiative Heat Transfer, Academic Press, New York, NY, USA, 2nd edition, 2003. [3] D. W. Larson and R. Viskanta, “Transient combined laminar free convection and radiation in a rectangular enclosure,” Journal of Fluid Mechanics, vol. 78, no. 1, pp. 65–85, 1976.l [19] V. A. F. Costa, “Heatline and massline visualization of laminar natural convection boundary layers near a vertical wall,” Inter- national Journal of Heat and Mass Transfer, vol. 43, no. 20, pp. 3765–3774, 2000. [4] G. Lauriat, “Gray fluids enclosed in vertical cavities,” Journal of Heat Transfer, vol. 104, no. 4, pp. 609–615, 1982. [5] C. Bouallou and J. F. Sacadura, “Thermal radiation, convection, and conduction in porous media contained in two-dimensional vertical cavities,” Journal of Heat Transfer, vol. 113, no. 1, pp. 255– 258, 1991. [20] Q. H. Deng and G. F. Tang, “Numerical visualization of mass and heat transport for mixed convective heat transfer by streamline and heatline,” International Journal of Heat and Mass Transfer, vol. 45, no. 11, pp. 2387–2396, 2002. [6] A. Draoui, F. Allard, and C. Beghein, “Numerical analysis of heat transfer by natural convection and radiation in participat- ing fluids enclosed in square cavities,” Numerical Heat Transfer A: Applications, vol. 20, no. 2, pp. 253–261, 1991. [21] M. A. Hossain, M. Saleem, and R. S. R. Gorla, “Surface- radiation effect on natural convection flow in a fluid-saturated non-Darcy porous medium enclosed by non-isothermal walls,” International Journal of Numerical Methods for Heat & Fluid Flow, vol. 23, no. 8, pp. 1320–1339, 2013. [7] A. G. Salinger, S. Brandon, R. Aris, and J. J. Derby, “Buoyancy- driven flows of a radiatively participating fluid in a vertical cylinder heated from below,” Proceedings Royal Society of London A, vol. 442, no. 1915, pp. 313–341, 1993. [22] M. Saleem, S. Asghar, and M. A. Mathematical Problems in Engineering Greek Letters 𝛼: Thermal diffusivity (𝑘/𝜌𝐶𝑝) 𝛼𝑚: Mean extinction coefficient 𝛽: Thermal expansion coefficient (K−1) 𝜖𝑖: Wall emissivity/absorption 𝜃: Nondimensional temperature 𝜇: Dynamic viscosity (m−1s−1) ]: Kinematic viscosity m2s−1Kg−1 𝜌: Density of fluid (Kgm−3) 𝜎: Stefan-Boltzmann constant Wm−2K−4 𝜏0: Optical thickness 𝜓: Stream function (m2s−1Kg−1) 𝜓: Nondimensional stream function Ω0: Scattering Albedo 𝜔: Dimensional vorticity function (s−1) 𝜔: Nondimensional vorticity function. Greek Letters 𝛼: Thermal diffusivity (𝑘/𝜌𝐶𝑝) 𝛼𝑚: Mean extinction coefficient 𝛽: Thermal expansion coefficient (K−1) 𝜖𝑖: Wall emissivity/absorption 𝜃: Nondimensional temperature 𝜇: Dynamic viscosity (m−1s−1) ]: Kinematic viscosity m2s−1Kg−1 𝜌: Density of fluid (Kgm−3) 𝜎: Stefan-Boltzmann constant Wm−2K−4 𝜏0: Optical thickness 𝜓: Stream function (m2s−1Kg−1) 𝜓: Nondimensional stream function Ω0: Scattering Albedo 𝜔: Dimensional vorticity function (s−1) N di i l i i f i [11] M. M. Molla, S. C. Saha, and M. A. Hossain, “Radiation effect on free convection laminar flow along a vertical flat plate with streamwise sinusoidal surface temperature,” Mathematical and Computer Modelling, vol. 53, no. 5-6, pp. 1310–1319, 2011. [12] S. Siddiqa, M. A. Hossain, and S. C. Saha, “Natural convection flow with surface radiation along a vertical wavy surface,” Numerical Heat Transfer A: Applications, vol. 64, no. 5, pp. 400– 415, 2013. [13] R. C. Aziz, I. Hashim, and S. Abbasbandy, “Effects of thermo- capillarity and thermal radiation on flow and heat transfer in a thin liquid film on an unsteady stretching sheet,” Mathematical Problems in Engineering, vol. 2012, Article ID 127320, 14 pages, 2012. [14] R. Nandkeolyar, M. Das, and P. Sibanda, “Unsteady hydro- magnetic heat and mass transfer flow of a heat radiating and chemically reactive fluid past a flat porous plate with ramped wall temperature,” Mathematical Problems in Engineering, vol. 2013, Article ID 381806, 12 pages, 2013. 𝜔: Dimensional vorticity function (s−1) 𝜔: Nondimensional vorticity function. [15] X. Liu, G. Gong, and H. Cheng, “Combined natural convection and radiation heat transfer of various absorbing-emitting- scattering media in a square cavity,” Advances in Mechanical Engineering, vol. 2014, Article ID 403690, 10 pages, 2014. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. [16] S. Kimura and A. Bejan, “The “Heatline” visualization of convective heat transfer,” ASME Journal of Heat Transfer, vol. 105, no. 4, pp. 916–919, 1983. 5. Conclusion Mathematical Problems in Engineering 12 2.8 40.3 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −6.0 (a) 0.8 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −14.5 −4.3 (b) 0.3 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −13.2 −4.3 (c) Figure 10: Heatlines for Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0 at (a) 𝑁CR = 0.2, (b) 𝑁CR = 2.0, and (c) 𝑁CR = 4.0. 2.8 40.3 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −6.0 (a) 0.8 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −14.5 −4.3 (b) 0.3 0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 0 1 −13.2 −4.3 (c) Figure 10: Heatlines for Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0 at (a) 𝑁CR = 0.2, (b) 𝑁CR = 2.0, and (c) 𝑁CR = 4.0. 5 10 15 20 0 5 10 15 20 25 NuR Nu Nuc NCR 5 10 15 20 0 5 10 15 20 25 NuR Nu Nuc NCR Figure 11: Heat transfer rate as a function of 𝑁CR at Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0. Figure 11: Heat transfer rate as a function of 𝑁CR at Ra = 5 × 105, Ω0 = 0.2, and 𝜏0 = 1.0. 13 [27] P. J. Roache, Computational Fluid Dynamics, Hermosa, Albu- querque, NM, USA, 2nd edition, 1988. References Submit your manuscripts at http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Problems in Engineering Hindawi Publishing Corporation http://www.hindawi.com Differential Equations International Journal of Volume 2014 Applied Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Physics Advances in Complex Analysis Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Optimization Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Combinatorics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Function Spaces Abstract and Applied Analysis Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Mathematics and Mathematical Sciences Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Algebra Discrete Dynamics in Nature and Society Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in Discrete Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Stochastic Analysis International Journal of Submit your manuscripts at http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Problems in Engineering Hindawi Publishing Corporation http://www.hindawi.com Differential Equations International Journal of Volume 2014 Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Algebra Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Problems in Engineering Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Algebra Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Differential Equations International Journal of Volume 2014 References Hossain, “Natural convection flow in an open rectangular cavity with cold sidewalls and constant volumetric heat source,” Proceedings of the Institution of Mechanical Engineers C: Journal of Mechanical Engineering Science, vol. 225, no. 5, pp. 1191–1201, 2011. [8] J. J. Derby, S. Brandon, and A. G. Salinger, “The diffusion and P1 approximations for modeling buoyant flow of an optically thick fluid,” International Journal of Heat and Mass Transfer, vol. 41, no. 11, pp. 1405–1415, 1998. [23] M. Saleem, M. A. Hossain, S. Mahmud, and I. Pop, “Entropy generation in Marangoni convection flow of heated fluid in an open ended cavity,” International Journal of Heat and Mass Transfer, vol. 54, no. 21-22, pp. 4473–4484, 2011. [9] E. H. Ridouane, M. Hasnaoui, and A. Campo, “Effects of surface radiation on natural convection in a rayleigh-benard square enclosure: steady and unsteady conditions,” Heat and Mass Transfer/Waerme- und Stoffuebertragung, vol. 42, no. 3, pp. 214– 225, 2006. [24] J. F. Hinojosa, R. E. Cabanillas, G. Alvarez, and C. E. Estrada, “Nusselt number for the natural convection and surface thermal radiation in a square tilted open cavity,” International Commu- nications in Heat and Mass Transfer, vol. 32, no. 9, pp. 1184–1192, 2005. [10] S. Shateyi, S. S. Motsa, and P. Sibanda, “The effects of thermal radiation, hall currents, soret, and dufour on MHD flow by mixed convection over a vertical surface in porous media,” Mathematical Problems in Engineering, vol. 2010, Article ID 627475, 20 pages, 2010. [25] H. Nouanegue, A. Muftuoglu, and E. Bilgen, “Conjugate heat transfer by natural convection, conduction and radiation in Mathematical Problems in Engineering 14 open cavities,” International Journal of Heat and Mass Transfer, vol. 51, no. 25-26, pp. 6054–6062, 2008. [26] E. M. Sparrow and J. P. Abraham, “A new buoyancy model replacing the standard pseudo-density difference for internal natural convection in gases,” International Journal of Heat and Mass Transfer, vol. 46, no. 19, pp. 3583–3591, 2003. [27] P. J. Roache, Computational Fluid Dynamics, Hermosa, Albu- querque, NM, USA, 2nd edition, 1988. The Scientific World Journal Hindawi Publishing Corporation http //www hindawi com Volume 2014 Submit your manuscripts at http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematics Journal of Applied Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Complex Analysis Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Optimization Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Function Spaces Abstract and Applied Analysis Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Mathematics and Mathematical Sciences Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Discrete Dynamics in Nature and Society Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Discrete Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Stochastic Analysis International Journal of
https://openalex.org/W4291131262	https://www.thno.org/v12p5931.pdf	English	null	Machine learning-based identification of tumor-infiltrating immune cell-associated lncRNAs for improving outcomes and immunotherapy responses in patients with low-grade glioma	Theranostics	2,022	cc-by	12,047	Ivyspring International Publisher Ivyspring International Publisher Theranostics 2022; 12(13): 5931-5948. doi: 10.7150/thno.74281 Abstract Rationale: Accumulating evidence demonstrated that long noncoding RNAs (lncRNAs) involved in the regulation of the immune system and displayed a cell-type-specific pattern in immune cell subsets. Given the vital role of tumor-infiltrating lymphocytes in effective immunotherapy, we explored the tumor-infiltrating immune cell-associated lncRNA (TIIClncRNA) in low-grade glioma (LGG), which has never been uncovered yet. Methods: This study utilized a novel computational framework and 10 machine learning algorithms (101 combinations) to screen out TIIClncRNAs by integratively analyzing the sequencing data of purified immune cells, LGG cell lines, and bulk LGG tissues. Results: The established TIIClnc signature based on the 16 most potent TIIClncRNAs could predict outcomes in public datasets and the Xiangya in-house dataset with decent efficiency and showed better performance when compared with 95 published signatures. The TIIClnc signature was strongly correlated to immune characteristics, including microsatellite instability, tumor mutation burden, and interferon γ, and exhibited a more active immunologic process. Furthermore, the TIIClnc signature predicted superior immunotherapy response in multiple datasets across cancer types. Notably, the positive correlation between the TIIClnc signature and CD8, PD-1, and PD-L1 was verified in the Xiangya in-house dataset. Conclusions: The TIIClnc signature enabled a more precise selection of the LGG population who we potential beneficiaries of immunotherapy. Key words: Immunotherapy, low-grade glioma, lncRNA, immune checkpoint, immune infiltration Machine learning-based identification of tumor-infiltrating immune cell-associated lncRNAs for improving outcomes and immunotherapy responses in patients with low-grade glioma Nan Zhang1,2,8#, Hao Zhang1,8,9#, Wantao Wu1,3,8, Ran Zhou4, Shuyu Li5, Zeyu Wang1,8, Ziyu Dai1,8, Liyang Zhang1,8, Fangkun Liu1,8, Zaoqu Liu6, Jian Zhang7, Peng Luo7, Zhixiong Liu1,8, Quan Cheng1,8 1. Department of Neurosurgery, Xiangya Hospital, Central South University, China. p g y, gy p , y, 2. One-third Lab, College of Bioinformatics Science and Technology, Harbin Medical University, China. p gy gy p y 4. Division of Neuroscience and Experimental Psychology, Faculty of Biology, Medicine and Health, University of Man 5. Department of Thyroid and Breast Surgery, Tongji Hospital, Tongji Medical College of Huazhon 6. Department of Interventional Radiology, The First Affiliated Hospital of Zhengzhou, China. p y g y, gj p , gj g g 6. Department of Interventional Radiology, The First Affiliated Hospital of Zhengzhou, China. p gy, p g 7. Department of Oncology, Zhujiang Hospital, Southern Medical University, China. p gy j g p y 8. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, China. 9 D f N Th S d Affili d H i l Ch i M di l U i i Chi 8. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, 9. Department of Neurosurgery, The Second Affiliated Hospital, Chongqing Medical University, China. , gy p , y, . Department of Neurosurgery, The Second Affiliated Hospital, Chongqing Medical University, China. #These authors contributed equally to this work. #These authors contributed equally to this work.  Corresponding authors: Dr. Zhixiong Liu, Department of Neurosurgery, Xiangya Hospital, Center South University, Address: Changsha 410008, Hunan, P. R. China. E-mail: zhixiongliu@csu.edu.cn; Dr. Quan Cheng, Department of Neurosurgery, Xiangya Hospital, Center South University, Address: Changsha 410008, Hunan, P. R. China. E-mail: chengquan@csu.edu.cn. © 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. Received: 2022.04.22; Accepted: 2022.07.28; Published: 2022.08.08 Key words: Immunotherapy, low-grade glioma, lncRNA, immune checkpoint, immune infiltration 5931 5931 Theranostics 2022, Vol. 12, Issue 13 1. Department of Neurosurgery, Xiangya Hospital, Central South University, China. LGG patient and tumor cell line cohorts collection Transcriptome data and clinical information of LGG patients were accessed from three databases, The Cancer Genome Atlas (TCGA, https://portal.gdc. cancer.gov) via Illumina-HiSeq platform, Chinese Glioma Genome Atlas (CGGA, http://www.cgga. org.cn/) via Illumina-HiSeq platform, and Gene Expression Omnibus (GEO, http://www.ncbi.nlm. nih.gov/geo) via Affymetrix Human Genome U133 Plus 2.0 Array platform. The total number of samples included in this study was 932, including 518 samples from the TCGA LGG dataset, 77 samples from the Xiangya in-house dataset, 169 samples from the CGGA LGG dataset, and 168 samples from the GSE108474 dataset. The immune-associated lncRNA signature was constructed through the TCGA LGG training dataset and subsequently validated using the Xiangya in-house dataset (Glioma tissues were collected and written informed consent was obtained from all patients. The included glioma tissues were approved by the Ethics Committee of Xiangya Hospital, Central South University). CGGA LGG and GSE108474 datasets were used as the external validating datasets. Transcriptome data of 10 LGG cell lines via the Affymetrix Human Genome U133 Plus 2.0 Array platform was accessed from GSE36133 (Cancer Cell Line Encyclopedia project (CCLE). Long noncoding RNAs (lncRNAs), a group of noncoding RNAs with more than 200 nucleotides, are closely related to diversified biological functions [6]. Many studies demonstrated that lncRNAs played a critical role in regulating cellular biological processes through modulating gene expression at transcriptional, post-transcriptional, and epigenetic levels [7]. Furthermore, the latest research has found that lncRNAs participated in the immune system modulation and showed a cell-type-specific pattern in immune cell subsets [8]. Ranzan et al. investigated over 500 lncRNAs and identified T cell-specific lncRNAs regulating cell differentiation [9]. Another study found lncRNA SATB2-AS1 as an essential regulator in colorectal cancer progression and immune cell density [10]. Given their close association with immune cell infiltration [8, 10], lncRNAs have colossal potential to evaluate immunotherapy response and predict clinical outcomes. Thus, it is promising to incorporate these powerful lncRNAs to develop prognostic biomarkers based on bioinformatics technology. Besides, as scientific research has entered the big data era with the fast development of high-throughput sequencing technologies, machine learning has been gradually widely applied to extract essential knowledge from big data bioinformatics. Purified immune cell line cohorts collection Transcriptome data of 115 purified cell lines from 19 major immune cell types via Affymetrix Human Genome U133 Plus 2.0 Array platform was accessed from 16 datasets, including GSE27291, GSE27838, GSE28490, GSE13906, GSE23371, GSE2 5320, GSE28698, GSE28726, GSE49910, GSE51540, GSE59237, GSE37750, GSE39889, GSE42058, GSE6863, GSE8059, and processed as previously described [8]. Transcriptome data of 115 purified cell lines from 19 major immune cell types via Affymetrix Human Genome U133 Plus 2.0 Array platform was accessed from 16 datasets, including GSE27291, GSE27838, GSE28490, GSE13906, GSE23371, GSE2 5320, GSE28698, GSE28726, GSE49910, GSE51540, GSE59237, GSE37750, GSE39889, GSE42058, GSE6863, GSE8059, and processed as previously described [8]. Introduction out of four glioma grades classified by the World Health Organization (WHO) [2]. Given the superior malignancy of glioblastoma (grade IV glioma), The Low-grade glioma (LGG) is a heterogeneous group of neuroepithelial tumors derived from supporting glial cells [1] and includes grades I and II https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5932 Cancer Genome Atlas (TCGA) database classifies grades II and III gliomas as LGG. The outcomes of LGG are highly variable, depending on age at diagnosis, histological subtype, tumor size, etc. [3]. Surgical resection with postoperative radiotherapy and chemotherapy is the conventional treatment for LGG patients [4]. Despite its relatively benign biological characteristics, LGG remains incurable and slowly develops till premature death [1]. Numerous recent studies have focused on the tumor immune microenvironment (TIME) and the interactions between tumor and immune cells [5]. Immunotherapy based on the immune response, including immune checkpoint blockade (ICB) and adoptive cell transfer (ACT), has revolutionized the therapeutic outcomes for tumor patients. Researchers have made unremitting efforts to find more precise targets for the further development of immunotherapy. However, studies on immunotherapy of LGG remain to be enriched. Materials and methods Tumor-infiltrating immune cell-associated lncRNA signature establishment Through integrative lncRNA profiling analysis of purified immune cells, LGG cell lines, and bulk LGG cancer tissues, a novel computational framework was utilized to identify a tumor-infiltrating immune cell-associated lncRNA (TIIClnc) signature on account of a couple of machine learning algorithms. The details are as follows (Figure 1): aj = δj - ∑ δ𝑘 𝑛− Γ𝑘 +1 Γ𝑗 𝑘=1 where Γ𝑘 = #[t : Tt ≤ Tk] and Γ𝑗 represents the index of the order for Tj. The log-rank score test came as follows: • The top 15% expressed lncRNAs (each immune cell line) were taken for candidate immune-related lncRNAs. TIIClnc signature = S (x, c) = ∑ (𝑎𝑗 −𝑛𝑙𝑎) 𝑥𝑘≤c ට𝑛𝑙ቀ1− 𝑛𝑙 𝑛ቁ𝑆𝑎2 • The tissue specificity index (TSI) proposed by Yanai et al. [12] was applied to calculate the expression specificity of candidate lncRNAs for each cell type: • The tissue specificity index (TSI) proposed by Yanai et al. [12] was applied to calculate the expression specificity of candidate lncRNAs for each cell type: where 𝑎 and 𝑠𝑎 2 represent the sample mean and sample variance of [aj : j = 1, . . . , n], respectively. The measure of node separation is determined using log-rank score splitting by \| S (x, c) \|. The best split is reached by maximizing this value over x and c. TSIlnc = ∑ (1−𝑥𝑙𝑛𝑐,𝑖) 𝑁 𝑖=1 𝑁−1 where N represents the total number of immune cell types and xlnc, i represents the expression level of immune cell i normalized by the maximal expression intensity of lncRNA in any immune cell type. TSI ranges from 0 to 1, in which lncRNA is defined as an immune cell-general lncRNA when the value is 0 or an immune cell-specific lncRNA when the value is 1. The highly expressed lncRNAs in all immune cell types were identified as immune-related intrinsic lncRNAs (ilncRNA). Preprocessing of transcriptome data The Robust Multi-array Average (RMA) algorithm [11] from the R package affy was used to perform quantile normalization, background correction, and log2 transformation of microarray data from the Affymetrix platform. The sequencing data fragments per kilobase million (FPKM) values were changed into transcripts per kilobase million (TPM) values. The probes of microarray data from the Affymetrix platform were renamed to obtain lncRNA expression profiles. After matching the annotation file of GENCODE (release 39) with the NetAffx annotation files (release 36), probe sets with Ensembl gene IDs as ‘long non-coding RNA’ was picked out. This study aimed to screen tumor-infiltrating immune cell-associated lncRNA (TIIClnc) through a novel computational framework. Via integrative analysis of sequencing data of purified immune cells, LGG cell lines, bulk LGG tissues, and machine learning algorithms, we established a TIIClnc signature to stratify LGG patients and predict the outcomes of immunotherapy. https://www.thno.org 5933 Theranostics 2022, Vol. 12, Issue 13 signature with the highest C-index. 1711 characteristic lncRNAs matching with 2019 probes in microarray data from the Affymetrix platform were selected for further analysis. Correspondingly, lncRNA expression profiles (IlluminaHiSeq platform) were accessed from the TCGA and CGGA databases. • The TIIClnc signature was established based on the combination of RSF and CoxBoost. CoxBoost algorithm was used to screen out the most valuable TIIClncRNAs. RSF algorithm was further used to filtrate the most reliable model. Log-rank score test for splitting survival trees was conducted as previously described [13]. First, the x-variable x was assumed to be ordered as x1 ≤ x2 ≤ … ≤ xn. Then, the “ranks” for each survival time Tj (j ∈ [1, …, n]) were computed. The obtained equation is as follows: • The TIIClnc signature was established based on the combination of RSF and CoxBoost. CoxBoost algorithm was used to screen out the most valuable TIIClncRNAs. RSF algorithm was further used to filtrate the most reliable model. Log-rank score test for splitting survival trees was conducted as previously described [13]. First, the x-variable x was assumed to be ordered as x1 ≤ x2 ≤ … ≤ xn. Then, the “ranks” for each survival time Tj (j ∈ [1, …, n]) were computed. The obtained equation is as follows: Annotation of immune-related characteristics for the TIIClnc signature Seven types of immune modulators were collected [14]. T cell-inflamed gene expression profile (GEP), Cytotoxic activity (CYT), and interferon γ (IFN-γ) were calculated [15, 16]. Tumor mutation burden (TMB), microsatellite instability (MSI), T cell receptor (TCR) richness, TCR Shannon, and SNV Neoantigen were collected from the TCGA database. GATK4 was used to search for the SNPs and indels from the RNA sequencing data of the Xiangya in-house dataset. ANNOVAR was used to annotate the mutation information based on CRCh38 [17]. The tmb function of the R package maftools was applied further to calculate the TMB value of the Xiangya in-house dataset. The R package PreMSIm was used to predict the MSI value of the Xiangya in-house dataset. Six immune subtypes and immunophenoscore (IPS) were determined as previously described [14, 18]. The Tumor Immune Estimation Resource (TIMER) algorithm [19], single cell gene set enrichment analysis (ssGSEA) algorithm [18], Microenvironment Cell Populations-counter (MCPcounter) algorithm [20], and Estimation of STromal and Immune cells in MAlignant Tumours using Expression data (ESTIMATE) algorithm [21] were applied for calculating the abundance of immune infiltrating cells and ESTIMATE score. • ilncRNAs differentially expressed between immune cell lines (upregulated) and LGG cell lines (downregulated) were determined as TIIClncRNAs. • Univariate Cox regression analysis was subsequently used to filtrate the TIIClncRNAs with prognostic potential in the TCGA LGG dataset. • 101 combinations of 10 machine learning algorithms, including Lasso, Ridge, stepwise Cox, CoxBoost, random survival forest (RSF), elastic network (Enet), partial least squares regression for Cox (plsRcox), supervised principal components (SuperPC), generalized boosted regression modeling (GBM), and survival support vector machine (survival-SVM) based on a 10-fold cross-validation were further used to screen out the most valuable TIIClnc https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5934 Cancer immunity cycle, displaying the functional status of chemokines and immunomodulators, and 114 metabolic pathways were collected and calculated using gene set variation analysis (GSVA) [22-24]. TIME signatures independently developed by Kobayashi [25] and Bagaev [26] were collected and calculated using GSVA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms were also quantified using GSVA and gene set enrichment analysis (GSEA). 3% bovine serum albumin (BSA) was used as a blocking reagent. The sections were incubated with primary antibodies against CD8 (Mouse, 1:1000, 66868-1-Ig, Proteintech, China), PD-1 (Rabbit, 1:800, 18106-1-AP, Proteintech, China), PD-L1 (Mouse, 1:1000, 66248-1-Ig, Proteintech, China). Annotation of immune-related characteristics for the TIIClnc signature The sections were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:200, GB23303, Servicebio, China). 3-3’-diaminobenzidine (G1211, Servicebio, China) was finally used for coloration, and hematoxylin was used for counterstaining cell nuclei. EdU assay The EdU (5-ethynyl-2'-deoxyuridine) assay was performed according to the manufacturer's instructions (BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488, China). Three groups of the THP-1 cells transfected with siRNAs were incubated overnight with 50 μL 50 μM EdU medium and then fixed with 50 μL 4% paraformaldehyde. 100 μL 1x Apollo reaction solution was added for incubation for 30 min. Finally, the cells were incubated with 1 mL 1 × Hoechst 33342 reaction solution for 10 min and observed with a confocal microscope. Immunohistochemistry staining Paraffin-embedded tumor tissues of 20 of 77 LGG samples used for RNA sequencing from the Xiangya in-house dataset were further collected for immunohistochemistry (IHC) staining. Briefly, tissue sections were placed in citric acid antigen repair buffer (pH 6.0) in a microwave oven for antigen retrieval. Slices were placed in a 3% hydrogen peroxide solution to block endogenous peroxidase. RT-qPCR assay The GSE35640 (melanoma) [27], GSE91061 (melanoma) [28], GSE78220 (melanoma) [29], Allen (melanoma) [30], Nathanson (melanoma) [31], IMvigor (urothelial carcinoma) [32], Braun (renal cell carcinoma) [33], GSE179351 (colorectal adenocarci- noma and pancreatic adenocarcinoma) [34], GSE165252 (esophageal adenocarcinoma) [35], and PRJNA482620 (glioblastoma) [36] datasets were used to predict the immunotherapy response, while the TIIClnc signature was calculated in each dataset. The GSE103668 (triple-negative breast cancer) [37] dataset was used to predict the targeted therapy response (cisplatin & bevacizumab). The subclass mapping was utilized to predict anti-PD-1 and anti-CTLA-4 immunotherapy responses [38]. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was also used in this section [39]. The primers of GAPDH (F ACAGCCTCAAG ATCATCAGC; R GGTCATGAGTCCTTCCACGAT), LOC101928134 (F GAGCGAGGGTGATTGTCCA; R GAAGAGGGGAAGGGGTTCTC), and LOC100133461 (F GAGAGACCTGCCCAAGCATT; R TCCAGGTTCTGCATGTGTCC) were designed using the primer 5.0. Total RNAs were extracted and reversely transcribed into cDNA by HiScript Q RT SuperMix for RT-qPCR. The expression levels of LOC101928134 and LOC100133461 were quantified using 2-ΔΔCT. RNA sequencing RNAstore-fixed tumor tissues of 77 LGG samples from the Xiangya in-house dataset (Table S1) were collected for RNA sequencing as previously described [40]. Briefly, total RNA was extracted from the tumor tissues using TRIzol (Sigma-Aldrich, CA, USA). RNA purity and RNA integrity were checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA). Cell Counting Kit-8 (CCK-8) assay The THP-1 cells were cultured in 1640 medium with 10% fetal bovine serum (FBS). The THP-1 cells were seeded in a 96-well plate at 104 cells/hole density. Three groups of the THP-1 cells transfected with siRNAs were cultured for 24 h, 48 h, and 72 h, respectively. Each group has four duplicated holes. The absorbance at 450 nm was measured after hatching under the condition of 37 ℃ and 5% CO2. the migrated THP-1 cells in the lower chamber were collected and counted by flow cytometry. he migrated THP-1 cells in the lower chamber were collected and counted by flow cy the migrated THP-1 cells in the lower chamber were collected and counted by flow cytometry. the migrated THP-1 cells in the lower chamber were igrated THP-1 cells in the lower chamber were collected and counted by flow cytometry. . The computational framework for establishing the TIIClnc signature. The top 15% expressed lncRNAs were taken for candidate immune-related mmune cell line. TSI was applied to calculate the expression specificity of candidate immune-related lncRNAs for each cell type. The highly expressed lncRN cell types were identified as immune-related ilncRNA. ilncRNAs significantly upregulated in immune cell lines and downregulated in LGG cell lines were d NAs. Univariate cox regression analysis was further used to screen out the prognostic TIIClncRNAs. All combinations of 10 machine learning algorithms, inclu so, Ridge, stepwise Cox, CoxBoost, plsRcox, SuperPC, GBM, and survival-SVM, based on a 10-fold cross-validation were further used to screen out the most gnature with the highest C-index. The TIIClnc signature was finally generated based on the combination of RSF and CoxBoost. The association between th prognosis, tumor immune microenvironment, and immunotherapy response was comprehensively investigated. Figure 1. The computational framework for establishing the TIIClnc signature. The top 15% expressed lncRNAs were taken for candidate immune-related lncRNAs for each immune cell line. TSI was applied to calculate the expression specificity of candidate immune-related lncRNAs for each cell type. The highly expressed lncRNAs in all immune cell types were identified as immune-related ilncRNA. ilncRNAs significantly upregulated in immune cell lines and downregulated in LGG cell lines were defined as TIIClncRNAs. Univariate cox regression analysis was further used to screen out the prognostic TIIClncRNAs. All combinations of 10 machine learning algorithms, including RSF, Enet, Lasso, Ridge, stepwise Cox, CoxBoost, plsRcox, SuperPC, GBM, and survival-SVM, based on a 10-fold cross-validation were further used to screen out the most valuable TIIClnc signature with the highest C-index. The TIIClnc signature was finally generated based on the combination of RSF and CoxBoost. The association between the TIIClnc signature, prognosis, tumor immune microenvironment, and immunotherapy response was comprehensively investigated. Figure 1. The computational framework for establishing the TIIClnc signature. The top 15% expressed lncRNAs were taken for candidate immune-related lncRNAs for each immune cell line. TSI was applied to calculate the expression specificity of candidate immune-related lncRNAs for each cell type. Transwell assay Three groups of the THP-1 cells transfected with siRNAs were centrifuged and resuspended using the serum-free medium. The density was adjusted to 105 cells/mL. 100 μL cell suspension was added to the upper chamber, and 500 μL 1640 with 10% FBS was added to the lower chamber. After culturing for 48 h, https://www.thno.org 5935 Theranostics 2022, Vol. 12, Issue 13 the migrated THP-1 cells in the lower chamber were collected and counted by flow cytometry. In accordance with our previous analyses, 16 TIIClncRNAs were generally expressed in all 19 immune cell types (Figure S1A). Besides, 16 TIIClncRNAs were significantly differentially expressed in immune cell lines (upregulation) and LGG cell lines (downregulation) (Figure S1B). Notably, LGG patients with high expression of 5 TIIClncRNAs had increased survival time, while LGG patients with high expression of 11 TIIClncRNAs had decreased survival time in the TCGA LGG dataset (Figure S2). Statistical analysis Differentially expressed lncRNAs between immune and LGG cell lines were extracted by R packages ‘limma’. Samples were grouped based on the cutoff value of the TIIClnc signature determined by the R package ‘survminer’. The Kaplan-Meier survival plots were applied to estimate overall survival (OS) between the two TIIClnc signature groups using the R package ‘survival.’ C-index of OS was performed on the individual clinical variables, including the TIIClnc signature. The calibration curves of the TIIClnc signature were generated using the R package ‘pec’. The predictive value of the TIIClnc signature for prognosis was measured with time-dependent receiver operating characteristic (ROC) curves using the R package ‘timeROC.’ For normally distributed variables, statistical differences between groups were determined by a two‐tailed t-test while a one‐way ANOVA test determined statistical differences among groups. For nonnormally distributed variables, statistic differences between groups were determined by a Wilcoxon test while a Kruskal–Wallis test determined statistic differences among groups. All the statistical analyses were performed in the R project, version 4.1.2. In vitro validation of TIIClncRNAs To comprehensively evaluate the immune cell-related lncRNA, 115 purified cell lines from 19 major immune cell types from 16 datasets were collected by searching for literature from 2007 to 2022 (Figure 1). 546 lncRNAs (top 15% expressed lncRNAs) were taken for candidate immune-related lncRNAs selection in each immune cell line. TSI score of the 546 lncRNAs was calculated to identify the ilncRNAs generally expressed in all 19 immune cell types (Table S2). Note that lncRNAs with a lower TSI score are generally highly expressed in different immune cell types, indicating their fundamental roles in immunity. 308 ilncRNAs were confirmed to be critical for regulating elemental immunity with the threshold of TSI < 0.2. By comparing the expression of 308 ilncRNAs in immune cell lines and LGG cell lines, 136 ilncRNAs were further found to be significantly upregulated in 115 immune cell lines and downregulated in 10 LGG cell lines. These 136 ilncRNAs were defined as LGG TIIClncRNAs. Among the 16 TIIClncRNAs, LOC100133461 and LOC101928134, with relatively higher expression in immune cell lines, were selected to further validate their potential roles in the TIME. RNA expression of LOC100133461 and LOC101928134 was significantly inhibited in three siRNAs groups of THP-1 cells, respectively (Figure S3A). The CCK-8 assay revealed the increased proliferation ability of THP-1 cells with inhibited expression of LOC100133461 and LOC101928134, respectively (Figure S3B and S3C). The EdU assay further proved the significantly increased proliferation ability of THP-1 cells with inhibited expression of LOC100133461 and LOC101928134, respectively (Figure S3D, S3E, S3F, S3G). Moreover, the Transwell assay revealed the significantly increased migration ability of THP-1 cells with inhibited expression of LOC100133461 and LOC101928134, respectively (Figure S3H, S3I, S3J, S3K). the migrated THP-1 cells in the lower chamber were collected and counted by flow cytometry. The highly expressed lncRNAs in all immune cell types were identified as immune-related ilncRNA. ilncRNAs significantly upregulated in immune cell lines and downregulated in LGG cell lines were defined as TIIClncRNAs. Univariate cox regression analysis was further used to screen out the prognostic TIIClncRNAs. All combinations of 10 machine learning algorithms, including RSF, Enet, Lasso, Ridge, stepwise Cox, CoxBoost, plsRcox, SuperPC, GBM, and survival-SVM, based on a 10-fold cross-validation were further used to screen out the most valuable TIIClnc signature with the highest C-index. The TIIClnc signature was finally generated based on the combination of RSF and CoxBoost. The association between the TIIClnc signature, prognosis, tumor immune microenvironment, and immunotherapy response was comprehensively investigated. Figure 1. The computational framework for establishing the TIIClnc signature. The top 15% expressed lncRNAs were taken for candidate immune-related lncRNAs for each immune cell line. TSI was applied to calculate the expression specificity of candidate immune-related lncRNAs for each cell type. The highly expressed lncRNAs in all immune cell types were identified as immune-related ilncRNA. ilncRNAs significantly upregulated in immune cell lines and downregulated in LGG cell lines were defined as TIIClncRNAs. Univariate cox regression analysis was further used to screen out the prognostic TIIClncRNAs. All combinations of 10 machine learning algorithms, including RSF, Enet, Lasso, Ridge, stepwise Cox, CoxBoost, plsRcox, SuperPC, GBM, and survival-SVM, based on a 10-fold cross-validation were further used to screen out the most valuable TIIClnc signature with the highest C-index. The TIIClnc signature was finally generated based on the combination of RSF and CoxBoost. The association between the TIIClnc signature, prognosis, tumor immune microenvironment, and immunotherapy response was comprehensively investigated. https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5936 learning algorithms, including RSF, Enet, stepwise Cox, CoxBoost, plsRcox, Lasso, Ridge, SuperPC, GBM, and survival-SVM, were combined based on a 10-fold cross-validation to identify the most robust TIIClnc signature with the highest C-index in the TCGA LGG training dataset, Xiangya in-house validating dataset, and two external validating datasets (CGGA LGG and GSE108474) (Figure 2A). A final TIIClnc signature with the best performance was established based on the combined RSF and CoxBoost algorithms, which CoxBoost algorithm identified the 16 most valuable TIIClncRNAs (C7orf13, LINC00628, LINC01121, LOC100133461, LINC01134, TP53TG3HP, SLCO4A1-AS1, C1RL-AS1, LOC284395, TMEM72- AS1, PSMB8-AS1, DKFZp779M0652, LOC100506142, RPARP-AS1, CARD8-AS1, LOC101928134) (Figure 2B, Table S3) and RSF algorithm filtrated the most reliable model (Figure 2C). Development of the TIIClnc signature LGG patients with high TIIClnc signature scores had decreased survival time in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets (Figure 2D). Consistently, time-dependent To develop a prognostic TIIClnc signature, univariate Cox regression analysis regarding OS confirmed 46 prognostic TIIClncRNAs from 136 ilncRNAs in the TCGA LGG dataset. 10 machine https://www.thno.org 5937 Theranostics 2022, Vol. 12, Issue 13 algorithm, the TIIClnc signature score was found to be positively correlated with almost all tumor immune infiltrating cells, such as T cells, T helper (Th) cells, natural killer (NK) cells, B cells, dendritic cell (DC), mast cells, myeloid-derived suppressor cell (MDSC), fibroblasts, macrophages, and regulatory T (Treg) cells in the TCGA LGG (Figure S4A), Xiangya in-house (Figure 4A), CGGA LGG (Figure S6A), and GSE108373 (Figure S7A) datasets. Based on the ESTIMATE algorithm, the TIIClnc signature score was positively correlated with a stromal score, immune score, and ESTIMATE score except for tumor purity in the TCGA LGG (Figure S4A), Xiangya in-house (Figure 4A), CGGA LGG (Figure S6A), and GSE108373 (Figure S7A) datasets. In addition, the TIIClnc signature score positively correlated with most immune modulators categorized into antigen presentation, cell adhesion, co-inhibitor, co-stimu- lator, ligand, receptor, and others in the TCGA LGG (Figure S4B), Xiangya in-house (Figure 4B), CGGA LGG (Figure S6B), and GSE108373 (Figure S7B) datasets. Notably, a strong correlation was observed between the TIIClnc signature score and classical immune checkpoint molecules, including IDO-1, LAG-3, PD-1, PD-L1, PD-L1, CTLA-4, BTLA, and TIGIT (Figure 4B, S4B, S6B, S7B). ROC curves of 1-year, 2-year, 3-year, 4-year, and 5-year OS in the TCGA LGG dataset (AUC values, 0.913, 0.932, 0.960, 0.947, 0.938, respectively), Xiangya in-house dataset (AUC values, 0.755, 0.772, 0.827, 0.810, 0.861, respectively), CGGA LGG dataset (AUC values, 0.749, 0.779, 0.816, 0.800, 0.812, respectively), and GSE108474 dataset (AUC values, 0.656, 0.707, 0.715, 0.645, 0.642, respectively) confirmed the prognostic value of the TIIClnc signature (Figure 2E). The C-index of clinical factors in LGG patients was calculated. The univariate and multivariate Cox regression analysis was performed on clinical factors. The TIIClnc signature was found to be an independent prognostic factor like age, gender, grade, isocitrate dehydrogenase (IDH) status, 1p/19q status, methylation of O6-methylguanine-DNA methyltrans- ferase (MGMT) status, with an advantage over all these clinical factors (Figure 3A, Table S4). Notably, the combination signature incorporating the TIIClnc signature, age, gender, grade, IDH status, 1p/19q status, and MGMT status showed better predictive efficacy in prognosis (Figure 3A). Development of the TIIClnc signature The calibration curves further proved the predictive accuracy of the TIIClnc signature (Figure 3B). The TIIClnc signature is a predictive biomarker for immunotherapy response Thanks to the development of next-generation sequencing and technologies for mining big data, predictive gene expression-based signatures have been widely explored and developed. For a comprehensive comparison of the performance of the TIIClnc signature with other signatures, the published signatures over the past ten years were systematically retrieved. Ultimately, 95 signatures (including mRNA and lncRNA signatures) were enrolled in this study (Table S5). These 95 signatures were closely related to different biological features, including immuno- therapy response, immune infiltration, autophagy, ferroptosis, pyroptosis, stemness, epithelial-mesen- chymal transition, hypoxia, glycolysis, epigenetics, N6-methyladenosine, and aging. Notably, the TIIClnc signature displayed better performance regarding C-index in the TCGA LGG (Figure 3C), Xiangya in-house (Figure 3D), CGGA LGG (Figure 3E), and GSE108474 (Figure 3F) datasets than almost all models. To further explore the role of the TIIClnc signature in immunotherapy response, the relationship between the TIIClnc signature and several immunotherapy predictors was explored (Table S6). Notably, the MSI (Figure S5A), TMB (Figure S5B), CYT (Figure S5C), GEP (Figure S5D), TCR richness (Figure S5E), TCR Shannon (Figure S5F), SNV Neoantigen (Figure S5G), and IFN-γ (Figure S5H) levels were all significantly higher in the high TIIClnc signature score group in the TCGA LGG dataset, all of which were determinants of better immunotherapy response. The significant expression differences of CYT (Figure 4C, S6C, S7C), GEP (Figure 4D, S6D, S7D), and IFN-γ (Figure 4E, S6E, S7E) between two TIIClnc signature score groups were also verified in the Xiangya in-house, CGGA LGG, and GSE108474 datasets. Furthermore, the MSI high group was significantly associated with high TIIClnc signature scores (Figure 4F), and the TMB had a substantially higher level in the high TIIClnc signature score group (Figure 4G). Besides, a high TIIClnc signature score was more associated with lymphocyte depleted and inflammatory immune subtypes and high IPS score in the TCGA LGG dataset (Figure S5I), which the significant expression difference of IPS score between the two TIIClnc Theranostics 2022, Vol. 12, Issue 13 the 8474 datasets (Figure 4H, S6F, S7F). al of 101 combinations of machine learning algorithms for the TIIClnc signatures via validation datasets, including TCGA LGG, Xiangya in-house, CGGA LGG, and GSE10847 st algorithm. C. The number of trees for determining the TIIClnc signature with minima hm. D. Kaplan-Meier survival curve of OS between patients with a high score of TIICln CGGA LGG, and GSE108474 datasets. E. Time-dependent ROC curves of 1-year, 2-ye d GSE108474 datasets. signature score groups was also verified in the Xiangya in-house, CGGA LGG, and GSE108474 signature score groups was also verified in the Xiangya in-house, CGGA LGG, and GSE108474 a in-house, CGGA LGG, and GSE108474 The prognostic value of the TIIClnc signature. A. A total of 101 combinations of machine learning algorithms for the TIIClnc signatures ation framework. The C-index of each model was calculated across validation datasets, including TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108 ibition of the 16 most valuable TIIClncRNAs based on the CoxBoost algorithm. C. The number of trees for determining the TIIClnc signature with minim ance of the 16 most valuable TIIClncRNAs based on the RSF algorithm. D. Kaplan-Meier survival curve of OS between patients with a high score of TIIC ow score of TIIClnc signature in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. E. Time-dependent ROC curves of 1-year, 2 The prognostic value of the TIIClnc signature. A. A total of 101 combinations of mac tion framework. The C-index of each model was calculated across validation datasets, including TC bition of the 16 most valuable TIIClncRNAs based on the CoxBoost algorithm C. The number of Figure 2. The prognostic value of the TIIClnc signature. A. A total of 101 combinations of machine learning algorithms for the TIIClnc signatures via a 10-fold cross-validation framework. The C-index of each model was calculated across validation datasets, including TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. B. The exhibition of the 16 most valuable TIIClncRNAs based on the CoxBoost algorithm. C. The number of trees for determining the TIIClnc signature with minimal error and the importance of the 16 most valuable TIIClncRNAs based on the RSF algorithm. D. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and with a low score of TIIClnc signature in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. E. Time-dependent ROC curves of 1-year, 2-year, 3-year, 4-year, and 5-year OS in the CGGA LGG, Xiangya in-house, TCGA LGG, and GSE108474 datasets. Figure 2. The prognostic value of the TIIClnc signature. A. A total of 101 combinations of machine learning algorithms for the TIIClnc signatures via a 10-fold cross-validation framework. The C-index of each model was calculated across validation datasets, including TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. B. The exhibition of the 16 most valuable TIIClncRNAs based on the CoxBoost algorithm. C. Immune characteristics related to the TIIClnc signature To assess the role of the TIIClnc signature in the TIME of LGG, the relationship between the TIIClnc signature and immune cell infiltration and immune modulators was explored. Based on the TIMER algorithm, MCPcounter algorithm, and ssGSEA https://www.thno.org 5938 Theranostics 2022, Vol. 12, Issue 13 signature score groups was also verified in the Xiangya in-house, CGGA LGG, and GSE108474 The number of trees for determining the TIIClnc signature with minimal error and the importance of the 16 most valuable TIIClncRNAs based on the RSF algorithm. D. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and with a low score of TIIClnc signature in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. E. Time-dependent ROC curves of 1-year, 2-year, 3-year, 4-year, and 5-year OS in the CGGA LGG, Xiangya in-house, TCGA LGG, and GSE108474 datasets. https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5939 Figure 3. Comparison between the TIIClnc signature and other models. A. The C-index of the TIIClnc signature, other clinical factors, and the combination signature in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. B. The 1-year, 2-year, and 3-year calibration curves of the TIIClnc signature in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. C. The C-index of the TIIClnc signature and other models developed in the TCGA LGG dataset. D. The C-index of the TIIClnc signature and other models developed in the Xiangya in-house dataset. E. The C-index of the TIIClnc signature and other models developed in the CGGA LGG dataset. F. The C-index of the TIIClnc signature and other models developed in the GSE108474 dataset. Figure 3. Comparison between the TIIClnc signature and other models. A. The C-index of the TIIClnc signature, other clinical factors, and the combination signature in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. B. The 1-year, 2-year, and 3-year calibration curves of the TIIClnc signature in the TCGA LGG, Xiangya in-house, CGGA LGG, and GSE108474 datasets. C. The C-index of the TIIClnc signature and other models developed in the TCGA LGG dataset. D. The C-index of the TIIClnc signature and other models developed in the Xiangya in-house dataset. E. The C-index of the TIIClnc signature and other models developed in the CGGA LGG dataset. F. The C-index of the TIIClnc signature and other models developed in the GSE108474 dataset. https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5940 https://www.thn gure 4. Immune-related characteristics of the TIIClnc signature in the Xiangya in-house dataset. A. Heatmap displaying the correlation between the nature and immune infiltrating cells. B. Heatmap displaying the correlation between the TIIClnc signature and immune modulator molecules. C. Box plot displaying th els between two TIIClnc signature score groups. D. Box plot displaying the GEP levels between two TIIClnc signature score groups. E. Box plot displaying the IFN- tween two TIIClnc signature score groups. F. Box plot displaying the TIIClnc levels between two MSI groups. G. Box plot displaying the TMB levels between two nature score groups. H. Box plot displaying the IPS levels between two TIIClnc signature score groups. Figure 4. Immune-related characteristics of the TIIClnc signature in the Xiangya in-house dataset. A. Heatmap displaying the correlation between the TIICln signature and immune infiltrating cells B Heatmap displaying the correlation between the TIIClnc signature and immune modulator molecules C Box plot displaying the CY Figure 4. Immune-related characteristics of the TIIClnc signature in the Xiangya in-house dataset. A. Heatmap displaying the correlation between the TIIClnc signature and immune infiltrating cells. B. Heatmap displaying the correlation between the TIIClnc signature and immune modulator molecules. C. Box plot displaying the CYT levels between two TIIClnc signature score groups. D. Box plot displaying the GEP levels between two TIIClnc signature score groups. E. Box plot displaying the IFN-γ levels between two TIIClnc signature score groups. F. Box plot displaying the TIIClnc levels between two MSI groups. G. Box plot displaying the TMB levels between two TIIClnc signature score groups. H. Box plot displaying the IPS levels between two TIIClnc signature score groups. https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5941 The insignificant statistical analysis could be attributed to the small sample size. Moreover, the TIIClnc signature was directly established in immunotherapy datasets to evaluate its predictive value. In the IMvigor dataset, urothelial carcinoma patients with high TIIClnc signature scores had a prolonged survival time (Figure 5A). As expected, urothelial carcinoma patients with high TIIClnc signature scores were more likely to respond to anti-PD-L1 immunotherapy (Figure 5B). In the Braun dataset, renal cell carcinoma patients with high TIIClnc signature scores had prolonged survival time (Figure 5D). As expected, renal cell carcinoma patients with high TIIClnc signature scores tended to respond to anti-PD-1 immunotherapy (Figure 5E). In addition, patients in the GSE179351 (colorectal adenocarcinoma and pancreatic adenocarcinoma) (Figure 5C), and GSE165252 (esophageal adenocarcinoma) (Figure 5G) datasets with high TIIClnc signature scores were also more likely to respond to immunotherapy. Notably, patients in the GSE103668 (triple-negative breast cancer) dataset with high TIIClnc signature scores were more likely to respond to targeted therapy (Figure 5F). In the Allen dataset, melanoma patients with high TIIClnc signature scores had prolonged survival time (Figure 5H). As expected, melanoma patients with high TIIClnc signature scores tended to respond to anti-CTLA-4 immunotherapy (Figure 5I). In the GSE78220 dataset, melanoma patients with high TIIClnc signature scores had prolonged survival time (Figure 5J). As expected, melanoma patients with high TIIClnc signature scores tended to respond to anti-PD-1 immunotherapy (Figure 5K). In the Nathanson dataset, melanoma patients with high TIIClnc signature scores had prolonged survival time (Figure 5L), and melanoma patients with high TIIClnc signature scores tended to respond to anti-CTLA-4 immunotherapy (Figure 5M). In addition, patients in the GSE35640 (melanoma) (Figure 5N) and GSE91061 (melanoma) (Figure 5O) datasets with high TIIClnc signature scores were also more likely to respond to immunotherapy. Based on the TIDE algorithm, a high TIIClnc signature score was significantly associated with immune checkpoint inhibitors response in the TCGA LGG (Figure S8A), Xiangya in-house (Figure 5P), CGGA LGG (Figure S8C), and GSE108474 (Figure S8E) datasets. Based on the submap analysis, a high TIIClnc signature score was related to anti-CTLA-4 and anti-PD-1 immunotherapy responses in the Xiangya in-house dataset (Figure 5Q). In contrast, a high TIIClnc signature score was mainly associated with anti-PD-1 immunotherapy response in the TCGA LGG (Figure S8B), CGGA LGG (Figure S8D), and GSE108474 (Figure S8F) datasets. Notably, in an immunotherapy dataset of glioblastoma patients, Subsequently, the predictive value of the TIIClnc signature in immunotherapy response was explored in the Xiangya in-house dataset. Scatter plots of the TIIClnc signature score and CD8, PD-1, and PD-L1 demonstrated a significant positive correlation (Figure 6A). IHC images (Figure 6B) and H-scores (Figure 6C) further proved that the protein expressions of CD8, PD-1, and PD-L1 were dramatically higher in the high TIIClnc signature score group. Our research verified that patients with a high TIIClnc signature score could benefit more from immunotherapy than those with a low TIIClnc signature score. Potential biological mechanisms related to the TIIClnc signature The cancer immunity cycle was calculated to explore the potential biological mechanisms related to the TIIClnc signature. Of note, all of the seven steps in the cancer immunity cycle were more activated in the high TIIClnc signature score group, including antigen release (Step 1), cancer antigen presentation (Step 2), priming and activation (Step 3), tumor immune infiltrating cells recruitment (Step 4), immune cells infiltration (Step 5), cancer cells recognition by T cells (Step 6), and cancer cells killing (Step 7) in the TCGA LGG (Figure S9A), Xiangya in-house (Figure 7A), CGGA LGG (Figure S10A), and GSE108474 (Figure S11A) datasets. The TIIClnc signature score positively correlated with the metabolic activity of metabolites, such as retinoid, glycan, galactose, or glutathione, in the TCGA LGG (Figure S9B), Xiangya in-house (Figure 7B), CGGA LGG (Figure S10B), and GSE108474 (Figure S11B) datasets. In the GSVA of GO and KEGG terms, the TIIClnc signature score positively correlated with T cell activity, macrophage activity, Treg differentiation, regulation of fibroblast proliferation, DC migration, and pathways in cancer (Figure S9B, 7B, S10B, S11B). In the analysis of TIME signatures, a high TIIClnc signature score was associated with higher levels of innate immunity, T cells, IFN-γ response, Treg, MDSC, recognition of tumor, proliferation, glycolysis developed by Kobayashi and antigen presentation, cytotoxic T and NK cells, anti-tumor microenvironment, checkpoint inhibition, Treg, granulocytes, MDSC, tumor promotive immune infiltrate, CAF, angiogenesis, tumor features developed by Bagaev in the TCGA LGG (Figure S9C and S9D), Xiangya in-house (Figure 7C and 7D), CGGA LGG (Figure S10C and S10D), and GSE108474 (Figure S11C and S11D) datasets. Additionally, in the GSEA of GO (Figure S9E, 7E, S10E, S11E) and KEGG (Figure S9F, 7F, S10F, S11F) https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5942 PI3K-Akt pathway, and NF-kappa B pathway, JAK-STAT pathway was more enriched in the high TIIClnc signature score group. PI3K-Akt pathway, and NF-kappa B pathway, JAK-STAT pathway was more enriched in the high TIIClnc signature score group. terms, T cell chemotaxis, T cell migration, response to IFN-γ, regulation of macrophage activation, PD-L1 expression and PD-1 checkpoint pathway in cancer, https://www.thno gure 5. Predictive value of the TIIClnc signature in immunotherapy response. A. Kaplan-Meier survival curve of OS between patients with a high score of TI nature and a low score of TIIClnc signature in the IMvigor dataset. B. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses i Figure 5. Potential biological mechanisms related to the TIIClnc signature Predictive value of the TIIClnc signature in immunotherapy response. A. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the IMvigor dataset. B. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the https://www.thno.org Figure 5. Predictive value of the TIIClnc signature in immunotherapy response. A. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the IMvigor dataset. B. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the https://www.thno.org https://www.thno.org 5943 Theranostics 2022, Vol. 12, Issue 13 Theranostics 2022, Vol. 12, Issue 13 Theranostics 2022, Vol. 12, Issue 13 IMvigor dataset. C. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE179351 dataset. D. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the Braun dataset. E. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the Braun dataset. F. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE103668 dataset. G. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE165252 dataset. H. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the Allen dataset. I. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the Allen dataset. J. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the GSE78220 dataset. K. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE78220 dataset. L. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the Nathanson dataset. M. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the Nathanson dataset. N. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE35640 dataset. O. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE91061 dataset. P. Potential biological mechanisms related to the TIIClnc signature Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the PRJNA482620 dataset. Figure 6. A. Scatter plot displaying the correlation between the TIIClnc signature score and CD8, PD-1, and PD-L1 in the Xiangya in-house dataset. B. Box plot displaying the H-score levels of CD8, PD-1, and PD-L1 based on IHC staining between two TIIClnc signature score groups in the Xiangya in-house dataset. The H-score was calculated by intensity score * quantity score. As for intensity scores, 0, 1, 2, and 3 were considered negative, weak, moderate, and strong, respectively. As for quantity scores, 0, 1, 2, 3, and 4 represented <10%, 10-25%, 25-50%, 50-75%, >75% proportion of stained cells, respectively. H-score ranges from 0 to 12. C. Representative IHC staining images of CD8, PD-1, and PD-L1 in two TIIClnc signature score groups in the Xiangya in-house dataset. Figure 6. A. Scatter plot displaying the correlation between the TIIClnc signature score and CD8, PD-1, and PD-L1 in the Xiangya in-house dataset. B. Box plot displaying the H-score levels of CD8, PD-1, and PD-L1 based on IHC staining between two TIIClnc signature score groups in the Xiangya in-house dataset. The H-score was calculated by intensity score * quantity score. As for intensity scores, 0, 1, 2, and 3 were considered negative, weak, moderate, and strong, respectively. As for quantity scores, 0, 1, 2, 3, and 4 represented <10%, 10-25%, 25-50%, 50-75%, >75% proportion of stained cells, respectively. H-score ranges from 0 to 12. C. Representative IHC staining images of CD8, PD-1, and PD-L1 in two TIIClnc signature score groups in the Xiangya in-house dataset. might benefit from immunotherapy is urgently needed. Potential biological mechanisms related to the TIIClnc signature Contingency table between immunotherapy responses and TIIClnc signature score groups based on TIDE algorithm in the Xiangya in-house dataset. Q. Contingency table between immunotherapy responses (anti-PD-1 and anti-CTLA-4) and TIIClnc signature score groups based on submap analysis in the Xiangya in-house dataset. R. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the PRJNA482620 dataset. IMvigor dataset. C. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE179351 dataset. D. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the Braun dataset. E. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the Braun dataset. F. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE103668 dataset. G. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE165252 dataset. H. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the Allen dataset. I. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the Allen dataset. J. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the GSE78220 dataset. K. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE78220 dataset. L. Kaplan-Meier survival curve of OS between patients with a high score of TIIClnc signature and a low score of TIIClnc signature in the Nathanson dataset. M. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the Nathanson dataset. N. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE35640 dataset. O. Box plot displaying the TIIClnc signature score in patients with different immunotherapy responses in the GSE91061 dataset. P. Contingency table between immunotherapy responses and TIIClnc signature score groups based on TIDE algorithm in the Xiangya in-house dataset. Q. Contingency table between immunotherapy responses (anti-PD-1 and anti-CTLA-4) and TIIClnc signature score groups based on submap analysis in the Xiangya in-house dataset. R. Discussion This study utilized a novel computational framework to identify a robust and stable TIIClnc signature. Firstly, TSI was applied to screen out the ilncRNAs generally expressed in immune cells, indicating their fundamental roles in immune system regulation. Secondly, 101 combinations of 10 machine learning algorithms were used to identify the combination of CoxBoost and RSF as the optimal model based on the prognostic TIIClncRNAs, significantly reducing the dimensionality of variables and revealing underlying patterns, contributing to a more simplified and translational model. Therefore, the established TIIClnc signature conceivably precisely predicts LGG patients’ prognosis and immune characteristics. Compared with glioblastoma patients, LGG patients experienced a relatively prolonged survival time of 5-10 years. However, the treatment options for LGG are still limited to chemoradiotherapy and target therapy of tyrosine kinase receptor pathway [41]. The insufficient treatment options could sometimes lead to the overtreatment or undertreatment of LGG patients. Immunotherapy has become a leading star in the treatment of solid tumors. Although PD-1 has been widely detected among LGG patients, anti-PD-1 and anti-PD-L1 immunotherapies have not widely entered clinical trials of LGG patients partly due to the poor immunotherapy response and the results of several clinical trials (NCT03718767) remain to be announced [42]. For this reason, establishing reliable prognostic biomarkers to stratify LGG patients who https://www.thno.org anostics 2022, Vol. 12, Issue 13 Theranostics 2022, Vol. 12, Issue 13 5944 Figure 7. Functional annotation of the TIIClnc signature in the Xiangya in-house dataset. A. Box plot displaying the cancer immunity cycle differences bet TIIClnc signature score groups. B. Butterfly plot displaying the correlation between the TIIClnc signature score and metabolic pathways, immune-related pathways GSVA of GO and KEGG terms. Immunogram radar plot displaying the correlation between the TIIClnc signature score and TIME signatures developed by C. Kobayas Bagaev. E. GSEA of GO terms for the TIIClnc signature score. F. GSEA of KEGG terms for the TIIClnc signature score. Figure 7. Functional annotation of the TIIClnc signature in the Xiangya in-house dataset. A. Box plot displaying the cancer immunity cycle differences between two TIIClnc signature score groups. B. Butterfly plot displaying the correlation between the TIIClnc signature score and metabolic pathways, immune-related pathways based on GSVA of GO and KEGG terms. Immunogram radar plot displaying the correlation between the TIIClnc signature score and TIME signatures developed by C. Kobayashi and D. Bagaev. E. GSEA of GO terms for the TIIClnc signature score. F. TIIClnc signature. g The TIME is closely associated with the prognosis of brain tumors and the efficacy of immunotherapy [55, 56]. LGG patients with high TIIClnc signature score presented abundant immune cell infiltration, including T cells, Th cells, NK cells, B cells, DC, mast cells, MDSC, fibroblasts, macrophages, and Treg cells, all of which were related to anti-tumor or pro-tumor immunity in immunotherapy [57-62]. Immunotherapy has demonstrated considerable benefits in cancer patients with MSI-H [63]. TMB could enhance tumor immunogenicity and activate cytotoxic T cells; cancer patients with high TMB also benefit more from immunotherapy [64]. In this study, LGG patients with high TIIClnc signature scores were prone to higher MSI and TMB. Meanwhile, LGG patients with high TIIClnc signature scores were also associated with higher levels of predictors for better immunotherapy responses, including CYT, GEP, TCR richness, TCR Shannon, SNV Neoantigen, IFN-γ, and IPS score. Moreover, the TIIClnc signature score was positively associated with classical immune checkpoint molecules, including IDO-1, LAG-3, PD-1, PD-L1, PD-L1, CTLA-4, BTLA, and TIGIT. For this reason, LGG patients with high TIIClnc signature scores are expected to benefit more from immunotherapy. The predictive analysis demonstrated that the TIIClnc signature was a hazardous marker of OS in LGG patients. The ROC analysis further proved that the TIIClnc signature possessed high accuracy in predicting 1-year, 2-year, 3-year, 4-year, and 5-year OS of LGG patients. The stable performance of the TIIClnc signature in the TCGA LGG training dataset, Xiangya in-house dataset, and CGGA LGG, GSE108474 external validating datasets indicated the massive potential for the clinical application of the TIIClnc signature. IDH, 1p/19q, and MGMT status have long been determined as risk biomarkers for evaluating clinical strategies and outcomes of glioma patients [52, 53]. Notably, the TIIClnc signature was an independent risk factor with significantly superior performance than the above three risk biomarkers and clinical factors: age, grade, and gender. In addition, 95 published signatures of various functional gene combinations were retrieved for comparison. Few of these 95 signatures have been put into clinical practice [54]. Besides, many models displayed exemplary performance in the training dataset but weak performance in the validating datasets, indicating these models’ poor universality and generalizability. Notably, according to the C-index assessment, the TIIClnc signature performed better than almost 95 signatures. Discussion GSEA of KEGG terms for the TIIClnc signature score. Among the 16 most valuable TIIClncRNAs identified for the TIIClnc signature, nine lncRNAs have been reported. C7orf13 was proved with an inverse correlation with DNA methylation in glioblastoma [43]. LINC00628 contributes to lung adenocarcinoma by epigenetically interacting with the https://www.thno.org 5945 Theranostics 2022, Vol. 12, Issue 13 LAMA3 promoter [44]. LINC01121 promotes cell proliferation, migration, and invasion of breast cancer via miR-150-5p/HMGA2 axis [45]. LINC01134 dictates hepatocellular carcinoma progression by interplaying with YY1 [46]. SLCO4A1-AS1 could predict poor prognosis and promote proliferation and metastasis of colorectal cancer via the EGFR/MAPK axis [47]. C1RL-AS1 drives the malignant phenotype of gastric cancer via the AKT/β-Catenin/c-Myc axis [48]. PSMB8-AS1 leads to pancreatic cancer progression via miR-382-3p/STAT1/PD-L1 axis [49]. RPARP-AS1/miR125a-5p axis promoted prolifera- tion, migration, and invasion of colon Cancer [50]. CARD8-AS1 was found to regulate the metastasis of glioma [51]. The detailed reports of other 7 lncRNAs are lacking. Our research revealed that these 16 TIIClncRNAs were highly expressed in immune cells. Notably, LOC100133461 and LOC101928134 were found to inhibit the proliferation and migration ability of THP-1 cells, which was in accordance with the finding that they were hazardous markers in LGG patients. Thus, LOC100133461 and LOC101928134 were likely to facilitate tumor progression by suppressing the activity of monocytes in the TIME of LGG. Abbreviations lncRNAs: long noncoding RNAs; TIIClncRNA: tumor-infiltrating immune cell-associated lncRNA; LGG: low-grade glioma; WHO: World Health Organization; TIME: tumor immune microenviron- ment; ICB: immune checkpoint blockade; ACT: adoptive cell transfer; TCGA: The Cancer Genome Atlas; CGGA: Chinese Glioma Genome Atlas; GEO: Gene Expression Omnibus; CCLE: Cancer Cell Line Encyclopedia project; RMA: Robust Multi-array Average; FPKM: fragments per kilobase million; TPM: transcripts per kilobase million; TSI: tissue specificity index; RSF: random survival forest; Enet: elastic network; plsRcox: partial least squares regression for Cox; SuperPC: supervised principal components; GBM: generalized boosted regression modeling; survival-SVM: survival support vector machine; GEP: T cell-inflamed gene expression profile; CYT: cytotoxic activity; IFN-γ: interferon γ; TMB: tumor mutation burden; MSI: microsatellite instability; TCR: T cell receptor; IPS: immunophenoscore; GSVA: gene set variation analysis; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: gene set enrichment analysis; OS: overall survival; ROC: receiver operating characteristic; BSA: bovine serum albumin; FBS: fetal bovine serum; TIMER: Tumor Immune Estimation Resource; MCPcounter: Microenvironment Cell Populations-counter; ssGSEA: single cell gene set enrichment analysis; ESTIMATE: Estimation of STromal and Immune cells in Supplementary Material Although the clinical significance of the TIIClnc signature in LGG is remarkable, some limitations in this study need to be issued. First, all included datasets were from single-center retrospective studies, and future TIIClnc signature validation should be performed in prospective multicenter cohorts. Second, the in-depth molecular mechanisms of how 16 identified most valuable TIIClncRNAs could influence the TIME and immunotherapy response of LGG should be further explored. Third, more immunotherapy cohorts of glioma patients are urgently needed to validate the TIIClnc signature in predicting immunotherapy response. Supplementary Material Supplementary figures. https://www.thno.org/v12p5931s1.pdf Supplementary table 1. https://www.thno.org/v12p5931s2.xlsx Supplementary table 2. https://www.thno.org/v12p5931s3.xlsx Supplementary table 3. https://www.thno.org/v12p5931s4.xlsx Supplementary table 4. https://www.thno.org/v12p5931s5.xlsx Supplementary table 5. https://www.thno.org/v12p5931s6.xlsx Supplementary table 6. https://www.thno.org/v12p5931s7.xlsx Supplementary Material Supplementary figures. https://www.thno.org/v12p5931s1.pdf Supplementary table 1. https://www.thno.org/v12p5931s2.xlsx Supplementary table 2. https://www.thno.org/v12p5931s3.xlsx Supplementary table 3. https://www.thno.org/v12p5931s4.xlsx Supplementary table 4. https://www.thno.org/v12p5931s5.xlsx Supplementary table 5. https://www.thno.org/v12p5931s6.xlsx Supplementary table 6. https://www.thno.org/v12p5931s7.xlsx In conclusion, by integrative analysis of sequencing data of purified immune cells, LGG cell lines, and bulk LGG tissues based on a wealth of machine learning algorithms, a stable and robust TIIClnc signature to stratify LGG patients and predict the outcomes of immunotherapy was developed. A TIIClnc signature is a promising tool for personalized treatment and clinical management for individual LGG patients. pp y https://www.thno.org/v12p5931s7.xlsx https://www.thno.org/v12p5931s7.xlsx Author contributions NZ, HZ, QC, ZL, and WW designed and drafted the manuscript. NZ, HZ, QC, JZ, PL, ZL, SL, FL, WW, RZ, and ZD wrote figure legends and revised the manuscript. NZ, HZ, and QC conducted data analysis. All authors have read and approved the final manuscript. Code availability Essential scripts for implementing machine learning-based integrative procedure are available on the Github website (https://github.com/AweKevin/ TIIClnc). Availability of data and materials All data used in this work can be acquired from the Gene Expression Omnibus (GEO; https://www. ncbi.nlm.nih.gov/geo/), the Cancer Genome Atlas (TCGA) datasets (https://xenabrowser.net/), the Chinese Glioma Genome Atlas (CGGA) datasets (http://www.cgga.org.cn/), and the Cancer Cell Line Encyclopedia (CCLE) project (https://portals. broadinstitute.org/ccle). Acknowledgements Financial support was provided by the National Natural Science Foundation of China (NO. 82073893, 82172685, and 81873635). Hunan Provincial Natural Science Foundation of China (NO.2022JJ20095). Hunan Provincial Health Committee Foundation of China (NO.202204044869). TIIClnc signature. It was conceivable that the feature gene selection and statistical prediction based on the best fit model performed by two combined machine learning algorithms ensured the stability and potential of our py The TIIClnc signature was directly established in immunotherapy datasets to verify this hypothesis. The predictive analysis demonstrated that the TIIClnc signature was a favorable marker of OS in urothelial carcinoma (IMvigor dataset), renal cell carcinoma (Braun dataset), and melanoma (Allen, GSE78220, and Nathanson datasets) patients. As expected, urothelial carcinoma, renal cell carcinoma, and melanoma patients with high TIIClnc signature scores were prone to benefit more from anti-PD-1, anti-PD-L1, or anti-CTLA-4 immunotherapy, respectively. Consis- tently, melanoma patients in the GSE35640 and GSE91061 datasets, colorectal adenocarcinoma and pancreatic adenocarcinoma patients in the GSE179351 dataset, and esophageal adenocarcinoma patients in the GSE165252 dataset with high TIIClnc signature scores also benefit more from immunotherapy. Besides, triple-negative breast cancer patients in the GSE103668 dataset with high TIIClnc signature scores benefit more from targeted therapy. More importantly, glioblastoma patients with high TIIClnc signature scores in the PRJNA482620 dataset were related to relatively reduced survival time, which the small sample size could partly explain the insignificant results. Furthermore, in our Xiangya in-house dataset, the protein expression level of CD8, PD-1, and PD-L1 also increased in the high TIIClnc signature score group, suggesting that the TIIClnc https://www.thno.org Theranostics 2022, Vol. 12, Issue 13 5946 MAlignant Tumours using Expression data. signature could potentially predict the response rate of immunotherapy in LGG. References 29. Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, et al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell. 2017; 168: 542. 1. Youssef G, Miller JJ. Lower Grade Gliomas. Curr Neurol Neurosci Rep. 2020; 20: 21. 1. Youssef G, Miller JJ. Lower Grade Gliomas. Curr Neurol Neurosci Rep. 2020; 20: 21. 30. Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, et al. Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science. 2015; 350: 207-11. 2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. 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https://openalex.org/W4224226098	https://orca.cardiff.ac.uk/id/eprint/149377/1/12883_2022_2634_OnlinePDF.pdf	English	null	MORe PREcISE: a multicentre prospective study of patient reported outcome measures in stroke morbidity: a cross sectional study	BMC neurology	2,022	cc-by	5,963	© 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. Corrigan et al. BMC Neurology _#####################_ https://doi.org/10.1186/s12883-022-02634-0 Abstract Background and Purpose: The use of patient reported outcomes measures (PROMs) may offer utility that are important for stroke survivors. This study assessed the PROMIS-10, which contains Mental health (MH) and Physical Health (PH) domains, with an additional five stroke specific questions. The aim of this study was to evaluate the asso- ciation between the MH and PH measures following a stroke and pre-existing health conditions. Methods: A multicentre prospective cohort study at 19 hospital sites across England and Wales during 2019 was conducted. The association between each PROMIS-10 domain and demographic and health conditions were calcu- lated using a multilevel multivariable linear and present the adjusted mean difference (aMD). Results: The study enrolled 549 stroke survivors within 14 days of the index event, 232 were women (42.3%) and with a mean age of 72.7 years (SD = 12.9, range 25 to 97). The MH domain was scored as poor in 3.9% of participants, and very good or excellent in almost a half (48.4%). In contrast the PH domain was scored as poor in 39.9%, compared to very good or excellent in 8.5%. The MH domain was associated with pre-existing diabetes (aMD = − 2.01; 95%CI -3.91, − 0.12; p = 0.04), previous stroke (aMD = − 3.62; 95%CI -5.86, − 1.39; p = 0.001), age (aMD = 0.07; 95%CI: 0.01, 0.14; p = 0.037), and female sex (aMD = 1.91; 95%CI 0.28, 3.54; p = 0.022). The PH domain was found to be associated with sex (female) (aMD = 2.09; 95%CI 0.54, 3.65; p = 0.008) and previous stroke (aMD = − 3.05; 95%CI -5.17, − 0.93; p = 0.005). Conclusions: Almost half of stroke survivors reported poor PH using a PROM with less reporting poor MH. age, and sex were associated with both MH and PH domains, and additionally pre-exising diabetes and stroke were associated with poorer MH. Clinical management offers an opportunity to investigate and intervene to prevent long term poorer health in stroke survivors. Keywords: Morbidity, Patient reported outcome, PROM, Stroke Neurological deficits that persist secondary to stroke are heterogeneous and vary across the patient population. Introductionh The World Health Organisation (WHO) estimates 15 million people suffer from stroke each year, with more than 5 million living with permanent disability [1–3]. The use of patient reported outcomes measures (PROMs), defined as questionnaires measuring views on health status from the perspective of the patient rather than the clinician, have grown in importance and signifi- cance. PROMs offer a way to measure specific functional domains in a way which is meaningful to the patient, encapsulating the patient’s own perspective of their Correspondence: hewittj2@cardiff.ac.uk 2 Division of Population Medicine, School of Medicine, Cardiff University, Cardiff, UK Full list of author information is available at the end of the article Ethical approval Whilst the GAD-7 [13] has not been validated within stroke use, it is a widely used self reported screening tool for generalised anxiety in primary care [14, 15]. All participants provided informed consent to participate for the study. All methods were conducted in accordance with relevant guidelines and regulations. Ethical approval was granted by the NHS Research Ethics Committee – Wales REC 3–18/WA/0299 - Health and Care Research Wales Support and Delivery Centre for all the sites. Demographic, lifestyle, and clinical measures During the baseline assessment the following were assessed: age; sex; stroke type; pre stroke smoking; alco- hol consumption; level of care: clinical characteristics Modified Rankin Scale (mRS)h The mRS [19] is clinician recorded and delineated using the Rankin Focussed Assessment (RFA), a questionnaire that allows global consideration of disability after the occurrence of stroke [20]. © 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. Corrigan et al. BMC Neurology _#####################_ Page 2 of 7 which included past medical history (hypertension, dia- betes; transient ischemic attacks and prior stroke). health [4, 5]. To improve the use of PROMs within the stroke community, a consensus Stroke Standard Set of outcome data has been developed which promotes the use of patient reported outcomes as part of a value-based assessment of care [4]. Patient Health Questionnaire‑9 (PHQ‑9)h The PHQ-9 [11] is a widely used self-reported primary care screening tool for depression and has previously been recommended in stroke, the instrument has strong psychometric properties [12]. Study designh The study protocol has previously been published [16]. The cohort was recruited between August 2018, and October 2019 from 19 hospital sites with acute and hyper-acute patient facilities in the UK. Data was assessed within 14 days of the index stroke event (the baseline period post-stroke). Data collection was con- ducted by trained and experienced research staff. Patient‑Reported Outcome Measures (PROM)h p ( ) The PROM is a combination of the PROMIS-10 and additional five stroke specific questions. The PROMIS-10 was developed by Cella et. al [7] and has been validated in previous stroke population [4]. The instrument has two domains of physical health (PH) and mental health (MH) and consists of 10 items. Raw domain scores are converted to T-Scores which are normed to the US pop- ulation. The PROMIS-10 is a well validated and estab- lished patient-reported outcome measure [3, 4, 6, 7, 17, 18]. The additional five stroke specific questions were added to include assessment of patient function, which included items on: walking; eating; toileting; dressing; and communication [4, 8, 9, 18]. These additional ques- tions were developed in order to add stroke specific reporting as well as improve functional-capacity report- ing within the PROMIS-10. Further, the PROMIS-10 in combination with the five additional questions has been shown to hold value within other common neurological conditions, such as Multiple sclerosis, Parkinson’s disease and Acquired brain injury [18]. The PROMIS-10 is a patient reported outcome meas- ure which allows components of both physical and men- tal health to be accessed from the patient perspective [6, 7]. Utilised in stroke, the PROMIS-10, allows considera- tion of functional and cognitive status post-stroke. It has previously been estimated that around 50% of survivors are chronically disabled [3]. Physically, survivors of stroke have been seen to suffer from impairments in language and speech, swallowing, vision, weakness, and paralysis. Mentally, survivors of stroke have been reported to expe- rience higher levels of anxiety and depression as well as greater levels of cognitive impairment [8–15]. However, the understanding of the impact of symptoms across mental and physical health domains, especially from the patient perspective, is lacking. Short‑form Montreal Cognitive Assessment (SF‑MoCA)h Short‑form Montreal Cognitive Assessment (SF‑MoCA) The SF-MoCA is an adapted shorter 10-point version of the 30 item Montreal Cognitive Assessment. This contains three sections, comprising of clock drawing, abstraction and 5-word recall. It is a clinician delivered tool which acts as an indicator of post-stroke cognitive impairment [10]. Aimsh The primary aim of this study was to assess the quality of life for stroke survivors throughout England and Wales. The primary objective was to determine the prevalence of mental and physical health outcome for stroke survivors, and secondarily, to report if there was any association with clinical and demographic risk factors. Statistical analysish The association between exposures and outcomes were fitted using a crude and multivariable multilevel linear model, where hospital site was fitted as a random effect. This utilised PH and MH domain T-scores. The mul- tivariable model was adjusted for: age, sex, pre-stroke hypertension, previous stroke event, previous TIA and pre-stroke diabetes diagnosis. Residuals were used to visually inspect the distributional assumptions from each linear model. The analysis presented the mean difference (MD) and adjusted mean difference (aMD) reported with associated 95% CI and P-values. We have reported sig- nificant differences of 2 (or more) as both statistically and clinically important differences to patients. Data analysis All data analysis were undertaken in Stata version 16.0. The measures were scored using the validated methods. Corrigan et al. BMC Neurology _#####################_ Page 3 of 7 Corrigan et al. BMC Neurology _#####################_ Covariatesh The following were fitted to assess any association with the outcomes: pre-stroke hypertension, previous TIA, previous stroke, pre-stroke diabetes, male sex, and age. f The other comparisons were not significant (p > 0.05). Study population Better mRS scores were associated with sex (aMD = − 0.26 95%CI -0.48, − 0.04; p = 0.018) and worse mRS scores were associated with age (aMD = 0.01; 95%CI 0.01, 0.02; p = 0.003) (Supplementary Table 5). Worse SF-MoCA scores were found to be associated with pre-stroke diabetes (aMD = − 0.59; 95%CI -1.15, − 0.03; p = 0.039) and age (aMD = − 0.03; 95%CI -0.06, − 0.02; p < 0.001) (Supplementary Table 6). From the 19 hospitals 550 participants consented into the study, and one subject withdrew prior to completing the baseline visit assessments, leaving 549. The population consisted of 232 women (42.3%) and 317 men (57.7%) aged between 25 to 97 (mean = 72.7, SD = 12.9) (Supple- mentary Table 1). PROMIS-10 MH was poor in 3.9% participants, very good in 29.3% and excellent in 19.1%, and within PH was with poor in 39.9%, very good 7.31% and excellent in 1.19% (Table 1). There were 253 (50.9%) of people were able to walk unaided, 214 (41.4%) who needed help going to the toilet, 220 (42.6%) who needed aid to dress, 31 (6.2%) who needed a tube for feeding and 97 (17.9%) who had problems communicating or understanding (Supple- mentary Table 2). Outcomesh The co-primary outcomes were the MH and PH domains of the PROMIS-10. Secondary outcomes included the GAD-7; PHQ-9; mRS, SF-MoCA and the additional 5 stroke specific questions (walking, toileting, dressing, tube feeding and communication). Demographic and clinical characteristics associated with PROMIS‑10 Item missingness (e.g. no more than 30%) within each measure (or domain) were pro-rata mean imputed [21]. Participants with over 30% of missing items were marked as missing. For each analysis the residuals were approximately nor- mally distributed with zero mean and constant variance. Multivariable analysis for MH was associated with pre- existing diabetes (aMD = − 2.01; 95%CI -3.91, − 0.12; p = 0.036, Table 2); female sex (aMD = 1.91; 95%CI 0.28, 3.54; p = 0.022); history of stroke (aMD = − 3.62; 95%CI -5.86, − 1.39; p = 0.001); and age (aMD = 0.07; 95%CI: 0.01, 0.14; p = 0.037). PH were associated with sex (aMD = 2.09; 95%CI 0.54, 3.65; p = 0.008), and his- tory of stroke (aMD = − 3.05; 95%CI -5.17, − 0.93; p = 0.005). Thus, worse mental health scores were asso- ciated previous stroke and pre-stroke diabetes and bet- ter mental health scores with male sex and age. Worse physical health scores were associated with previous stroke and better physical health scores were associated with male sex. Previous stroke and pre-stroke diabetes were both statistically significant and clinically important differences. Demographic and clinical characteristics associated with clinical outcomes For each analysis the residuals were approximately nor- mally distributed with zero mean and constant variance. Generalised anxiety using GAD-7 was not found to be associated with any demographic or clinical char- acteristics (p > 0.05) (Supplementary Table 3). Worse PHQ-9 scores were associated with pre-stroke diabetes (aMD = 2.34; 95%CI 1.17, 3.50; p < 0.001). Lower, and thus better scores, in the PHQ-9 were associated with sex (aMD = − 1.27; 95%CI -2.28, − 0.27; p = 0.013), and age (aMD = − 0.08; 95%CI -0.12, − 0.04), p < 0.001). Patients with diabetes had increased depression, male exhibited lower depression, and increasing age had lower depres- sion (Supplementary Table 4). Discussion We included 549 stroke survivors and found almost half reported poor physical health. Poorer mental health (MH) outcomes were associated with age, female sex, previous stroke and pre-stroke diabetes and worse physi- cal health (PH) outcomes with female sex and age. The Corrigan et al. BMC Neurology _#####################_ Page 4 of 7 Table 1 Domain specific breakdown of PRO – Breakdown of Mental and Physical domain of the 10 Question PRO across sub-groups of patients. Sub-groups encompass age groups, sex (male, female), patients who report pre-stroke hypertension, previous TIA, previous stroke and pre-stroke diabetes were reported (N = 549). Results are reported as poor, fair, good, very good and excellent. Discussion BMC Neurology _#####################_ Corrigan et al. BMC Neurology _#####################_ Page 5 of 7 Table 2 Mean difference of hypertension, TIA, previous stroke, diabetes, sex and age associated with PRO mental health domain. The mean difference (MD) and adjusted MD are reported with associated p-values and intervals. Statistically significant p -values are reported in bold. As a negative score is associated with worse outcome – a negative value indicates a factor resulting in worse outcome PROMIS-10 Mental Health domain Mean differences MD P-value (95% CI) Adjusted MD P-value (95% CI) Pre-Stroke Hypertension 0.24 0.768 (−1.36, 1.84) 0.65 0.435 (−0.97, 2.27) Previous TIA −2.01 0.067 (−4.16, 0.14) −1.67 0.130 (−3.84, 0.49) Previous stroke −3.75 0.001 (−5.95, − 1.56) − 3.62 0.001 (−5.86, − 1.39) Pre-Stroke Diabetes −2.20 0.022 (−4.09, − 0.31) − 2.01 0.036 (− 3.91, − 0.12) Sex (Male) 1.64 0.048 (0.01, 3.25) 1.91 0.022 (0.28, 3.54) Age 0.06 0.060 (−0.01, 0.12) 0.07 0.037 (0.01, 0.14) PROMIS-10 Mental Health domain Mean differences pre-stroke diabetes and a worse MoCA and PHQ9 scores across the cohort at the baseline period (Supple- mentary Table 4 and 6). A diagnosis of diabetes is con- sidered a high-risk factor for stroke and confers worse outcomes in terms of overall morbidity, functional out- comes and readmission or recurrence [32]. Diabetes, as a comorbidity, has been accepted as being highly related to overall outcome [27, 33] and the reflection within the PROMIS-10 measures is not surprising. stroke specific questions [4] suggested that stroke survi- vors had a high degree of stroke specific comorbidity. g g pi y The PROMIS-10 has been shown as a feasible instru- ment in stroke survivors [22–27] and exhibited the components of patient reported morbidity. Particularly when considering the high post-stroke prevalence of PROMIS-10 poor outcomes immediately after stroke. The PROMIS-10 represents an outcome measure that is real and accessible to the stroke survivor. Work by Phillipp et al. also demonstrates the instrument across stroke survivor populations, suggesting it was a valid and reliable instrument in German stroke surviors [28]. Previously, measures accessing quality of life, such as the HRQOL, in stroke, has demonstrated that physi- cal domains are adversely impacted by stroke in diverse communities [29, 30, 31]. Further assessment of the PROMIS-10 in broader populations is needed to inte- grate the tool to wider clinical populations. The study demonstrated that sex was associated with different outcomes. Discussion Domain specific cut offs (normed against the US population) as delineated by Hays, Spritzer, Thompson and Cella, 2015; Hays, Schalet, Spritzer and Cella, 2017 – Domain Table of morbidity outcome domains within PRO Scoring across age, sex and pre-stroke conditions (Hypertension, TIA, stroke and diabetes) Mental Health domain Physical health domain Poor (< 29) Fair (30–40) Good (41–48) Very Good (49–56) Excellent (> 56) Missing Poor (< 35) Fair (36–42) Good (43–50) Very Good (51–58) Excellent (< 59) Missing Total 20 (13%) 101 (18.4%) 141 (25.7%) 149 (27.1%) 97 (17.7%) 41 (7.5%) 202 (36.8%) 122 (22.2%) 139 (25.3%) 37 (6.7%) 6 (1.1%) 43 (7.8%) Age 18–49 1 (0.2%) 8 (1.4%) 8 (1.4%) 8 (1.4%) 6 (1.1%) 3 (0.5%) 12 (2.2%) 6 (1.1%) 10 (1.8%) 3 (0.5%) – 3 (0.5%) 50–64 8 (1.4%) 18 (3.2%) 29 (5.3%) 21 (3.8%) 16 (2.9%) 3 (0.5%) 43 (7.8%) 24 (4.4%) 19 (3.4%) 6 (1.1%) 2 (0.4%) 1 (0.2%) 65–74 4 (0.7%) 27 (4.9%) 40 (7.3%) 38 (6.9%) 28 (5.1%( 4 (0.7%) 50 (9.1%) 36 (6.5%) 40 (7.3%) 8 (1.4%) 2 (0.4%) 5 (0.9%) 75–84 5 (0.9%) 37 (6.7%) 41 (7.5%) 53 (9.7%) 33 (6%) 17 (3.1%) 65 (11.8%) 39 (7.1%) 49 (8.9%) 13 (2.3%) 2 (0.4%) 18 (3.2%) > 85 2 (0.4%) 11 (2%) 21 (3.8%) 27 (4.9%) 14 (2.6%) 14 (2.6%) 31 (5.6%) 15 (2.7%) 20 (26.6%) 7 (9.4%) – 16 (2.9%) Sex Female 10 (1.8%) 45 (8.2%) 60 (10.9%) 59 (10.7%) 34 (6.2%) 24 (4.4%) 88 (16%) 53 (9.7%) 53 (9.7%) 14 (2.6%) 1 (0.2%) 23 (4.2%) Male 10 (1.8%) 56 (10.2%) 81 (14.7%) 90 (16.4%) 63 (11.4%) 17 (3.1%) 114 (20.7%) 69 (12.6) 86 (15.7%) 23 (4.2%) 5 (0.9%) 20 (3.6%) Pre-Stroke Hypertension Yes 9 (1.6%) 58 (10.6%) 73 (13.3%) 78 (14.2%) 52 (9.5%) 19 (3.5%) 100 (18.2%) 76 (13.8%) 71 (12.9%) 19 (3.5%) 1 (0.2%) 22 (4%) No 11 (2%) 43 (7.8%) 68 (12.4%) 71 (12.9%) 45 (8.2%) 22 (4%) 102 (18.5%) 46 (8.4%) 68 (12.4%) 18 (3.3%) 5 (0.9%) 19 (2%) Previous TIA Yes 3 (0.5%) 20 (3.6%) 29 (5.3%) 19 (3.5%) 12 (2.2%) 12 (2.2%) 35 (6.4%) 21 (3.8%) 24 (4.4%) 3 (0.5%) – 12 (2.2%) No 17 (3.1%) 81 (14.8%) 112 (20.4%) 130 (23.7%) 85 (15.5%) 29 (5.3%) 167 (30.4%) 101 (18.4%) 115 (20.9%) 34 (6.2%) 6 (1.1%) 31 (5.6%) Previous stroke Yes 8 (1.4%) 16 (2.9% 22 (4%) 23 (4.2) 8 (1.4%) 5 (0.9%) 37 (6.7%) 25 (4.6%) 12 (2.2%) 1 (0.2%) 1 (0.2%) 6 (1.1%) No 12 (2.2%) 85 (15.5%) 119 (21.7%) 126 (22.9%) 89 (16.2%) 36 (6.6%) 165 (30.1%) 97 (17.7%) 127 (23.1%) 36 (6.6%) 5 (0.9%) 37 (6.7%) Pre-Stroke Diabetes Yes 6 (1.1%) 30 (5.5%) 38 (6.9%) 36 (6.6%) 13 (2.3%) 1 0.2%) 54 (9.8%) 32 (5.8%) 26 (4.7%) 5 (0.9%) 3 (0.5%) 1 (0.2%) No 14 (2.6%) 71 (12.9%) 103 (18.8%) 113 (20.6%) 84 (15.3%) 40 (7.3%) 148 (26.9%) 90 (16.4%) 113 (20.5%) 32 (5.8%) 3 (0.5%) 42 (7.7%) Corrigan et al. Discussion When using the PROMIS-10 as an outcome measure, male sex was associated with bet- ter cognitive and physical domain scores post-stroke (Tables 2 and 3). This was comparable with the modi- fied Rankin scale, (Supplementary Table 3), where male sex was shown to be associated with better physical functioning. This is consistent with the literature; worse post-stroke outcomes have been previously reported across female patients [34–36]. One large scale study of post stroke outcomes including > 19,000 people reported that 3–6 months after stroke women are more likely to experience disability and worse quality of life [37]. Further, the PROMIS-10 aligns with clinical risk fac- tors of stroke. A pre-stroke diagnosis of diabetes is significantly associated with worse MH PROMIS-10 outcome scores following a stroke. This is mirrored within the association with other outcome measures; Table 3 Association of hypertension, TIA, previous stroke, diabetes, sex and age on PRO physical health domain. Both crude and adjusted results are reported with associated p-values and intervals. Statistically significant p-values are reported in bold. As a negative score is associated with worse outcome – a negative value indicates a factor resulting in worse outcome Table 3 Association of hypertension, TIA, previous stroke, diabetes, sex and age on PRO physical health domain. Both crude and adjusted results are reported with associated p-values and intervals. Statistically significant p-values are reported in bold. As a negative score is associated with worse outcome – a negative value indicates a factor resulting in worse outcome PROMIS 10 Physical health domain Mean differences MD P-value (95% CI) Adjusted MD P-value (95% CI) Pre-Stroke Hypertension −0.22 0.774 (−1.72, 1.28) 0.19 0.808 (− 1.34, 1.72) Previous TIA −1.65 0.111 (−3.67, 0.38) − 1.25 0.234 (− 3.31, 0.81) Previous stroke −3.17 0.003 (−5.27,-1.08) −3.05 0.005 (− 5.17, −0.93) Pre-Stroke Diabetes −1.64 0.071 (−3.44, 0.134) −1.48 0.107 (−3.27, 0.32) Sex (Male) 2.02 0.009 (0.50, 3.54) 2.09 0.008 (0.54, 3.65) Age 0.01 0.724 (−0.05, 0.07) 0.02 0.472 (−0.04, 0.09) PROMIS 10 Physical health domain Mean differences PROMIS 10 Physical health domain Mean differences Corrigan et al. BMC Neurology _#####################_ Corrigan et al. BMC Neurology _#####################_ Corrigan et al. BMC Neurology _#####################_ Page 6 of 7 This study offers a novel insight to the use of a PROMIS-10 to assess post-stroke quality of life. We demonstrate the patient outcomes of MH and PH and highlight association with clinical and demographic risk factors. Discussion While it must be noted that the PROMIS-10 is not a direct measure of morbidity, adverse scores in this instrument globally is likely to correlate strongly with high morbidity, thus this adds value for the use of PROMs in clinical practice for the feasible measurement of patient-reported outcomes. This is important to consider in the clinical context and may allow a way to understand physical and mental outcomes, that are significant to a patient, in a global manner. By using the PROMIS-10 cli- nicians may potentially enact a more sensitive measure to indicate morbidity, especially as perceived by the stroke survivor. In turn, this is likely to add understanding of individual patient needs and improve quality of care in conjunction with improvement in quality of life. Additional file 3: Supplementary Table 3. Association of hypertension, TIA, previous stroke, diabetes, sex and age on clinical outcome measure – GAD7 . Both crude and adjusted results are reported with associated p-values and intervals. Statistically significant p -values are reported in bold. As a higher score is associated with worse outcome – a positive value indicates a factor resulting in worse outcome. Additional file 3: Supplementary Table 3. Association of hypertension, Additional file 4: Supplementary Table 4. Association of hypertension, TIA, previous stroke, diabetes, sex and age on clinical outcome measure – PHQ9 . Both crude and adjusted results are reported with associated p-values and intervals. Statistically significant p -values are reported in bold. As a higher score is associated with worse outcome – a positive value indicates a factor resulting in worse outcome. Additional file 5: Supplementary Table 5. Association of hypertension, TIA, previous stroke, diabetes, sex and age on clinical outcome meas- ure – mRS Both crude and adjusted results are reported with associated p-values and intervals. Statistically significant p -values are reported in bold. As a higher score is associated with worse outcome – a positive value indicates a factor resulting in worse outcome. Additional file 6: Supplementary Table 6. Association of hypertension, TIA, previous stroke, diabetes, sex and age on clinical outcome measure – SF-MoCA . Both crude and adjusted results are reported with associated p-values and intervals. Statistically significant p -values are reported in bold. As a lower score is associated with worse outcome – a negative value indicates a factor resulting in worse outcome. Ethics approval and consent to participate f All participants provided the informed consent to participate for the study. All methods were conducted in accordance with relevant guidelines and regula- tions. Ethical approval was granted by the NHS Research Ethics Committee – Wales REC 3–18/WA/0299 - Health and Care Research Wales Support and Delivery Centre for all the sites. Authors’ contributions JH conceived the study and received funding for the study. AP, AS, BC, and JH designed the study. AP was the study coordinator supported by AS. AC and BC developed the statistical analysis plan. Data analysis and interpretation was carried out by AC and BC. The first draft of the manuscript was carried out by AC, BC and JH. All authors have drafted final manuscript. The authors read and approved the final manuscript. Discussion This was a large UK wide prospective multicentre study that assessed patient reported outcomes. The limitations to our study are that PROMIS-10 may only be useful in patients with mild to moderate impairment [28, 38]. While 146 of our subjects (26.9%) reported post-stroke aphasia, the degree and severity of impairment was not noted. Therfore, it is unclear if the functional impairment in communication may hinder the completion of the PROMIS-10. The use of patient reported tools in patients that struggle with communication is a current limitation of all self-reporting measures and should continue to be considered. Further, validation and standardisation of the 15-question measure, the PROMIS-10 and five additional reported questions, would be beneficial and allow a more specific measure of physical health, metal health and functional-capacity that is specific to a stroke population. Acknowledgements We would like to acknowledgements the More Precise study team – consist- ing of Amber Corrigan, Alexander Smith, Anna Pennington, Ben Carter and Jonathan Hewitt. We would further like to acknowledge the lead site investigations at each hos- pital site; Louise Coombe, Sarah Jones, Jayne Thomas, Sharon Storton, Richard Dewar, Lindsay Tarasconi, Benjamin Jelly, Walee Sayeed, Maureen Bartley, Kerry Smith, Sarah Dunne, Sarah Board, Katherine Ahlquist, Angela Kulendran, Ravneeta Singh, Asis Kumar, Deborah Ward, Rachel Teal, Ken Fotherby and Radim Licenik. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12883-022-02634-0. Conclusions PROMs such as the PROMIS-10 offer a feasible way to measure patient reported quality of life. Consent for publication Additional file 1: Supplementary Table 1. Reported Means, Standard deviation and missingness of measures. Patients were screened for PHQ-9 and GAD-7 with the PHQ-4 as per the methods. The total missing- ness of the PHQ-9 and GAD-7 are reported in regard to the population as a total, yet not all participants met the requirement for PHQ-9 or GAD-7. Declarations Future research is needed to compare a wider set of PROMs. Future clinical practice should investigate comorbidity of mental health in stroke survivors. References Turner A, Hambridge J, White J, Carter G, Clover K, Nelson L, et al. Depres- sion Screening in Stroke. Stroke. 2012;43(4):1000–5. 12. Turner A, Hambridge J, White J, Carter G, Clover K, Nelson L, et al. Depres- sion Screening in Stroke. Stroke. 2012;43(4):1000–5. 37. Carcel C, Wang X, Sandset E, Delcourt C, Arima H, Lindley R, et al. Sex differences in treatment and outcome after stroke. Neurology. 2019;93(24):e2170–80. 13. Spitzer R, Kroenke K, Williams J, Löwe B. A brief measure for assessing generalized anxiety disorder. Arch Intern Med. 2006;166(10):1092. 13. 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The Riks-stroke story: building a sustainable National Register for quality assessment of stroke care. Int J Stroke. 2010;6(2):99–108. 34. Niewada M, Kobayashi A, Sandercock P, Kamiński B, Członkowska A. Influ- ence of gender on baseline features and clinical outcomes among 17,370 patients with confirmed Ischaemic stroke in the international stroke trial. Neuroepidemiology. 2005;24(3):123–8. q y 9. Bevans M, Ross A, Cella D. Patient-reported outcomes measurement information system (PROMIS): efficient, standardized tools to measure self-reported health and quality of life. Nurs Outlook. 2014;62(5):339–45. gy 35. Gall S, Phan H, Madsen T, Reeves M, Rist P, Jimenez M, et al. Focused update of sex differences in patient reported outcome measures after stroke. Stroke. 2018;49(3):531–5. 10. Mai L, Oczkowski W, Mackenzie G, Shuster A, Wasielesky L, Franchetto A, et al. Screening for cognitive impairment in a stroke prevention clinic using the MoCA. Can J Neurol Sci. 2013;40(2):192–7. 36. Reeves M, Bushnell C, Howard G, Gargano J, Duncan P, Lynch G, et al. Sex differences in stroke: epidemiology, clinical presentation, medical care, and outcomes. Lancet Neurol. 2008;7(10):915–26. 11. Kroenke K, Spitzer R. The PHQ-9: a new depression diagnostic and sever- ity measure. Psychiatr Ann. 2002;32(9):509–15. 12. Competing interests p g JH is funded by the Stroke Research and Innovation Fund (SRIF) of the Stroke Implementation Group (SIG) [Welsh Government] - 03 ABUHB. AS is funded by the Stroke Association (SA PGF 18\100029 Stroke Association Post-Graduate Fellowship). Additional file 2: Supplementary Table 2. Proportion of responses to additional five questions, as per Martins et al, in the PRO. AC, BC, AP declare no funding associated with this study. AC, BC, AP, JH and AS report no other non-financial conflicts of interests. AC, BC, AP declare no funding associated with this study. AC, BC, AP, JH and AS report no other non-financial conflicts of interests. Corrigan et al. BMC Neurology _#####################_ Page 7 of 7 Corrigan et al. BMC Neurology _#####################_ 22. Lam K, Kwa V. Validity of the PROMIS-10 Global Health assessed by telephone and on paper in minor stroke and transient ischaemic attack in the Netherlands. BMJ Open. 2018;8(7):e019919. Received: 21 August 2021 Accepted: 24 February 2022 Received: 21 August 2021 Accepted: 24 February 2022 24. Alonso J, Bartlett S, Rose M, Aaronson N, Chaplin J, Efficace F, et al. The case for an international patient-reported outcomes measurement information system (PROMIS®) initiative. Health Qual Life Outcomes. 2013;11:210. Author details 1 1 Department of Biostatistics and Health Informatics, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, UK. 2 Division of Population Medicine, School of Medicine, Cardiff University, Cardiff, UK. 3 Aneurin Bevan University Health Board, Newport, South Wales, UK. 23. 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Zsuga J, Gesztelyi R, Kemeny-Beke A, Fekete K, Mihalka L, Adrienn S, et al. Different effect of hyperglycemia on stroke outcome in non-diabetic and diabetic patients — a cohort study. Neurol Res. 2012;34(1):72–9. 3. Donkor E. Stroke in the21stCentury: A snapshot of the burden, epidemi- ology, and quality of life. Stroke Res Treat. 2018;2018:1–10. 3. Donkor E. Stroke in the21stCentury: A snapshot of the burden, epidemi- ology, and quality of life. Stroke Res Treat. 2018;2018:1–10. 28. Philipp R, Lebherz L, Thomalla G, Härter M, Appelbohm H, Frese M, et al. Psychometric properties of a patient-reported outcome set in acute stroke patients. Brain Behav. 2021;11(8):e2249. 4. Martins S, Norrving B, Salinas J, Sprinkhuizen S, Schwamm L. Defining an international standard set of patient-centered outcome measures after stroke. J Neurol Sci. 2015;357:e394. 4. Martins S, Norrving B, Salinas J, Sprinkhuizen S, Schwamm L. Defining an international standard set of patient-centered outcome measures after stroke. J Neurol Sci. 2015;357:e394. 29. Owolabi M. Impact of stroke on health-related quality of life in diverse cultures: the Berlin-Ibadan multicenter international study. Health Qual Life Outcomes. 2011;9(1):81. 5. Reeves M, Lisabeth L, Williams L, Katzan I, Kapral M, Deutsch A, Prvu- Bettger J. Patient-reported outcome measures (PROMs) for acute stroke: rationale Methods and Future Directions. Stroke. 2018;49(6):1549–56. 5. Reeves M, Lisabeth L, Williams L, Katzan I, Kapral M, Deutsch A, Prvu- Bettger J. Patient-reported outcome measures (PROMs) for acute stroke: rationale Methods and Future Directions. Stroke. 2018;49(6):1549–56. 5. Reeves M, Lisabeth L, Williams L, Katzan I, Kapral M, Deutsch A, Prvu- Bettger J. Patient-reported outcome measures (PROMs) for acute stroke: rationale Methods and Future Directions. Stroke. 2018;49(6):1549–56. 6. Hays R, Spritzer K, Thompson W, Cella D. U.S. general population esti- mate for “excellent” to “poor” self-rated health item. J Gen Intern Med. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 16. Smith A, Bains N, Copeland L, Pennington A, Carter B, Hewitt J. Morbidity prevalence estimate at 6 months following a stroke: protocol for a cohort study. JMIR Res Protoc. 2020;9(6):e15851. 17. Hays R, Schalet B, Spritzer K, Cella D. Two-item PROMIS® global physical and mental health scales. J Patient Report Outcomes. 2017;1(1). 18. Carter B, Hayes C, Smith A, Pennington A, Price M, Pearson O, et al. A sin- gle patient reported outcome measure for acquired brain injury, multiple sclerosis & Parkinson’s disease. PLoS One. 2021;16(6):e0251484. 19. Bloch R. 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https://openalex.org/W2056165775	https://zenodo.org/records/1656622/files/article.pdf	English	null	A Portable Capillary Electrometer	Proceedings of the Physical Society of London	1,902	public-domain	3,369	Home Search Collections Journals About Contact us My IOPscience A Portable Capillary Electrometer A Portable Capillary Electrometer A Portable Capillary Electrometer This content has been downloaded from IOPscience. Please scroll down to see the full text. 1902 Proc. Phys. Soc. London 18 377 (http://iopscience.iop.org/1478-7814/18/1/334) View the table of contents for this issue, or go to the journal homepage for more Download details: Please note that terms and conditions apply. ON A PORTABLE CAPILLARY ELECTROMETER. 377 XXXIV. A Poytable Capillary Electgeometer. B y S. W. J. SMITH, M.A., Demonstrator in Physbs, Rsyal CoUegc o j Science, .London.* * Rend December 12, 1902, [Plate V.] [Plate V.] TITIS instrument is a modification of the form of capillary electrometer represented in the first figure, and consisting of two wide tubes joined across by a capillary tube which is cylindrical, and may be horizontal or may slope upwards y y y p p Fig. 1. Fig. 2 v- at any angle from b towards u. The apparatus contains mercury and sulphuric acid of about maximum conductivity distributed roughly as shown. A spring-key, like that repre- sented in the figure, is commonly used with the instrument, and keeps the platinum terminals PI and P, at the same potential unless the lever S is depressed. When the lever is depressed thc potentials of the terminals beconic El aud * Rend December 12, 1902, VOL XVIII 2 D Fig. 2 Fig. 1. Fig. 2 v- g at any angle from b towards u. The apparatus contains mercury and sulphuric acid of about maximum conductivity distributed roughly as shown. A spring-key, like that repre- sented in the figure, is commonly used with the instrument, and keeps the platinum terminals PI and P, at the same potential unless the lever S is depressed. When the lever is depressed thc potentials of the terminals beconic El aud * Rend December 12 1902 2 D VOL. XVIII. 378 MR. S. W. J. SMITH ON A EB, which inay, for ex~inple, be the potentials of two points in a potentiometer circuit. It is the function of the instru- ment to determine whether these potentials are the same or different. The nature of the modifications inade in the present electro- meter is shown in the second figure. To prevent evaporation of the sulphuric acid solution, without preventing free motion of the liquids within the apparatus, the wide tubes are closed at the top; but are joined across by another tube opening into thein as shown. With this arrangement the apparatus is made air-tight, and can, if desired, be inade air-free by exhaustion of the appa- ratus before sealing. It is obvious also that the appnratus can be upset without spilling of the liquid. The two liinbs are of equal size, and the capillary passes froin the middle of one to the middle of the other. The apparatus contains approximately enough mercury to completely fill one limb, and about half as much sulphuric acid solution. [Plate V.] By suitably adjusting the distribution of the inercury and the solution in the two liinbs, the apparatus can be arranged for use with the capillary tube either horizontal or tilted upwards at R Considerable angle. The maximum angle of tilt avnilnlole is increased by increasing the lengths of the limbs in com- parison with the length of the capillary. It is easy to construct : ~ n electrometer which can be used with the capillary at almost any inclination between horizontal and vertical. y The distribution of the mercury can be altered most easily by means of a cross-piece provided with a tap, as represented in the figure by dotted lines. When the tap is open there is free communication between the mercury in the two limbs, and the relative amount in each can be altered by tilting the apparatus. When the tap is closed the two quantities of mercury are insulated from each other. The addition of this arrangement tends to complicate the construction of the instrument, and, although it is very convenient in practice, it is not indispensable. Any desired changes in the distribu- tion of the mercury and the solution can usually be effected without much trouble, by means of the capillary and the upper cross-tube. To prevent the plittinnm wires forming the clectrodes being wdted by the acid <olntion, if the npparatm sho~ld acci- 379 PORTABLE CAPILLARY ELECTROMPTEX. dentally, or during transit, be laid on its side or turned up- side down, the lower ends of the tubes may be drawn out, :IS shown at P,' and P,' in the second figure, and slightly constricted above the ends of the platinum terminals. This precaution may Le dispensed with if the electrodes are formed by welding pieces of platinum foil, which are afterwards amalgamated, on to the ends of the wires PI and P,. The apparatus can then be turned upside down and shaken, so that the amalgamated foil becomes wetted by the acid, with- out any further ill-effect being produced than an occasional slight wandering of the zero of the instrument for a short time after it is righted again. g g Thus, if it is made of brass, the contacts frequently become unsatis- factory through surface tarnishing, and if, to avoid this, the hearing surfaces are made of platinnin the key soriletimes sh0~~7s pronounced thermoelectric effects. [Plate V.] Hence inensuremeiits can be made with the instrument even if the zero is altering fairly rapidly. With a key of this kind it is also obvious that the observa- tion of the meniscus through the inicroscope can receive a very near approach to un- divided attention. Cornmutators and keys which ninlie a set of connexioiis in a prescribed order cnii be con- structed on the same principlc th ti B b Fig. 3. y g y g - . between the t u o extreme plati- num wires, the change of potential at Pi from E, to E, and conversely, can be made almost instantaneously. Hence inensuremeiits can be made with the instrument even if the zero is altering fairly rapidly. With a key of this kind it is also obvious that the observa- tion of the meniscus through the inicroscope can receive a very near approach to un- divided attention. Cornmutators and keys which ninlie a set of connexioiis in a prescribed order cnii be con- structed on the same principlc as the pneumatic Bey above described. The third figure represents a combined cominu- tator and electrometer key. Fig. 3. The U-tube to the left is the key already described, aiid the double U-tube to the right is the commutator. The positions of the different wires are so arrtinged with respect to the levels of the mercury, that it requires greater pressure to change the contacts in the double U-tube, than in the tube to the left. The first effect of pressure is to throw the eleciro- meter into the potentiometer or other circuit, coiinecting PI Nith El and P2 with E2. On increasing the pressure, the connexions in the double U-tube arc reversed and PI is coii- Fig. 3. Fig. 3. Fig. 3. Cornmutators and keys which ninlie a set of connexioiis in a prescribed order cnii be con- structed on the same principlc as the pneumatic Bey above described. The third figure represents a combined cominu- tator and electrometer key. The U-tube to the left is the key already described, aiid the double U-tube to the right is the commutator. The positions of the different wires are so arrtinged with respect to the levels of the mercury, that it requires greater pressure to change the contacts in the double U-tube, than in the tube to the left. [Plate V.] Further-and this is a point of some importance in a portable instrument-the key cannot conveniently be fastened on to the same stand as tho rest of the instrument, for, unless the stand and the snpport on which it rests are very rigid, the pressure iiecessary to depress the spring produces sufficient movement of tho meniscus, by change in the inclination of the capillary tube dnring the act of depression, to render the detection of niinutc changes of surface-tension impossible. The usual spring-key has several disadvantages. g p The mercury-key, represented in the second figure, is free froni these disadvantages. It consists, as shown, of a U-tube closed at one end and cominunicating at the other with a pneumatic-pressure ball a i d con tainiag niercury in the bend. Three platinum wires are fused into the tube and connected as shown. It is obvious that the same change of contacts is produced by squeezing the ball B, as by depressing tho lever S in the spring-key. The mercury in this key takes the place of the lever in the spring-key, and the two different contacts between it and El and E, reapwtivcly are hcbre quite definite and practically independent of the amount of pressure exerted upon the ball B. Further, the contacts are not exposed directly to the laboratory atmosphere. The thermoelectric effects are very small since the changing contacts take place between platinum and mercury which are almost identical thermoel(~ct~ic:i11~. The warmth com- 2 D 2 380 MR. S. TV. J. SMITII ON A municated to the lrey from thc hand of the operator cnn bc neglected, and the heat produced by the compression of the air in the lrey need only be very small. The key can Le fixed to the same stand as the rest of the apparatus, for even if the pressure which changes the contacts is npplicd as suddenly as possible the maximum vertical pressure upon the stand is only a few grams, whereas in the case of the ordinary spring-key it inay be 500 grains or inorc. By adjusting the length of the mercury column in thc kcy so that it is only slightly leas than the length of tlic U-tube - . between the t u o extreme plati- num wires, the change of potential at Pi from E, to E, and conversely, can be made almost instantaneously. * Since this paper NVILS read I have fbuud that mercury keys (similar in principle to those referred to above) in which tlie tlLermoelectric effects are redneed to a minimum^ nre clcscribcd by Ihinerlingh Onnr,a, Legdm C~mmunicnti~s, NO. 27, p. 37 , 1896. [Plate V.] The first effect of pressure is to throw the eleciro- meter into the potentiometer or other circuit, coiinecting PI Nith El and P2 with E2. On increasing the pressure, the connexions in the double U-tube arc reversed and PI is coii- 351 PORTABLE CAPILLARP ELECTROMETER. iioctecl with E, ancl P, with E,. Hence, when the contacts c h n g e in tho commutator, we g d a motion of the electrometer meniscus corresponding to approxiinately twice the difference of potential between E, and E2, and so, in this way, the sensitiveness of the instrument is doubled. While it is clear that to get the minimum thermoelectric effect in mercury-platinum keys, it would be necessary to use a method of changing the contacts in which compression of air in the key does not take place, yct the thermoelectric electromotive forces which occur in the pneumatic keys described are much too small to produce an observable effect upon the readings of the most sensitive capillary electro- meter . The sensitiveness of the electrometer, using the simple key first descrihcd, is such that when the diameter of the wide tubcs is about 1 cm. and the diameter of the capillary is about 1 min., a movement of the meniscus perceptible with certainty in a microscope magnifying 100 times is produced by a potential-difference equal to .0001 volt. The actual extent of the movement is somewhat variable, and amounts usually to about 001 mm. The following numbers, obtained with apparatus previously described (Phil. Trans. A. 189’3, vol. cxciii. p. 63), show the effect of comparatively large polarizing electromotive forces upon the surface-tension between mercury and sulphuric acid solution of density 1.17 :- 1.17 :- 1.17 :- EMF. Volts. Surface-Tension. 0 Y 0.0202 1.021 y 0.0404 1.040 y 00807 1.080 y 0.0605 1,059 y 01009 1.097 y y is the ‘‘ naturai” surface-tension between the mercury and the solution. Its approximate value is 300 ergs per sq. em. Judging from these numbors, it would seem that the surface- teiision is altered by about one part in 10,000 by a polarizing y is the ‘‘ naturai” surface-tension between the mercury and the solution. Its approximate value is 300 ergs per sq. em. Judging from these numbors, it would seem that the surface- teiision is altered by about one part in 10,000 by a polarizing 382 BlR. S. W. J. SMITH ON h E.M.F. [Plate V.] of -0001 volt. I n the case in which tho capillary- tube is horizontal and the wide tubes are vertical, the relation between the motion of the meniscus ax and the corresponding change in surface-tension 6y is expressed approximately by a9p g 6x=Ac6y if we assume that the capillary and wide tubes are of uniform cross-section-a being the' area of cross-section of the capil- lary, c its circumference, A the area of cross-section of the wide tubes, and p the sum of the densities of the mercury and the solution. From this formula it would appear possible to produce il much larger movement of the meniscus than -01 Rim. by means of a potential-difference of ,0001 volt by using a very narrow capillary, especially if the cross- section of the capillary were elliptical instead of circular. It is found in practice, however, that the motion of the meniscus under minute polarizing forces is controlled very largely by stickiness and accompanying changes in the angle of contact between the liquids and the glass, and by variation in the cross-section of the tube at the place where the meniscus rests. The Sensitiveness of the instru- nient to small electromotive forces is practically as great when the diameter of the capillary is 1 min. as when it is very much less. A very narrow capillary is less easy to manipulate than a comparatively wide one (about 1 mm. in diameter) and is only advantageous when it is desirable that the capacity oE the instrument should be as small as possible, or that its action should be as rapid as possible. In the latter case the length of the capillary should also be small. g p y I f the capillary-tube, instead of being horizontal, is inclined sat an angle 8 to the horizontal, the equation given above becomes ag{(pl + p&/A . COS 8 + (pl -pz) sin 0) 6z= c ay, in which pl is the density of the mercury, and pa that of the solution. [Plate V.] From this equation it follows : (1) that if 8 is posi- tive (mercury thread sloping upwards towards the meniscus) the sensitiveness is not increased appreciably by making A very large in comparison with a, unless 6 is very small ; and (2) that if 8 is negative (meniscus at lowest part of thread) the mercury becomes unstable when 8 i 5 numerically greater than tan-' (pl + pz)u/(p, - p z ) A, i. e. when 8 is numerically PORTABLE CAPILLART ELECTROMETER. 383 greater than ufA approximntely, since in order that the polarization inay be practically confined to the capillary electrode, the ratio u/A must be small. It also follou~ that if it were proposed to design an instrument which should have the utmost sensitiveness possible, attention would have to be paid to the straightness of the capillary as well as to the uniformity of its cross-section. I t is not necessary, however, to take tho precautions here indicated in order to obtain the sensitiveness already quoted j it may be obtained with almost any capillary-tube chosen at random, and with 0 having such a positive value that the restoring force for a small displacement is considerable enough to render the instruincnt cnsy to work with. The sensitiveness of 0001 volt, which is obtainable with- out any difficulty if the mercury is clean, is sufficient for a great many measuroments in which the electrometer can be einployed, and for these the electrometer (which for the pur- pose in qucstion is really a surface-tension galvanometer) is mow conveiiieiit than an ordinary galvanometer with a sus- pended magnetic system. The electrometer is much more easily set up than an ordinary galvanometer. There is no suspension, no lamp and scale, and, practically, no levelling ; but it is advisable when working with the instrument that the potential of El should never be more than a few tenths of n volt less, or more than about a volt greater, than the potential of E2. These conditions of working are, in general, not difficult to satisfy. If the potential-differences applied exceed either of the limits above mentioned, it is sninetimes necessary to run some of the mercury through the capillary tube in order to get the instruineiit again into its best working orclcr. RABCAB=VA-VB+EAB. . . . . (2) * Read January 33, 1903. [Plate V.] The capillary electrometer is already used almost universally in physico-chemical laboratories, and the modi- fications herein suggested may, perhaps, have the effect of slightly extoilcling the sphere of its usefulness. The accoin- panying Plate V., taken from a photograph, represciits one forin of the instrument clescribcd. Its size is about two-fifths of that of the actual instrument. Tho arrangeincat of the different parts will be obvious from tlic doscription alrencly given. The illumination of the end of tho mercury-thread is: effected by means of a concave mirror a t t d ~ e d to the base of the instrument. 384 PROF. L. R. WILBEHFORCE : ELEMENTARY and the application of Ohm's Inw gives us IL series whose Q ( ) type is DISCUSSIO Dr. R. T. GLAZEBROOK congratulated the author, and said that workers with the capillary electrometer would appre- ciate the improvements introduced. He said that the author had given the minimum E.M.F. which the instrument could detect, and asked what was the maximum E.M.F. which could be safely applied. H e referred to the advantages of the mercury keys exhibited, and remarked that, by their use, many difficulties were obviated. y Dr. LEHFELDT said the mercury keys were improvements on those used by Prof. Onnes. In these keys the contacts were chmged by the motion of mercury brought about by tilting. The tubes containing the mercury were exhausted to prevent oxid a t' ion. p Mr. S I V I I ~ , in reply to Dr. Glazebrook, said that, roughly, the maxiinum E.M.F. should not exceed half a volt. xxxv. Note on an Elementary Treatment of Conducting B y I;. R. WILBERFORCIE, JLA., Pvofessor of Networks. Physics at University College, Liverpool . IT may be worth while to point out that the well-known reciprocal relations between the parts of a conducting net- work can be readily established without an appeal to the properties of determinants. p p Let A, B, C, D,. . .,be a number of points connected by conductors AB, AC, AD,, . ,, BC, BD, . , ., CD, . . ,, of resist- ances RAD, ..., and suppose that currents &A, , .., are led into the network at the points A, . . ., from without, subject to the condition &A + QB + . . . =0, and that internal electro- motive forces, EBB, . . ., act in the conductors in the directions AB,. . .. Let the currents in the conductors bo CAB, . . ., and let the potentials at A, . . ., be VA, . . .. The fact that there is no continuous accumulation of electricity at any point gives us a series of equations whose type is :- q yp QA= CAB + CAC + . . . . . . . . (1) q yp QA= CAB + CAC + . . . . . . . . (1) and the application of Ohm's Inw gives us IL series whose ( ) type is (2) i ’ , ~ l l l ~ . i~ld.l,$. “%V. I-,4/ .y j - / J J , PI. 1,.
https://openalex.org/W4313361335	https://www.mdpi.com/2075-5309/13/1/89/pdf?version=1672383146	English	null	Nonlinear Stochastic Seismic Response Analysis of Three-Dimensional Reinforced Concrete Piles	Buildings	2,022	cc-by	15,108	Article Nonlinear Stochastic Seismic Response Analysis of Three-Dimensional Reinforced Concrete Piles Hongqiang Hu 1,2,3,4, Gang Gan 2,3,, Yangjuan Bao 5,, Xiaopeng Guo 6, Min Xiong 7, Xu Han 8, Kepeng Chen 2,3,4 , Linyong Cui 7 and Lin Wang 7 1 College of Engineering, Zhejiang University City College, Hangzhou 310015, China 2 The Architectural Design & Research Institute of Zhejiang University Corporation Limited, Hangzhou 310028, China g 3 Center for Balance Architecture, Zhejiang University, Hangzhou 310028, China 3 Center for Balance Architecture, Zhejiang University, Hangzhou 310028, China 4 College of Civil Engineering and Architecture, Zhejiang University, Hangzhou 310058, China Center for Balance Architecture, Zhejiang University, Hangzhou 310028, China 4 College of Civil Engineering and Architecture, Zhejiang University, Hangzhou 310058, China 5 School of Civil Engineering and Architecture, Zhejiang University of Science & Technology, Hangzhou 310023, China 5 School of Civil Engineering and Architecture, Zhejiang University of Science & Technology, Hangzhou 310023, China 6 Xi’an Center of Geological Survey, China Geological Survey, Xi’an 710054, China 7 Department of Geotechnical Engineering College of Civil Engineering Tongji University 6 Xi’an Center of Geological Survey, China Geological Survey, Xi’an 710054, China 6 Xi’an Center of Geological Survey, China Geological Survey, Xi’an 710054, China 7 Department of Geotechnical Engineering, College of Civil Engineering, Tongji University, Shanghai 200092, China 8 8 China Railway Siyuan Survey and Design Group Corporation Limited, Wuhan 430063, China 8 China Railway Siyuan Survey and Design Group Corporation Limited, Wuhan 430063, China * Correspondence: gang@zuadr.com (G.G.); yangjuanbao@zust.edu.cn (Y.B.) * Correspondence: gang@zuadr.com (G.G.); yangjuanbao@zust.edu.cn (Y.B.) Abstract: A reliable assessment and design of engineering structures requires appropriate estimation and consideration of different sources of uncertainty. The randomness of seismic ground motion is one major uncertainty that needs to be considered from the perspective of performance-based earth- quake engineering. To properly account for this uncertainty and its corresponding effect on pile foundations, a stochastic seismic response analytical framework based on a probability density evolution method and a stochastic ground motion model is proposed for the nonlinear stochastic seismic response analysis of pile foundations. A three-dimensional ﬁnite element model of a re- inforced concrete pile with a large diameter embedded in the soil foundation is ﬁrstly established and calibrated using the full-scale lateral load test results from the literature, in which the nonlinear behavior of soil, the soil–pile interaction, the concrete damaged plasticity, and the steel yielding of the pile are properly modeled. Article Nonlinear Stochastic Seismic Response Analysis of Three-Dimensional Reinforced Concrete Piles Then, the calibrated three-dimensional numerical model is em- ployed as an illustrative example for the stochastic seismic response analysis using the proposed framework. The mean, standard deviation, and probability density function of pile settlement and the dynamic reliabilities of the pile concerning various performance requirements are obtained in the present study. The settlements of the pile show great variability under the excitation of various stochastic ground motions, and the maximum mean value of the pile settlements is about 8 mm. The changing shape of the settlement probability density functions in every moment demonstrates that it is unreasonable to use assumed probabilistic distribution models to characterize the seismic responses of pile foundations during a seismic reliability analysis. Based on the proposed method, it is found that the dynamic reliabilities of the selected reinforced concrete pile concerning four different performance levels are 0, 0.1293, 0.8247, and 1, respectively. The proposed stochastic seismic response analytical method in the present study can provide a proper and comprehensive way to quantify various uncertainties and their corresponding effects on the seismic performance of pile foundations. It can also be used to estimate the actual reliability level of pile foundations that are designed by the current codes when they are subjected to seismic loads. buildings buildings buildings buildings Keywords: reinforced concrete pile; soil-pile interaction; stochastic ground motion; nonlinear stochas- tic dynamic analysis; three-dimensional ﬁnite element model Citation: Hu, H.; Gan, G.; Bao, Y.; Guo, X.; Xiong, M.; Han, X.; Chen, K.; Cui, L.; Wang, L. Nonlinear Stochastic Seismic Response Analysis of Three-Dimensional Reinforced Concrete Piles. Buildings 2023, 13, 89. https://doi.org/10.3390/ buildings13010089 Citation: Hu, H.; Gan, G.; Bao, Y.; Guo, X.; Xiong, M.; Han, X.; Chen, K.; Cui, L.; Wang, L. Nonlinear Stochastic Seismic Response Analysis of Three-Dimensional Reinforced Concrete Piles. Buildings 2023, 13, 89. https://doi.org/10.3390/ buildings13010089 Received: 27 October 2022 Revised: 27 November 2022 Accepted: 2 December 2022 Published: 30 December 2022 Received: 27 October 2022 Revised: 27 November 2022 Accepted: 2 December 2022 Published: 30 December 2022 1. Introduction Pile foundation has been widely used in the design of various kinds of engineering structures and infrastructures, and the performance assessment of pile foundations is consequently of great signiﬁcance [1]. However, much of the literature has shown that the bearing capacity and performance of piles are greatly affected by various sources of uncertainty (e.g., soil properties, structural parameters, geometrical parameters, and external loading conditions), and it is better to resort to probability theory and reliability methods for the quantiﬁcation of these effects. In recent decades, the bearing capacity and the response of pile foundations under the effects of various sources of uncertainty have been well investigated. Various probabilistic methods, such as approximate moment methods [2–6], response surface methods [3,7–9], and Monte Carlo simulation [5,10–12], have been used to combine different empirical methods, analytical methods, and numerical models of pile foundations to analyze the probabilistic characteristics of piles under axially/laterally loads. The uncertainty of soil properties is the main factor that has been considered in these studies by modeling soil properties as random variables [3,7–9] and random ﬁelds [4,10,11,13,14]. The uncertainties of the pile parameters and loading conditions (horizontal and axial loads) have also been taken into account by including them as random variables (e.g., [3,6,15]). For instance, Huh et al. [3] performed the probabilistic reliability estimation of an axially loaded pile by integrating the response surface method, the ﬁnite difference method, and the ﬁrst-order reliability method, in which the uncertainties of the load condition, the pile material, the sectional property of the pile, and the soil properties were explicitly considered. Haldar and Babu [10] used the Monte Carlo simulation approach combined with the ﬁnite difference program (FLAC) to analyze the effect of soil spatial variability on the responses of a laterally loaded pile in undrained clay. Kozubal et al. [8] evaluated the reliability of a pile under lateral loads by varying soil properties, in which linear elastic soil constitutive model and a random variable model were used. Zhang et al. [11,12] studied the effect of spatial variability of undrained shear strength of soil on the bearing capacity of piles under horizontal loading. In most of previous studies, the pile is usually regarded as the beam placed vertically in the soil foundation, and the soil around the pile is usually represented by a series of springs and the soil–structure interaction is investigated by analyzing the load–displacement curves. 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/). Keywords: reinforced concrete pile; soil-pile interaction; stochastic ground motion; nonlinear stochas- tic dynamic analysis; three-dimensional ﬁnite element model https://www.mdpi.com/journal/buildings Buildings 2023, 13, 89. https://doi.org/10.3390/buildings13010089 Buildings 2023, 13, 89 2 of 19 1. Introduction However, regarding the reliability analysis of pile foundations, most studies only focus on the static performance of axially/laterally loaded piles. The seismic reliability analysis of pile foundations is still limited. For instance, Liu and Zhang [16] employed the random field finite element method to examine the seismic responses of a pile–raft system by considering the randomness in the maximum shear modulus of soils using three-dimensional random fields. Only one input ground motion was input in their study, and the statistical results were analyzed using the Monte Carlo simulation. Esposito et al. [17] assessed and compared the reliability of single vertical pile foundations subjected to various seismic loads. They also investigated if the seismic geotechnical design in Canadian code achieved similar reliability levels as those aimed at by other static designs. However, in the study of Esposito et al. [17], only ten realizations of the site were produced in the seismic reliability analysis of axially loaded piles to represent the soil variability at the site. On the one hand, in most of the existing studies, the reliability analysis of pile founda- tions is conducted at a static level. However, in the seismic fortiﬁcation areas, the reliability of piles needs to be well understood and investigated under seismic loading condition. Under the seismic condition, there is a serious coupling effect of material nonlinearity with randomness, which signiﬁcantly improves the difﬁculty of calculating the seismic reliability of pile. Furthermore, most of the existing reliability analysis of pile founda- tions only considered the uncertainty of soil parameters. The randomness of earthquake loading is another important factor that cannot been neglected in the seismic performance analysis of pile foundations because lots of studies have revealed that the main source of uncertainty and the crucial uncertain factor in the seismic performance evaluation of Buildings 2023, 13, 89 3 of 19 3 of 19 geotechnical structures is the randomness of ground motion [17–24]. For example, in the study of Esposito et al. [17], only ten realizations of the site were produced in the seismic reliability analysis of axially loaded vertical piles to represent the soil variability at the site, and they thought it was sufﬁcient because the ground motion variability had the largest impact on the site response and the pile performance. 1. Introduction As a consequence, the understanding of the effect of ground motion randomness on the seismic performance of pile foundations is far behind the understanding of the effect of soil variability on the static and dynamic behaviors of pile foundations. Additionally, among the studies concerning the reliability analysis of pile foundations, the pile is always treated as a structural beam or entity element with elastic properties, without considering the physical and mechanical characteristics of concrete and rebar. Under a huge lateral load and an earthquake load, plasticity and damage behaviors (e.g., plastic strains, concrete cracking, and steel yielding) will develop in this system according to post-earthquake investigations, experiments, and numerical simulations (e.g., [25–31]). Neglecting such effect might not acquire the realistic results with regard to the reliability analysis of pile foundations. g y y p On the other hand, the approximate moment methods and the response surface methods are usually used in the static reliability analysis of piles, which need to obtain the limit state functions of piles. It can generally obtain satisfactory and reliable results in static cases. However, under a seismic condition, the coupling effect of randomness and soil nonlinearity will lead to a complex analysis of structural seismic performance and it is quite difﬁcult to obtain the explicit expression of the limit functions of pile foundations. Although the Monte Carlo simulation avoids the inﬂuence of nonlinear limit state equation as long as there have been sufﬁcient runs of the simulation, it has low computational efﬁciency and has the problem of random convergence. The calculation burden will increase sharply if it needs to be combined with the dynamic time history analysis for the seismic dynamic problem. As a consequence, it is necessary to employ a more efﬁcient and reliable stochastic dynamic analytical method to investigate the stochastic seismic responses and the seismic reliability of this kind of complicated soil–structure system. In the present study, to avoid the deﬁciencies mentioned above for the dynamic reliability analysis of pile foundations, an efﬁcient stochastic dynamic method, namely the probability density evolution method (PDEM), is proposed for the nonlinear stochastic seismic response analysis of pile foundations. Firstly, the proposed framework for the stochastic dynamic analysis of pile foundations, including the theory of PDEM and the quantiﬁcation of the uncertainty of seismic ground motions, is introduced. 1. Introduction Secondly, a three-dimensional ﬁnite element model of a reinforced concrete (RC) pile embedded in sandy soils under horizontal loading is modeled to calibrate the model using the results from a full-scale ﬁeld load test. This calibrated model is then extended to earthquake scenarios for the stochastic dynamic analysis in the present study. Finally, the stochastic seismic responses under the effect of earthquake randomness, including probabilistic settlement of pile and dynamic reliability, are presented. 2. Framework of Stochastic Dynamic Response Analysis 2.1. Probability Density Evolution Method 2. Framework of Stochastic Dynamic Response Analysis 2.1. Probability Density Evolution Method A pile foundation with various sources of uncertainty under the excitation of uncertain load can be regard as a multiple-degree-of-freedom stochastic dynamical system according to stochastic dynamics, and its balance equation can be written as follows: M(Θ .. )Z + D(Θ) . Z + R(Θ)Z = F(Θ, t) (1) M(Θ .. )Z + D(Θ) . Z + R(Θ)Z = F(Θ, t) (1) where M and D are the mass and the damping matrix, respectively; R is the linear or nonlinear restoring force vector; F is the random excitation vector; and .. Z, . Z and Z are the seismic accel- eration, velocity, and displacement vectors of the system, respectively. Θ is the parameter that reflects the uncertainties in the system and external excitation. It is a multi-dimensional ran- dom parameter vector with joint probability density function of ρΘ(θ), and θ = (θ1, θ2, . . . θn,) where M and D are the mass and the damping matrix, respectively; R is the linear or nonlinear restoring force vector; F is the random excitation vector; and .. Z, . Z and Z are the seismic accel- eration, velocity, and displacement vectors of the system, respectively. Θ is the parameter that reflects the uncertainties in the system and external excitation. It is a multi-dimensional ran- dom parameter vector with joint probability density function of ρΘ(θ), and θ = (θ1, θ2, . . . θn,) Buildings 2023, 13, 89 4 of 19 is the sample realization of the random parameter vector Θ. Only the uncertainty of seismic load was considered in the present study; thus, the Θ only denotes the randomness of input ground motions, and it can be used to combine with stochastic ground motion model in the following section to quantify the randomness of earthquake. g q y q The solution for the above equation Z(t) exists and depends on the basic random variable Θ, time, and initial condition. The most reﬁned stochastic seismic response analysis of a stochastic system is from the perspective of probability density function (PDF). According to the theory of description of random events, which is based on the principle of probability conservation, the generalized probability density evolution (GPDE) equation that governs the joint PDF of pile foundations can be written as follows [32–36]: ∂ρZΘ(z, θ, t) ∂t + ∑ m q=1 . Zq(θ, t)∂ρZΘ(z, θ, t) ∂zq = 0 (2) (2) where ∂ρZΘ(z, θ, t) is the joint PDF of Z and Θ at time t; z is the realization of Z; and . Zq(θ, t) is the velocity in the condition of {Θ = θ} and can also be expressed as . Zq(θ, t) = ∂Z(θ,t) ∂t . M(Θ .. )Z + D(Θ) . Z + R(Θ)Z = F(Θ, t) The initial condition of the GPDE equation is as follows: ρZΘ(z, θ, t)\|t=0 = δ(z −z0)ρZΘ(θ) (3) (3) where δ denotes Dirac’s delta function, and z0 is the initial value of Z. If only one seismic response of pile foundations is of interest in each stochastic analysis, the dimension of Equation (2) is reduced to a one-dimensional GPDE equation (q = 1): ∂ρZΘ(z, θ, t) ∂t + . Z(θ, t)∂ρZΘ(z, θ, t) ∂z = 0 (4) (4) By solving the equation, the instantaneous PDF ρZ(z, t) of seismic response Z(t) can be acquired as follows: By solving the equation, the instantaneous PDF ρZ(z, t) of seismic response Z(t) can be acquired as follows: ρZ(z, t) = Z ΩΘ ρZΘ(z, θ, t)dθ (5) (5) where ΩΘ is the distribution space of Θ. However, the closed-form solution of Equations (2) and (4) is only available for simple cases, for instance, the single-degree- of-freedom system. For the most complicated systems, the closed-form solution of the GPDE equation is difﬁcult to obtain because it is difﬁcult and sometimes even impossible to acquire the analytical solution for the seismic response of multidimensional nonlinear system under seismic excitation [36–38]. A numerical strategy, such as the ﬁnite difference method [34,36], is consequently usually employed to solve the GPDE equation to obtain the stochastic seismic results of the system. More information about the solving process can be found in Li and Chen [36]. where ΩΘ is the distribution space of Θ. However, the closed-form solution of Equations (2) and (4) is only available for simple cases, for instance, the single-degree- of-freedom system. For the most complicated systems, the closed-form solution of the GPDE equation is difﬁcult to obtain because it is difﬁcult and sometimes even impossible to acquire the analytical solution for the seismic response of multidimensional nonlinear system under seismic excitation [36–38]. A numerical strategy, such as the ﬁnite difference method [34,36], is consequently usually employed to solve the GPDE equation to obtain the stochastic seismic results of the system. More information about the solving process can be found in Li and Chen [36]. By combining the equivalent extreme-value event and the first-passage probability [39,40] with the GPDE equation, the PDF and cumulative distribution function (CDF) of the extreme value of the seismic responses of a system can also be obtained and, thus, can be used to analyze the seismic reliability of pile foundations. 2.2. Quantiﬁcation of Randomness of Input Ground Motions The randomness of seismic load is always quantiﬁed by a set of ground motions selected from an earthquake database or generated by stochastic ground motion models. However, it is difﬁcult to select a suite of seismic records that can meet the requirement of the same local site conditions for stochastic analysis. As a consequence, stochastic seismic ground motion models are often used to generate a suite of artiﬁcial seismic ground motions, that can easily meet some requirements of site conditions, for the stochastic seismic analysis of various structural systems. In the present study, a random-function-based spectral representation method [34,41,42], combined with a design spectrum in Chinese seismic code, was used to generate 144 fully non-stationary synthetic motions for the stochastic Buildings 2023, 13, 89 5 of 19 seismic response analysis of the pile foundation in the present study. Using this method, the fully non-stationary stochastic ground motions G(t) can be modeled as follows: seismic response analysis of the pile foundation in the present study. Using this method, the fully non-stationary stochastic ground motions G(t) can be modeled as follows: G(t) = ∑N k=1 p 2SG(t, ωk)∆ω ∗[cos(ωkt)Xk + sin(ωkt)Yk] (6) (6) where SG is the evolutionary power spectral density function; ωk = k∆ω and ∆ω is the frequency interval; t is the time; and {Xk, Yk}(k = 1, 2, 3 . . . N) is a set of standard orthogonal random variable that can be deﬁned as the function of basic random variables based on the theory of stochastic function [41]: Xk = √ 2 cos(kθ + π/4) Yk = √ 2 cos(kθ + π/4) k = 1, 2, · · · , N (7) (7) where θ is a basic random variable satisfying the uniform distribution in the interval [−π, π]. To account for the nonstationary characteristics of amplitude and frequency, the evolu- tionary power spectral density function needs to add an intensity modulation function: ( f (t) = t c exp 1 −t c d SG(t, ω) = f (t) ∗Sg(t, ω) (8) (8) where Sg(t, ω) is the power spectral density function that has not considered the nonsta- tionary characteristics of amplitude and frequency; f(t) is the intensity modulation function; c is the time of peak value; and d is the shape control index of f(t). 2.2. Quantiﬁcation of Randomness of Input Ground Motions If the error ε is larger than 0.05, the following response spectrum correction formula can be used to perform a series of iteration until the error ε is less than 0.05 [41,42]: 𝜀= ቚ ௌഀ(ఠ,క) ௌೖ(ఠ,క) ௌഀ(ఠ,క) ቚ (12) where 𝑆ఈ(𝜔, 𝜉) is the standard response spectrum and 𝑆௞(𝜔, 𝜉) is the average response spectrum of generated stochastic ground motions, respectively. The error ratio 𝜀 is con- trolled to be less than 0.05 during the generation of stochastic ground motions [41,42]. If the error 𝜀 is larger than 0.05, the following response spectrum correction formula can be used to perform a series of iteration until the error 𝜀is less than 0 05 [41 42]: where Sα(ω, ξ) is the standard response spectrum and Sk(ω, ξ) is the average response spectrum of generated stochastic ground motions, respectively. The error ratio ε is con- trolled to be less than 0.05 during the generation of stochastic ground motions [41,42]. If the error ε is larger than 0.05, the following response spectrum correction formula can be used to perform a series of iteration until the error ε is less than 0.05 [41,42]: 𝜀= ቚ ௌഀ(ఠ,క) ௌೖ(ఠ,క) ௌഀ(ఠ,క) ቚ (12) where 𝑆ఈ(𝜔, 𝜉) is the standard response spectrum and 𝑆௞(𝜔, 𝜉) is the average response spectrum of generated stochastic ground motions, respectively. The error ratio 𝜀 is con- trolled to be less than 0.05 during the generation of stochastic ground motions [41,42]. If the error 𝜀 is larger than 0.05, the following response spectrum correction formula can be d t f i f it ti til th i l th 0 05 [41 42] S(i+1) g (ω, ξ) = S(i) g (ω, ξ) ∗S2 α(ω, ξ)/S2 k(ω, ξ) (13) [ ] 𝑆௚ (௜ାଵ)(𝜔, 𝜉) = 𝑆௚ (௜)(𝜔, 𝜉) ∗𝑆ఈ ଶ(𝜔, 𝜉)/𝑆௞ ଶ(𝜔, 𝜉) (13) (13) With the above process, a set of fully non-stationary stochastic ground motions that are consistent with the target seismic responses spectrum in the Chinese seismic code can be generated to quantify the randomness of earthquake. To reﬂect local site effect on the seismic ground motions, the seismic response spectrum of a site type similar to the site where the adopted RC pile is located is used to acquire the power spectral density function for the generation of seismic ground motions, because the seismic response spectrum (Equation (10)) has already considered site effect in the Chinese seismic code. 2.2. Quantiﬁcation of Randomness of Input Ground Motions To generate a set of fully non-stationary random ground motions consistent with the target seismic response spectrum, the power spectral density function that is transformed from the response spectrum in the Chinese seismic code [43] is utilized based on the following transforming equation [44]: Sg(t, ω) = −ξ πω S2 α(ω, ξ) ln −π ωb ln(1 −p) (9) (9) where Sα(ω, ξ) is the absolute acceleration response spectrum with natural frequency ω = 2π/T0 and damping ratio ξ; T0 is the natural vibration period of single-degree-of- freedom structure; b is the duration of earthquake; and p is the exceeding probability of response spectrum. p p In the Chinese seismic code [43], the seismic response spectrum can be determined as follows: Sα(ω, ξ) = Sα(T0, ξ) =          [0.45 + (10η2 −4.5)T]αmax ∗g 0 < T ≤0.1 η2αmax ∗g 0.1 < T ≤Tg g ∗ Tg T γ η2αmax ∗g Tg < T ≤5Tg η2 · 0.2γ −η1 T −5Tg αmax ∗g 5Tg < T (10) (10) where T is the natural period of system; g is the gravitational acceleration; Tg is the natural period of site; αmax is the maximum value of seismic inﬂuence coefﬁcient; η2 is the damping adjustment coefﬁcient; η1 is the slope adjustment coefﬁcient; and γ is the attenuation index. η2, η1 and γ can be determined as follows:        η2 = 1 + 0.05−ξ 0.08+1.6ξ η1 = 0.02 + 0.05−ξ 4+32ξ γ = 0.9 + 0.05−ξ 0.3+6ξ (11) (11) The error ratio between the standard response spectrum and the average response spectrum of generated stochastic ground motions can be defined as the following fitting error: ε = Sα(ω, ξ) −Sk(ω, ξ) Sα(ω, ξ) (12) (12) Buildings 2023, 13, 89 6 of 19 where Sα(ω, ξ) is the standard response spectrum and Sk(ω, ξ) is the average response spectrum of generated stochastic ground motions, respectively. The error ratio ε is con- trolled to be less than 0.05 during the generation of stochastic ground motions [41,42]. 2.2. Quantiﬁcation of Randomness of Input Ground Motions In the present study, a suite of seismic ground motions consisting of 144 acceleration time histories with a mean peak ground acceleration of 0.1 g are generated to quantify the randomness of input seismic ground motions. Figure 1 presents four typical generated acceleration time histories, and it shows that different ground motions have different intensities and spectral characteristics. Figure 2 shows the comparison between the average response spectrum of 144 stochastic ground motions and the target standard response spectrum in the Chinese seismic code [43]. The relatively good consistency between these two lines validates the reasonability of the generated seismic ground motions used in the present study. With the above process, a set of fully non-stationary stochastic ground motions that are consistent with the target seismic responses spectrum in the Chinese seismic code can be generated to quantify the randomness of earthquake. To reflect local site effect on the seismic ground motions, the seismic response spectrum of a site type similar to the site where the adopted RC pile is located is used to acquire the power spectral density function for the generation of seismic ground motions, because the seismic response spectrum (Equation (10)) has already considered site effect in the Chinese seismic code. In the pre- sent study, a suite of seismic ground motions consisting of 144 acceleration time histories with a mean peak ground acceleration of 0.1 g are generated to quantify the randomness of input seismic ground motions. Figure 1 presents four typical generated acceleration time histories, and it shows that different ground motions have different intensities and spectral characteristics. Figure 2 shows the comparison between the average response spectrum of 144 stochastic ground motions and the target standard response spectrum in the Chinese seismic code [43]. The relatively good consistency between these two lines validates the reasonability of the generated seismic ground motions used in the present study. Figure 1. Four typical acceleration time histories generated in the present study. 0 5 10 15 -200 -100 0 100 200 Acceleration(cm/s2) Synthetic motion 1 0 5 10 15 -200 -100 0 100 200 Acceleration(cm/s2) Synthetic motion 48 0 5 10 15 -200 -100 0 100 200 Time (s) Acceleration(cm/s2) Synthetic motion 96 0 5 10 15 -200 -100 0 100 200 Time (s) Acceleration(cm/s2) Synthetic motion 144 Figure 1. Four typical acceleration time histories generated in the present study. 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model To illustrate the application of PDEM in the nonlinear stochastic seismic response analysis of RC piles, a large-diameter bored RC pile documented in the literature is adopted for the case study. It has to be noted that it is better to use the shaking table test results to calibrate the ﬁnite element model for the dynamic behavioral analysis of pile foundations. However, most of the shaking table tests of pile foundations have been conducted in model scale and cannot fully reﬂect the seismic behavior of prototype pile foundations. In addition, model-scale tests have usually neglected the characteristics of concrete and rebar by simply using elastic materials without rebars. Thus, to investigate the seismic behavior of full-scale pile foundations with consideration of site effect and the prototypical characteristics of soil, concrete, and rebar, the prototype concrete pile with ﬁeld measurement results from the full-scale lateral loading tests in Huang et al. [27] is consequently adopted to calibrate and validate the three-dimensional ﬁnite element model of the RC pile developed in the present study; this is because cyclic horizontal load test can also be used to investigate the seismic behavior of pile [45–47]. The available experimental results from the full-scale horizontal loading tests are ﬁrstly used to calibrate the parameters of soil and the RC pile in the three-dimensional ﬁnite element model. Then, a suite of stochastic ground motions (144 stochastic ground motions described in Section 2.2) is input into the numerical model for the nonlinear stochastic seismic response analysis. A set of full-scale lateral loading tests of bored and driven precast piles has been performed by Huang et al. [27]. In the present study, the results of the horizontal loading test of a single bored pile B7 are selected. Pile B7 is a large-diameter bored pile with a diameter of 1.5 m and a height of 34.9 m, in which 0.9 m of the pile is above the ground level. The 52φ32 steel bars are arranged in two rings to reinforce the concrete and the corresponding reinforcement ratio is 0.025. According to the site investigation results in the document of Huang et al. [27], the subsoil within 80 m depth can generally be classiﬁed as silty sand with layers of sandy silt and can be classiﬁed into six layers, which is illustrated in Figure 3. 2.2. Quantiﬁcation of Randomness of Input Ground Motions VIEW 0 5 10 15 -200 -100 0 100 200 Acceleration(cm/s2) Synthetic motion 1 0 5 10 15 -200 -100 0 100 200 Time (s) Acceleration(cm/s2) Synthetic motion 96 EW 0 5 10 15 -200 -100 0 100 200 Acceleration(cm/s2) Synthetic motion 48 0 5 10 15 -200 -100 0 100 200 Time (s) Acceleration(cm/s2) Synthetic motion 144 Acceleration(cm/s2) Figure 1. Four typical acceleration time histories generated in the present study. Figure 1. Four typical acceleration time histories generated in the present study. Figure 2. Comparison of the average response spectrum of 144 stochastic ground motions and the target standard response spectrum in the Chinese seismic code. 3 M d l D l t d C lib ti 0 1 2 3 4 5 6 0 0.05 0.1 0.15 0.2 0.25 Period (s) Spectral Accleration (g) Mean of 144 samples Chinese seismic code Figure 2. Comparison of the average response spectrum of 144 stochastic ground motions and the target standard response spectrum in the Chinese seismic code. 0 1 2 3 4 5 6 0 0.05 0.1 0.15 0.2 0.25 Period (s) Spectral Accleration (g) Mean of 144 samples Chinese seismic code Figure 2. Comparison of the average response spectrum of 144 stochastic ground motions and the target standard response spectrum in the Chinese seismic code. Figure 2. Comparison of the average response spectrum of 144 stochastic ground motions and the target standard response spectrum in the Chinese seismic code. 7 of 19 7 of 19 Buildings 2023, 13, 89 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model EW 8 of 20 main physical and mechanical parameters of the soil are listed in Table 1 based on the parameters provided in the studies by Huang et al. [27] and Conte et al. [25]. Figure 3. A diagrammatic sketch of a soil foundation model for the numerical simulation in the present study [25,27]. 1.5 m 5 m 4.5 m 11 m 7 m H=34.9 m Silt Silt sand Sandy silt Silty sand Sandy silt Silty sand D=1.5 m Figure 3. A diagrammatic sketch of a soil foundation model for the numerical simulation in the present study [25,27]. Figure 3. A diagrammatic sketch of a soil foundation model for the numerical simulation in the present study [25,27]. Figure 3. A diagrammatic sketch of a soil foundation model for the numerical simulation in the present study [25,27]. Table 1. The physical parameters of soil properties [25,27]. Soil Layers Elastic Modulus (Mpa) Poisson’s Ratio Cohesion (kPa) Friction Angle (°) N 1 Silt 80 08 0 3 0 33 In the numerical simulation, a linear elastic perfectly plastic model with the Mohr– Coulomb failure criterion and the ﬂow rule of non-associated type is employed to charac- terize the nonlinear behavior of soil. This model requires few parameters as input data, and the parameters can be readily determined from conventional geotechnical tests. The main Buildings 2023, 13, 89 8 of 19 physical and mechanical parameters of the soil are listed in Table 1 based on the parameters provided in the studies by Huang et al. [27] and Conte et al. [25]. physical and mechanical parameters of the soil are listed in Table 1 based on the parameters provided in the studies by Huang et al. [27] and Conte et al. [25]. physical and mechanical parameters of the soil are listed in Table 1 based on the parameters provided in the studies by Huang et al. [27] and Conte et al. [25]. Table 1. The physical parameters of soil properties [25,27]. Table 1. The physical parameters of soil properties [25,27]. Table 1. The physical parameters of soil properties [25,27]. 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model Soil Layers Elastic Modulus (Mpa) Poisson’s Ratio Cohesion (kPa) Friction Angle (◦) No.1: Silt 80.08 0.3 0 33 No.2: Silt sand 150.02 0.3 0 34 No.3: Sandy silt 150.28 0.3 0 28 No.4: Silty sand 228.28 0.3 0 33 No.5: Sandy silt 228.28 0.3 0 28 No.6: Silty sand 228.28 0.3 0 30 To simulate the nonlinear behavior and the damage characteristics of concrete pile under external load, the concrete damaged plasticity (CDP) model is adopted. This model fully considers the anisotropy and stiffness recovery effect of concrete under both ten- sion and compression states. On the one hand, tensile and compressive damage factors (dt and dc) are deﬁned in this model to simulate the stiffness degradation of concrete under tension and compression states, respectively, which will be introduced by the stress–strain relationship of concrete in the next paragraph. On the other hand, the stiffness recovery factor is also set to simulate the recovery ability of concrete stiffness in the CDP model. The stress–strain relationship of concrete under a tension condition is shown in Figure 4. When the stress exceeds the elastic limit stress σt0, it will enter the softening stage, and micro cracks will be formed in the concrete material, resulting in certain tensile damage. In the CDP model of concrete, the concrete tension damage characteristic can be characterized by the following stress–strain relationship: σt = (1 −dt)E0 εt −εpl t (14) (14) where σt is the tension stress of concrete; εt is the tension strain of concrete; dt is the tensile damage factor; εpl t is the tension plastic strain; and E0 is the initial elastic modulus of concrete. The tension crack strain εck t that is needed for the input in the numerical simulation can be determined as follows: IEW 9 of 20 εck t = εpl t + dtσt (1 −dt)E0 (15) 𝜀௧ ௖௞= 𝜀௧ ௣௟+ ௗ೟ఙ೟ (ଵିௗ೟)ாబ (15) (15) (15) Figure 4. Stress–strain relationship of concrete under the tension condition considered in the pre- sent study. Strain Stress Figure 4. Stress–strain relationship of concrete under the tension condition considered in the present study. Strain Strain Figure 4. Stress–strain relationship of concrete under the tension condition considered in the pre- sent study. Figure 4. Stress–strain relationship of concrete under the tension condition considered in the present study. Figure 4. 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model The compression inelastic strain εin c input in the numerical models can be determined as follows: 𝜎௖= (1 −𝑑௖)𝐸଴(𝜀௖−𝜀௖ ௣) (16) where 𝜎௖ is the compression stress of concrete; 𝜀௖ is the compression strain of concrete; 𝑑௖ is the compressive damage factor; 𝜀௖ ௣௟ is the compression plastic strain; and 𝐸଴ is the initial elastic modulus of concrete. The compression inelastic strain 𝜀௖ ௜௡ input in the nu- εin c = εpl c + dcσc (1 −d0)E0 (17) ed as follows: 𝜀௖ ௜௡= 𝜀௖ ௣௟+ ௗ೎ఙ೎ (ଵିௗబ)ாబ (17) (17) (17) Strain Stress Figure 5. Stress–strain relationship of concrete under the compression condition considered in the present study. Strain Figure 5. Stress–strain relationship of concrete under the compression condition considered in the present study. The CDP model under the tension–compression cyclic load considered in the present study is shown in Figure 6. The OABCDEF curve is on cyclic load, and the OABH and OI are the stress strain curves of uniaxial tension and uniaxial compression conditions respectively. The tension and compression stiffness recovery factors Wt and Wc are in- troduced to illustrate the CDP stress–strain relationship under the tension-compression load of the CDP model to, respectively, simulate the recovery ability of the tensile and the compressive stiffness of the concrete material under tension and compression cyclic loading. The concrete material will produce microcracks under tension, resulting in a reduction of its stiffness. At this time, when it is compressed, these microcracks will be closed again, and the compressive stiffness will recover (Wc = 1). On the contrary, when the concrete is compressed, it will produce crushing cracks, which will also reduce its stiffness. Meanwhile, even if it is under reverse tension, these crushing cracks still exist, and the tensile stiffness will not recover (Wt = 0). If tensile stiffness is assumed to be completely recovered (Wt = 1), the stress–strain relationship will become the curve OABCDEG, not the OABCDEF curve. More detailed information about the selected CDP model for the concrete pile used in this study can be found in the Abaqus manuals [48]. In the present study, the speciﬁc stress–stain relationship for concrete in the Code for the design of concrete structures (GB50010–2010) [49] is used to combine with the above CDP model to calculate the input parameters for the numerical simulation of RC pile in Abaqus. Other parameters of concrete are consistent with those used by Huang et al. 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model Stress–strain relationship of concrete under the tension condition considered in the pre- sent study Figure 4. Stress–strain relationship of concrete under the tension condition considered in the present study. Buildings 2023, 13, 89 9 of 19 The stress–strain relationship of concrete under the compression condition considered in the present study is shown in Figure 5. When the stress exceeds the elastic limit stress σc0, it will enter the strengthening stage. Thereafter, the stress increases gradually until it exceeds the limit stress σcu, and it will enter the softening stage, and concrete compression damage will happen. The concrete compression damage characteristic can be characterized by the following stress–strain relationship: g p p sent study. The stress–strain relationship of concrete under the compression condition consid- ered in the present study is shown in Figure 5. When the stress exceeds the elastic limit stress σc0, it will enter the strengthening stage. Thereafter, the stress increases gradually until it exceeds the limit stress σcu, and it will enter the softening stage, and concrete com- d ll h Th d h b σc = (1 −dc)E0 εc −εpl c (16) . The concrete compression damage characteristic can be stress–strain relationship: (1 𝑑)𝐸( ௣௟) (16) (16) where σc is the compression stress of concrete; εc is the compression strain of concrete; dc is the compressive damage factor; εpl c is the compression plastic strain; and E0 is the initial elastic modulus of concrete. The compression inelastic strain εin c input in the numerical models can be determined as follows: 𝜎௖= (1 −𝑑௖)𝐸଴(𝜀௖−𝜀௖ ௣) (16) where 𝜎௖ is the compression stress of concrete; 𝜀௖ is the compression strain of concrete; 𝑑௖ is the compressive damage factor; 𝜀௖ ௣௟ is the compression plastic strain; and 𝐸଴ is the initial elastic modulus of concrete. The compression inelastic strain 𝜀௖ ௜௡ input in the nu- i l d l b d i d f ll where σc is the compression stress of concrete; εc is the compression strain of concrete; dc is the compressive damage factor; εpl c is the compression plastic strain; and E0 is the initial elastic modulus of concrete. 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model [27], namely Young’s modulus Buildings 2023, 13, 89 10 of 19 alculate ameters 10 of 19 alculate ameters Ec = 32,173 MPa, Poisson’s ratio υc = 0.2, compressive strength f c = 27.5 MPa, and tensile strength f ct = 2.7 MPa. Ec = 32,173 MPa, Poisson s ratio υc = 0.2, compressive strength fc = 27.5 MPa, and tensile strength fct = 2.7 MPa. Ec = 32,173 MPa, Poisson’s ratio υc = 0.2, compressive strength f c = 27.5 MPa, and tens strength f ct = 2.7 MPa. Ec 32,173 MPa, Poisson s ratio υc 0.2, compressive strength fc 27.5 MPa, and tens strength fct = 2.7 MPa. Figure 6. The concrete damaged plasticity model for the reinforced concrete pile. ( =1 =0 =0 =1 O Figure 6. The concrete damaged plasticity model for the reinforced concrete pile. EVIEW 11 o The finite element meshes of soil, RC pile, and reinforcement are shown in Figur A 45 m-depth soil domain with a diameter of 48 m (about 32D, where D is the pile dia Figure 6. The concrete damaged plasticity model for the reinforced concrete pile. Figure 6. The concrete damaged plasticity model for the reinforced concrete pile. The finite element meshes of soil, RC pile, and reinforcement are sh A 45 m-depth soil domain with a diameter of 48 m (about 32D, where D The behavior of the steel bars is characterized by the elastic perfectly plastic consti- tutive model, which is the same as the model in Conte et al. [25] and is shown in Figure 7. The main parameters of steel bars used in the numerical simulation are Young’s modulus Es = 210,000 MPa and yield strength fy = 471 Mpa. The tension and compression behavior of the steel bars is identical in this model. The steel bars are embedded into the concrete pile and are assumed to be perfectly bonded to the surrounding concrete. Th li d d i i i b h RC il d il i id d The behavior of the steel bars is characterized by the elastic perfectly plastic constitu- tive model, which is the same as the model in Conte et al. [25] and is shown in Figure 7. The main parameters of steel bars used in the numerical simulation are Young’s modulus Es = 210,000 MPa and yield strength fy = 471 Mpa. 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model Finite element model of (a) soil foundation (b) pile and (c) reinforcement Figure 8. Finite element model of (a) soil foundation, (b) pile, and (c) reinforcement. To extend this model to earthquake scenarios for the stochastic dynamic analysis, the finite elements are employed in the Abaqus to reduce the seismic wave refection and oundary effect during the dynamic analyses. To model the vertical load on the RC pile nd the inertial effect from the superstructure, a mass block with a volume of 8 m3 and a ensity of 36,750 kg/m3 is assumed and tied at the pile head to simulate a vertical load of 40 kN and an inertial effect in earthquake cases To extend this model to earthquake scenarios for the stochastic dynamic analysis, the inﬁnite elements are employed in the Abaqus to reduce the seismic wave refection and boundary effect during the dynamic analyses. To model the vertical load on the RC pile and the inertial effect from the superstructure, a mass block with a volume of 8 m3 and a density of 36,750 kg/m3 is assumed and tied at the pile head to simulate a vertical load of 2940 kN and an inertial effect in earthquake cases. 3. Model Development and Calibration 3.1. Three-Dimensional Finite Element Model The tension and compression behavior of the steel bars is identical in this model. The steel bars are embedded into the concrete pile and are assumed to be perfectly bonded to the surrounding concrete. eter) is constructed to avoid the boundary effect from the bottom and the lateral sides. The 34,320, 5183, and 3728 finite elements are generated in the soil domain, the pile, and the rebar, respectively. Based on the above model, the nonlinear behavior of soil, the concrete damaged plasticity, the steel yielding of the pile, and the occurrence of slip and gap at the soil–pile interface can all be properly and comprehensively simulated. The complicated dynamic interaction between the RC pile and soil is considered as the “surface to surface” type in the Abaqus software with the shear-strength criterion of Mohr–Coulomb [25,48] in the 3D numerical model, in which slip and gap can occur at the pile–soil interface. Figure 7. Stress–strain relationship of steel bars. Strain Stress Figure 7. Stress–strain relationship of steel bars. Figure 7. Stress–strain relationship of steel bars Figure 7. Stress–strain relationship of steel bars. Figure 7. Stress–strain relationship of steel bars. Figure 7. Stress–strain relationship of steel bars. The complicated dynamic interaction between the RC pile and soil is considered as the “surface to surface” type in the Abaqus software with the shear-strength criterion of Mohr–Coulomb [25,48] in the 3D numerical model, in which slip and gap can occur at the pile–soil interface. The ﬁnite element meshes of soil, RC pile, and reinforcement are shown in Figure 8. A 45 m-depth soil domain with a diameter of 48 m (about 32D, where D is the pile diameter) is constructed to avoid the boundary effect from the bottom and the lateral sides. The 34,320, 5183, and 3728 ﬁnite elements are generated in the soil domain, the pile, and the rebar, respectively. Based on the above model, the nonlinear behavior of soil, the concrete Buildings 2023, 13, 89 11 of 19 11 of 19 damaged plasticity, the steel yielding of the pile, and the occurrence of slip and gap at the soil–pile interface can all be properly and comprehensively simulated. ure 7. Stress–strain relationship of steel bars. gure 8. Finite element model of (a) soil foundation, (b) pile, and (c) reinforcement. Figure 8. Finite element model of (a) soil foundation, (b) pile, and (c) reinforcement. gure 8. q 2 M d l C lib ti ith Fi ld L t l L d T t 3.2. Model Calibration with Field Lateral Load Tests 2. Model Calibration with Field Lateral Load Tests The field measurement results of the full-scale lateral loading tests of B7 bored pile ported by Huang et al. [27] are selected to calibrate and validate the three-dimensional The ﬁeld measurement results of the full-scale lateral loading tests of B7 bored pile reported by Huang et al. [27] are selected to calibrate and validate the three-dimensional ﬁnite element model of RC pile. The horizontal loads are applied to the pile head in the model in a similar way to the ﬁeld lateral loading tests of the pile. The lateral load– displacement curve from the full-scale ﬁeld tests and the nonlinear numerical simulation are presented and compared in Figure 9, which shows a fairly good agreement between the ﬁeld tests and the simulation. The comparison of the deﬂection results of the pile along the pile length from the ﬁeld tests and the numerical simulation under different values of horizontal loading is also illustrated in Figure 10, which also shows a relatively good agreement between them. The comparison results in Figures 9 and 10 corroborate the selected parameters of soil, pile, and reinforcement used in the present study for the numerical simulation. In Figure 10, the horizontal displacement of the pile mainly occurs at a depth of 0 to 10 m, which indicates that the inﬂuence of depth of the horizontal loads on the pile head is about 10 m (about 6.5D). Using a nonlinear constitutive model, the nonlinear and damage characteristics of the RC pile under the horizontal loads can be revealed. Figure 11 shows the distribution of plastic strain and concrete tension damage at the upper part of the pile body under 814 kN horizontal load. The plastic strain and concrete tension damage mainly occur at a depth of 2.4~6.9 m, indicating the development and distribution of the cracks in the upper portion of the pile under the horizontal loads. 12 of 19 12 of 19 Buildings 2023, 13, 89 Figure 9. Comparison of the lateral load–displacement curve at the pile head from the field tests and from the numerical simulation. 0 10 20 30 40 50 60 0 250 500 750 1000 1250 1500 1750 2000 Lateral load (kN) Horizontal displacement (mm) Field test Numerical calculation Figure 9. Comparison of the lateral load–displacement curve at the pile head from the ﬁeld tests and from the numerical simulation. q 2 M d l C lib ti ith Fi ld L t l L d T t 3.2. Model Calibration with Field Lateral Load Tests -5 0 5 10 15 20 25 30 35 40 45 50 55 60 -35 -30 -25 -20 -15 -10 -5 0 Depth (m) Deflection (mm) Numerical calculation (814 kN) Numerical calculation (1462 kN) Numerical calculation (1903 kN) Field test (814 kN) Field test (1462 kN) Field test (1903 kN) In Figure 10, the horizontal displacement of the pile main 10 m, which indicates that the influence of depth of the horizo is about 10 m (about 6.5D). Using a nonlinear constitutive mod age characteristics of the RC pile under the horizontal loads shows the distribution of plastic strain and concrete tension d the pile body under 814 kN horizontal load. The plastic strain age mainly occur at a depth of 2.4~6.9 m, indicating the devel the cracks in the upper portion of the pile under the horizonta -5 0 5 10 15 20 25 30 35 40 45 50 55 60 -35 -30 -25 -20 -15 -10 -5 0 Depth (m) Deflection (mm) Numerical calculation (814 kN) Numerical calculation (1462 kN) Numerical calculation (1903 kN) Field test (814 kN) Field test (1462 kN) Field test (1903 kN) -5 0 5 10 15 20 25 30 35 40 45 50 55 60 -35 -30 -25 -20 -15 -10 -5 0 Depth (m) Deflection (mm) Numerical calculation (814 kN) Numerical calculation (1462 kN) Numerical calculation (1903 kN) Field test (814 kN) Field test (1462 kN) Field test (1903 kN) igure 10. Comparison of the deflection results of the pile along the pile body between the field tests nd the numerical simulation under different values of horizontal load. Figure 10. Comparison of the deﬂection results of the pile along the pile body between the ﬁeld tests and the numerical simulation under different values of horizontal load. Figure 10. Comparison of the deflection results of the pile along the pile body between the field tests and the numerical simulation under different values of horizontal load. Figure 11. Distribution of (a) plastic strain, and (b) concrete tension damage at the upper part of the pile body under 814 kN horizontal load. 4. Stochastic Seismic Response Analysis of RC Pile Figure 11. Distribution of (a) plastic strain, and (b) concrete tension damage at the upper part of the pile body under 814 kN horizontal load. 4 St h ti S i i R A l i f RC Pil Figure 11. q 2 M d l C lib ti ith Fi ld L t l L d T t 3.2. Model Calibration with Field Lateral Load Tests VIEW 13 of 20 EVIEW 13 of 20 0 10 20 30 40 50 60 0 250 500 750 1000 1250 1500 1750 2000 Lateral load (kN) Horizontal displacement (mm) Field test Numerical calculation Figure 9. Comparison of the lateral load–displacement curve at the pile head from the field tests and from the numerical simulation. Figure 9. Comparison of the lateral load–displacement curve at the pile head from the ﬁeld tests and from the numerical simulation. EW 13 of 20 IEW 13 of 20 In Figure 10, the horizontal displacement of the pile mainly occurs at a depth of 0 to 10 m, which indicates that the influence of depth of the horizontal loads on the pile head is about 10 m (about 6.5D). Using a nonlinear constitutive model, the nonlinear and dam- age characteristics of the RC pile under the horizontal loads can be revealed. Figure 11 shows the distribution of plastic strain and concrete tension damage at the upper part of the pile body under 814 kN horizontal load. The plastic strain and concrete tension dam- age mainly occur at a depth of 2.4~6.9 m, indicating the development and distribution of the cracks in the upper portion of the pile under the horizontal loads. Figure 10. Comparison of the deflection results of the pile along the pile body between the field tests and the numerical simulation under different values of horizontal load. -5 0 5 10 15 20 25 30 35 40 45 50 55 60 -35 -30 -25 -20 -15 -10 -5 0 Depth (m) Deflection (mm) Numerical calculation (814 kN) Numerical calculation (1462 kN) Numerical calculation (1903 kN) Field test (814 kN) Field test (1462 kN) Field test (1903 kN) Figure 10. Comparison of the deﬂection results of the pile along the pile body between the ﬁeld tests and the numerical simulation under different values of horizontal load. Figure 10. Comparison of the deflection results of the pile along the pile body between the field tests and the numerical simulation under different values of horizontal load. 4. Stochastic Seismic Response Analysis of RC Pile In the study by Huang et al. [27], the large-diameter RC piles are planned for the construction of a high-speed railway system [27]. To ensure the safety of the train, the settlement of pile foundation is strictly controlled. Therefore, the settlement at the pile head is consequently selected as the seismic performance index to analyze the stochastic seismic responses and the seismic reliability of the RC pile under different seismic loads. Based on the framework of the method stated in Section 2.2, the generated 144 stochastic ground motions are input into the calibrated finite element model of the RC pile for a set of dynamic time history analysis. Then, the 144 time histories of settlement at the pile head are extracted for the stochastic analysis by combining with the GPDE equation (Equation (4)). VIEW 14 of 20 y y g q q Figure 12 shows the mean value, the standard deviation, and the 144 deterministic results of seismic settlement time histories at the pile head under various stochastic ground motions. It can be seen from the ﬁgure that the seismic settlements at the pile head under various seismic ground motions show a similar trend. They increase gradually and then become basically stable at certain permanent values due to the nonlinear characteristics of soil and RC pile. Some maximum settlements of the pile reach about 11 mm under the stochastic ground motion excitation, while some maximum settlements of the pile are only about 3 mm, which demonstrates that the seismic responses of the RC pile under stochastic ground motions have great variability. Thus, it is necessary to resort to stochastic methods to quantify theses great uncertainties. The mean value and the standard deviation of the seismic settlements are also illustrated in Figure 12. They all increase with time because of the increase in accumulative settlement under earthquakes. The maximum mean value of the pile settlement is about 8 mm. ground motions. It can be seen from the figure that the seismic settlements at the pile head under various seismic ground motions show a similar trend. They increase gradually and then become basically stable at certain permanent values due to the nonlinear character- istics of soil and RC pile. 4. Stochastic Seismic Response Analysis of RC Pile Some maximum settlements of the pile reach about 11 mm under the stochastic ground motion excitation, while some maximum settlements of the pile are only about 3 mm, which demonstrates that the seismic responses of the RC pile under stochastic ground motions have great variability. Thus, it is necessary to resort to stochas- tic methods to quantify theses great uncertainties. The mean value and the standard de- viation of the seismic settlements are also illustrated in Figure 12. They all increase with time because of the increase in accumulative settlement under earthquakes. The maxi- mum mean value of the pile settlement is about 8 mm. Figure 12. Mean value, standard deviation, and 144 deterministic results of the seismic settlement at the pile head under stochastic ground motions. 0 5 10 15 0 2 4 6 8 10 12 Time(s) Settlement (mm) 144 deteministic results Mean Standard deviation Figure 12. Mean value, standard deviation, and 144 deterministic results of the seismic settlement at the pile head under stochastic ground motions. Settlement (mm) Time(s) Figure 12. Mean value, standard deviation, and 144 deterministic results of the seismic settlement at the pile head under stochastic ground motions. Figure 12. Mean value, standard deviation, and 144 deterministic results of the seismic settlement at the pile head under stochastic ground motions. The PDEM has the ability to acquire instantaneous PDFs at different time points and reveal the propagation of uncertainty in nonlinear stochastic systems. The PDFs of the seismic settlements at three different time points (namely 0.6 s, 1.3 s, and 2.8 s) are illus- trated in Figure 13. An unusual PDF shape can be observed in this figure, which demon- strates that using ordinary assumed distribution models (e.g., normal distribution, lognor- mal distribution, and extreme value distribution) to represent the seismic responses of the system for the dynamic reliability analysis of engineering structures is unreasonable and imprecise. The distribution ranges of PDFs at these three time points are enlarged with time, and it is because the accumulative settlement of the pile increases under earth- quakes. The enlargement of the distribution ranges of the PDF also indicates that the var- iability of seismic settlements also increases with time, which is similar to the results ob- The PDEM has the ability to acquire instantaneous PDFs at different time points and reveal the propagation of uncertainty in nonlinear stochastic systems. q 2 M d l C lib ti ith Fi ld L t l L d T t 3.2. Model Calibration with Field Lateral Load Tests Distribution of (a) plastic strain, and (b) concrete tension damage at the upper part of the pile body under 814 kN horizontal load. Figure 11. Distribution of (a) plastic strain, and (b) concrete tension damage at the upper part of the pile body under 814 kN horizontal load. Figure 11. Distribution of (a) plastic strain, and (b) concrete tension damage at the upper part of the pile body under 814 kN horizontal load. Figure 11. Distribution of (a) plastic strain, and (b) concrete tension damage at the upper part of the pile body under 814 kN horizontal load. Buildings 2023, 13, 89 13 of 19 4. Stochastic Seismic Response Analysis of RC Pile The PDFs of the seismic settlements at three different time points (namely 0.6 s, 1.3 s, and 2.8 s) are illustrated in Figure 13. An unusual PDF shape can be observed in this ﬁgure, which demonstrates that using ordinary assumed distribution models (e.g., normal distribution, lognormal distribution, and extreme value distribution) to represent the seismic responses of the system for the dynamic reliability analysis of engineering structures is unreasonable and imprecise. The distribution ranges of PDFs at these three time points are enlarged with time, and it is because the accumulative settlement of the pile increases under earthquakes. The enlargement of the distribution ranges of the PDF also indicates that the variability of seismic settlements also increases with time, which is similar to the results observed Buildings 2023, 13, 89 14 of 19 in Figure 12. The propagation characteristic of the uncertainty in the nonlinear stochastic dynamic system of the RC pile is presented as the settlement PDF surface and the PDF contour in Figure 14. The settlement PDF surface is like a rolling hill and the high points denote the high probabilities of seismic response values. The settlement PDF contour is like ﬂowing water, and it can be seen that the settlement distribution range also increases due to the increase in accumulative settlement under the seismic ground motions. VIEW 15 of 20 15 of 20 Figure 13. PDF of seismic settlement at three different time points. -2 0 2 4 6 8 10 0 0.5 1 1.5 2 2.5 Settlement (mm) PDF PDF at 0.6 sec PDF at 1.3 sec PDF at 2.8 sec Figure 13. PDF of seismic settlement at three different time points. F of seismic settlement at three different time points. 0 2 4 6 8 10 Settlement (mm) PDF at 0.6 sec PDF at 1.3 sec PDF at 2.8 sec Figure 13. PDF of seismic settlement at three different time points. Figure 13. PDF of seismic settlement at three different time points. of seismic settlement at three different time points. (a) (b) Figure 14. (a) Settlement PDF surface and (b) PDF contour at a time interval of 2~3 s at the RC pile head. 4. Stochastic Seismic Response Analysis of RC Pile 2 2.2 2.4 2.6 2.8 3 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 Time (s) Settlement (mm) PDF 0 0.1 0.2 0.3 0.4 0.5 Time(s) Settlement(mm) 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 (a) (b) ) Settlement PDF surface and (b) PDF contour at a time interval of 2~3 s at the RC pile 2 2.2 2.4 2.6 2.8 3 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 Time (s) Settlement (mm) PDF 0 0.1 0.2 0.3 0.4 0.5 Time(s) Settlement(mm) 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 Figure 14. (a) Settlement PDF surface and (b) PDF contour at a time interval of 2~3 s at the RC pile head. (a) 2 2.2 2.4 2.6 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 Time ( Settlement (mm) PDF (a) 2 2.2 2.4 2.6 2.8 3 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 Time (s) Settlement (mm) PDF 0 0.1 0.2 0.3 0.4 0.5 (a) (a) (b) Time(s) Settlement(mm) 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 1 1.5 2 2.5 3 3.5 4 4.5 5 (b) Time(s) Settlement(mm) 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 (b) ( ) Figure 14. (a) Settlement PDF surface and (b) PDF contour at a time interval of 2~3 s at the RC pile h d ettlement PDF surface and (b) PDF contour at a time interval of 2~3 s at the RC pile Figure 14. (a) Settlement PDF surface and (b) PDF contour at a time interval of 2~3 s at the RC pile head. 4. Stochastic Seismic Response Analysis of RC Pile Buildings 2023, 13, 89 x FOR PEER REVIEW 15 of 19 15 of 19 To acquire the dynamic reliability of the RC pile under earthquake, the equivalent extreme event principle [39] combined with the GPDE equation in the PDEM is used to obtain the PDF and CDF of maximum settlement for the dynamic reliability analysis of the RC pile. The maximum settlement PDF and CDF of the RC pile at pile head are illustrated in Figure 15. The distribution range of settlement PDF and CDF is about 2~14 mm, which denotes that the maximum settlement value of the RC pile is about 2~14 mm. The determination of the settlement thresholds concerning various seismic performance requirements is of great signiﬁcance for the dynamic reliability assessment of pile foundations from the perspective of performance-based earthquake engineering [50]. Because the adopted large-diameter RC piles from the literature [27] are planned for a high-speed railway system, the settlement criteria for different performance levels and operation conditions of high-speed railway [51,52] are adopted for the dynamic reliability analysis of the RC pile in the present study, which is shown in Table 2 and also plotted in Figure 15b. From the ﬁgure, it can be observed that the dynamic reliability of the RC pile concerning four seismic settlement thresholds under the suite of stochastic ground motions are 0, 0.1293, 0.8247, and 1, respectively. The corresponding failure probabilities concerning these four seismic settlement thresholds are 1 (settlement thresholds = 2 mm), 0.8707 (settlement thresholds = 6 mm), 0.1853 (settlement thresholds = 10 mm), and 0 (settlement thresholds = 15 mm), respectively. e the dynamic reliability of the RC pile under earthquake, the equivalent principle [39] combined with the GPDE equation in the PDEM is used to F and CDF of maximum settlement for the dynamic reliability analysis of he maximum settlement PDF and CDF of the RC pile at pile head are illus- e 15. The distribution range of settlement PDF and CDF is about 2~14 mm, that the maximum settlement value of the RC pile is about 2~14 mm. The of the settlement thresholds concerning various seismic performance re- of great significance for the dynamic reliability assessment of pile founda- perspective of performance-based earthquake engineering [50]. 4. Stochastic Seismic Response Analysis of RC Pile Performance Levels Recommended Control Values and Corresponding Train Operation Status Settlement Thresholds (mm) Travel Speed Thresholds (km/h) Safety Levels Train Operation Status 1 2 160 Safe Normal operation 2 6 120 Low risk Comfortable 3 10 80 Medium risk Warning 4 15 60 High risk Operation with low speed To better illustrate the merits of the PDEM in the stochastic seismic response analyses of RC piles, the maximum settlement PDF and the maximum settlement CDF at the RC pile head obtained using the PDEM, histogram, empirical distribution, normal distribution, and Gumbel distribution are compared in Figure 16. The results obtained using the direct statistical methods (histogram and empirical distribution) are more precise because they can reﬂect the real structure of the original data. From the ﬁgure, it can be seen that the shape of the PDF and the CDF obtained using the PDEM is closest to that obtained using the direct statistical methods, when compared to the normal distribution and Gumbel distribution models. Some non-unimodal and detailed change characteristics in the PDF curves are also presented and reﬂected by the PDEM, which demonstrate that the PDEM has the ability to more precisely estimate the PDF and CDF compared to other speciﬁc assumed distribution forms. on safety control of a high-speed railway system concerning subgrade settlement Recommended Control Values and Corresponding Train Operation Status Settlement hresholds (mm) Travel Speed Thresholds (km/h) Safety Levels Train Operation Status 2 160 Safe Normal operation 6 120 Low risk Comfortable 10 80 Medium risk Warning 15 60 High risk Operation with low speed lustrate the merits of the PDEM in the stochastic seismic response analyses maximum settlement PDF and the maximum settlement CDF at the RC ned using the PDEM, histogram, empirical distribution, normal distribu- bel distribution are compared in Figure 16. The results obtained using the methods (histogram and empirical distribution) are more precise because the real structure of the original data. From the figure, it can be seen that e PDF and the CDF obtained using the PDEM is closest to that obtained statistical methods, when compared to the normal distribution and Gum- models. Some non-unimodal and detailed change characteristics in the also presented and reflected by the PDEM, which demonstrate that the ability to more precisely estimate the PDF and CDF compared to other d distribution forms. Table 2. Operation safety control of a high-speed railway system concerning subgrade settlement [51,52]. 4. Stochastic Seismic Response Analysis of RC Pile Performance Levels Recommended Control Values and Corresponding Train Operation Status Settlement Thresholds (mm) Travel Speed Thresholds (km/h) Safety Levels Train Operation Status 1 2 160 Safe Normal operation 2 6 120 Low risk Comfortable 3 10 80 Medium risk Warning 4 15 60 High risk Operation with low speed n safety control of a high-speed railway system concerning subgrade settlement Recommended Control Values and Corresponding Train Operation Status Settlement hresholds (mm) Travel Speed Thresholds (km/h) Safety Levels Train Operation Status 2 160 Safe Normal operation 6 120 Low risk Comfortable 10 80 Medium risk Warning 15 60 Hi h i k O i i h l d Table 2. Operation safety control of a high-speed railway system concerning subgrade settlement [51,52] f t t l f hi h d il t i b d ttl t To better illustrate the merits of the PDEM in the stochastic seismic response analyses of RC piles, the maximum settlement PDF and the maximum settlement CDF at the RC pile head obtained using the PDEM, histogram, empirical distribution, normal distribution, and Gumbel distribution are compared in Figure 16. The results obtained using the direct statistical methods (histogram and empirical distribution) are more precise because they can reﬂect the real structure of the original data. From the ﬁgure, it can be seen that the shape of the PDF and the CDF obtained using the PDEM is closest to that obtained using the direct statistical methods, when compared to the normal distribution and Gumbel distribution models. Some non-unimodal and detailed change characteristics in the PDF curves are also presented and reﬂected by the PDEM, which demonstrate that the PDEM has the ability to more precisely estimate the PDF and CDF compared to other speciﬁc assumed distribution forms. 15 60 High risk Operation with low speed ustrate the merits of the PDEM in the stochastic seismic response analyses maximum settlement PDF and the maximum settlement CDF at the RC ed using the PDEM, histogram, empirical distribution, normal distribu- el distribution are compared in Figure 16. The results obtained using the methods (histogram and empirical distribution) are more precise because the real structure of the original data. From the figure, it can be seen that PDF and the CDF obtained using the PDEM is closest to that obtained statistical methods, when compared to the normal distribution and Gum- models. 4. Stochastic Seismic Response Analysis of RC Pile Because the diameter RC piles from the literature [27] are planned for a high-speed rail- he settlement criteria for different performance levels and operation condi- peed railway [51,52] are adopted for the dynamic reliability analysis of the present study, which is shown in Table 2 and also plotted in Figure 15b. e, it can be observed that the dynamic reliability of the RC pile concerning ettlement thresholds under the suite of stochastic ground motions are 0, and 1, respectively. The corresponding failure probabilities concerning mic settlement thresholds are 1 (settlement thresholds = 2 mm), 0.8707 (set- olds = 6 mm), 0.1853 (settlement thresholds = 10 mm), and 0 (settlement 5 mm), respectively. ), p y (a) (b) Maximum settlement PDF and (b) maximum settlement CDF at the RC pile head. 0 2 4 6 8 10 12 14 16 0 0.05 0.1 0.15 0.2 0.25 Settlement (mm) PDF 0 2 4 6 8 10 12 14 16 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Settlement (mm) CDF Settlement threshold: 2mm Settlement threshold: 6mm Settlement threshold: 10mm Settlement threshold: 15mm Figure 15. (a) Maximum settlement PDF and (b) maximum settlement CDF at the RC pile head. 0 2 4 6 8 10 12 14 16 0 0.05 0.1 0.15 0.2 0.25 Settlement (mm) PDF (a) (b) 0 2 4 6 8 10 12 14 16 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Settlement (mm) CDF Settlement threshold: 2mm Settlement threshold: 6mm Settlement threshold: 10mm Settlement threshold: 15mm (b) aximum settlement PDF and (b) maximum settlement CDF at the RC pile head. Figure 15. (a) Maximum settlement PDF and (b) maximum settlement CDF at the RC pile head. Buildings 2023, 13, 89 16 of 19 Table 2. Operation safety control of a high-speed railway system concerning subgrade settlement [51,52]. 4. Stochastic Seismic Response Analysis of RC Pile Some non-unimodal and detailed change characteristics in the also presented and reflected by the PDEM, which demonstrate that the ability to more precisely estimate the PDF and CDF compared to other d distribution forms (a) (b) 0 2 4 6 8 10 12 14 16 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Settlement (mm) PDF Frequency histogram Normal distribution Gumbel distribution PDEM 0 2 4 6 8 10 12 14 16 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Settlement (mm) CDF Empirical distribution Normal distribution Gumbel distribution PDEM Figure 16. Comparison of the (a) maximum settlement PDF and (b) maximum settlement CDF at the RC pile head obtained using the PDEM and various distribution models. (a) 0 2 4 6 8 10 12 14 16 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Settlement (mm) PDF Frequency histogram Normal distribution Gumbel distribution PDEM PDF (a) (b) 0 2 4 6 8 10 12 14 16 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Settlement (mm) CDF Empirical distribution Normal distribution Gumbel distribution PDEM (b) Figure 16. Comparison of the (a) maximum settlement PDF and (b) maximum settlement CDF at the RC pile head obtained using the PDEM and various distribution models. Buildings 2023, 13, 89 17 of 19 By using the proposed method, the actual seismic reliability of pile foundations that are designed using the current seismic codes can be estimated and determined. Furthermore, the proposed method can also be used to quantify the seismic fragility and the seismic risk of pile foundations according to performance-based earthquake engineering and the probabilistic seismic risk assessment framework. In addition, it has to be noted that, although only a concrete pile example is adopted as the case study, the proposed method is applicable for various kinds of pile foundations because this stochastic method can combine various deterministic numerical ﬁnite element methods and the solution of the GPDE equation. 5. Conclusions In the present study, a probability density evolution method (PDEM), combined with a stochastic ground motion model, is proposed for the stochastic seismic response analysis of reinforced concrete (RC) piles. The randomness of earthquake and corresponding effect are properly quantiﬁed, and the nonlinear characteristics of soil, pile, and rebar and the soil–pile interaction are also properly modeled. With the proposed method, the settlement time histories of the pile head, the instanta- neous settlement probability density function (PDF), the cumulative distribution function (CDF), and the dynamic reliabilities of the pile concerning different performance levels under the excitation of a suite of ground motions are obtained. The results demonstrate the necessity of using stochastic methods to quantify the variability of RC piles under the effect of ground motion randomness. The shape of settlement PDF and CDF of the pile also demonstrates that it is unreasonable to use assumed probabilistic distribution models to characterize the seismic responses of pile foundations during a seismic reliability analysis. Author Contributions: Conceptualization, H.H., G.G. and M.X.; methodology, H.H., Y.B. and M.X.; software, H.H. and K.C.; validation, H.H. and M.X.; formal analysis, H.H. and Y.B.; investigation, H.H., Y.B., X.G., X.H., K.C., L.C. and L.W.; resources, H.H., G.G., X.H., K.C. and L.C.; data curation, H.H., X.G. and X.H.; writing—original draft preparation, H.H.; writing—review and editing, H.H., Y.B. and X.G.; visualization, H.H., Y.B., L.C. and L.W.; supervision, H.H. and G.G.; project adminis- tration, H.H. and G.G.; funding acquisition, H.H., G.G., M.X. and L.W. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the fellowship of China Postdoctoral Science Foundation (Grant No. 2021M702791), the projects from the Center for Balance Architecture in Zhejiang University (Grant Nos. K20203330C and K20212159), the National Natural Science Foundation of China (Grants Nos. 42272323, 42002272, 41902274 and 42202304), the project from the Ministry of Housing and Urban-Rural Development of China (Grant NO. 2021-K-030), and the project from the Department of Construction of Zhejiang Province, China (Grant NO. 2021K157). Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Author Contributions: Conceptualization, H.H., G.G. and M.X.; methodology, H.H., Y.B. and M.X.; software, H.H. and K.C.; validation, H.H. and M.X.; formal analysis, H.H. and Y.B.; investigation, H.H., Y.B., X.G., X.H., K.C., L.C. and L.W.; resources, H.H., G.G., X.H., K.C. and L.C.; data curation, H.H., X.G. and X.H.; writing—original draft preparation, H.H.; writing—review and editing, H.H., Y.B. and X.G.; visualization, H.H., Y.B., L.C. and L.W.; supervision, H.H. and G.G.; project adminis- tration, H.H. and G.G.; funding acquisition, H.H., G.G., M.X. and L.W. All authors have read and agreed to the published version of the manuscript. g g g y y p g p g 7. Chan, C.L.; Low, B.K. Reliability analysis of laterally loaded piles involving nonlinear soil and pile behavior. J. Geotech. Geoenviron. Eng. 2009, 135, 431–443. [CrossRef] References Zhu, C.; Cheng, H.; Bao, Y.; Chen, Z.; Huang, Y. Shaking table tests on the seismic response of slopes to near-fault ground motion. Geomech. 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https://openalex.org/W2070597361	https://www.epj-conferences.org/10.1051/epjconf/20135101003/pdf	French	null	Innovative SiC/SiC Composite for Nuclear Applications	EPJ web of conferences	2,013	cc-by	3,022	Centre of Excellence for Nuclear Materials Workshop Materials Innovation for Nuclear Optimized Systems December 5-7, 2012, CEA – INSTN Saclay, France Laurent CHAFFRON et al. CEA (France) Innovative SiC/SiC Composite for Nuclear Applications Workshop organized by: Christophe GALLÉ, CEA/MINOS, Saclay – christophe.galle@cea.fr Constantin MEIS CEA/INSTN Saclay constantin meis@cea fr Centre of Excellence for Nuclear Materials Workshop Materials Innovation for Nuclear Optimized Systems December 5-7, 2012, CEA – INSTN Saclay, France Laurent CHAFFRON et al. CEA (France) Innovative SiC/SiC Composite for Nuclear Applications Workshop organized by: Christophe GALLÉ, CEA/MINOS, Saclay – christophe.galle@cea.fr Constantin MEIS CEA/INSTN Saclay constantin meis@cea fr Innovative SiC/SiC Composite for Nuclear Applications Laurent CHAFFRON1, Cédric SAUDER1, Christophe LORRETTE1, Laurent BRIOTTET2, Aurore MICHAUX1, Lionel GÉLÉBART1, Aurélie COUPÉ1, Maxime ZABIEGO3, Marion LE FLEM1, Jean-Louis SÉRAN1 1CEA-DEN-DMN, Service de Recherche Métallurgiques Appliquées, SRMA (Saclay, France) 2 CEA-DRT, Laboratoires d’Innovation pour les Technologies des Energies, LITEN (Grenoble, France) 3 CEA-DEN-DEC, Service d’Etudes et de Simulation du Comportement des Combustibles, SESC (Cadarache, France) Among various refractory materials, SiC/SiC ceramic matrix composites (CMC) are of prime interest for fusion and advanced fission energy applications, due to their excellent irradiation tolerance and safety features (low activation, low tritium permeability,…). Initially developed as fuel cladding materials for the Fourth generation Gas cooled Fast Reactor (GFR), this material has been recently envisaged by CEA for different core structures of Sodium Fast Reactor (SFR) which combines fast neutrons and high temperature (500°C). Regarding fuel cladding generic application, in the case of GFR, the first challenge facing this project is to demonstrate the feasibility of a fuel operating under very harsh conditions that are (i) temperatures of structures up to 700°C in nominal and over 1600°C in accidental conditions, (ii) irradiation damage higher than 60 dpaSiC, (iii) neutronic transparency, which disqualifies conventional refractory metals as structural core materials, (iv) mechanical behavior that guarantees in most circumstances the integrity of the first barrier (e.g.: > 0.5%), which excludes monolithic ceramics and therefore encourages the development of new types of fibrous composites SiC/SiC adapted to the fast reactor conditions. No existing material being capable to match all these requirements, CEA has launched an ambitious program of development of an advanced material satisfying the specifications [1]. This project, that implies many laboratories, inside and outside CEA, has permitted to obtain a very high quality compound that meets most of the challenging requirements. We present hereinafter few recent results obtained regarding the development of the composite. One of the most relevant challenges was to make a gastight compo- site up to the ultimate rupture. Indeed, multicraking of the matrix is the counterpart of the damageable behavior observed in these amazing compounds. Among different solutions envisaged, an innovative one has been successful. It consists of inserting a metallic layer between two tubes of CMC [2]. The concept, illustrated in figure 1, guaranties a perfect helium tightness up to fracture of the CMC. Fig. 1: Sandwich cladding concept: tightness is ensured up to CMC failure thanks to the elastic metallic layer. Fig. 2: Sandwich cladding Cross section (metal is in white). This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fig. 2: Sandwich cladding Cross section (metal is in white). Fig. 2: Sandwich cladding Cross section (metal is in white). Centre of Excellence for Nuclear Materials Workshop Materials Innovation for Nuclear Optimized Systems December 5-7, 2012, CEA – INSTN Saclay, France Laurent CHAFFRON et al. CEA (France) Innovative SiC/SiC Composite for Nuclear Applications Workshop organized by: Christophe GALLÉ, CEA/MINOS, Saclay – christophe.galle@cea.fr Constantin MEIS, CEA/INSTN, Saclay – constantin.meis@cea.fr Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20135101003 Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20135101003 Workshop Materials Innovation for Nuclear Optimized Systems December 5-7, 2012, CEA – INSTN Saclay, France Workshop Materials Innovation for Nuclear Optimized Systems December 5-7, 2012, CEA – INSTN Saclay, France Workshop Materials Innovation for Nuclear Optimize December 5-7, 2012, CEA – INSTN S Another challenge was to prepare a representative cladding with very strict geometrical tolerances. Revisiting the fabrication of the entire breading process has allowed to ensure a perfect geometry of the final tube. Thanks to the high quality of manufacture and the high level of purity of composite materials manufactured at CEA, few tens of CMC objects (tubes, disks and plates) have been prepared in order to be irradiated in the Russian reactor “BOR 60”. For the first time, composite materials will be submitted to swift neutrons at very high damaging doses (up to 80 dpaSiC) between 400 and 520°C. Post irradiation examinations expected for 2015 should give reliable results on the behavior of this multi-materials component. In parallel, other basic researches are conducted to improve the properties of the CMC and round off the understanding [3, 4, 5]. Some new results allowed to extend the field of use of the CMC through an optimization of the interphase of the composite. The figure 4 shows the relative elongation of a CMC after a two hours dwell time annealing in argon at different temperatures: optimized composite can sustain very high temperature without drastic drop of its mechanical properties. Fig. 3: CMC specimen prepared for BOR60 irradiation Fig. 4: Evolution of the relative elongation of two composites with the annealing temperature: optimized CVI conditions to improve mechanical properties. Fig. 4: Evolution of the relative elongation of two composites with the annealing temperature: optimized CVI conditions to improve mechanical properties. Fig. 3: CMC specimen prepared for BOR60 irradiation Fig. 3: CMC specimen prepared for BOR60 irradiation Fig. 4: Evolution of the relative elongation of two composites with the annealing temperature: optimized CVI conditions to improve mechanical properties. Fig. 3: CMC specimen prepared for BOR60 irradiation Innovative SiC/SiC Composite for Nuclear Applications Laurent CHAFFRON1, Cédric SAUDER1, Christophe LORRETTE1, Laurent BRIOTTET2, Aurore MICHAUX1, Lionel GÉLÉBART1, Aurélie COUPÉ1, Maxime ZABIEGO3, Marion LE FLEM1, Jean-Louis SÉRAN1 1CEA-DEN-DMN, Service de Recherche Métallurgiques Appliquées, SRMA (Saclay, France) 2 CEA-DRT, Laboratoires d’Innovation pour les Technologies des Energies, LITEN (Grenoble, France) 3 CEA-DEN-DEC, Service d’Etudes et de Simulation du Comportement des Combustibles, SESC (Cadarache, France) Fig. 1: Sandwich cladding concept: tightness is ensured up to CMC failure thanks to the elastic metallic layer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. References [1] L. Chaffron, J. L. Séran, C. Sauder, C. Lorrette, A. Michaux, L. Gélébar1, A. Coupé, SiC/SiC Composite Materials for Fast Reactor Applications. Proceedings of ICAPP 2011, Nice, France, May 2-5, 2011, Paper 11433. [1] L. Chaffron, J. L. Séran, C. Sauder, C. Lorrette, A. Michaux, L. Gélébar1, A. Coupé, SiC/SiC Composite Materials for Fast Reactor Applications. Proceedings of ICAPP 2011, Nice, France, May 2-5, 2011, Paper 11433. y , , p [2] M. Zabiégo, C. Sauder, C. Lorrette, P. Guédeney, Tube multicouche amélioré en matériau composite à matrice céramique, gaine de combustible nucléaire en résultant et procédés de fabrication associés.Patent submitted 1 August 2011, in French. y p [2] M. Zabiégo, C. Sauder, C. Lorrette, P. Guédeney, Tube multicouche amélioré en matériau composite à matrice céramique, gaine de combustible nucléaire en résultant et procédés de fabrication associés.Patent submitted 1 August 2011, in French. g , [3] C. Sauder, J. Lamon, Influence of fiber surface roughness on mechanical behaviour of SiC/SiC minicomposites with Hi-Nicalon S and SA3 reinforcement. 35ème International Congress on Advanced Ceramic and Composites, Daytona beach 25 Janvier 2011. p y [4] E. Buet, C. Sauder, S. Poissonnet, P. Brender, R. Gadiou, C. Vix-Guterl, Influence of chemical and physical properties of the last generation of silicon carbide fibres on the mechanical behaviour of SiC/SiC composite. Journal of the European Ceramic Society, 2012. 32(3): p. 547-557. p p y ( ) p [5] A. Coupé, H. Maskrot, E. Buet, A. Renault, P.J. Fontaine, L. Chaffron, Dispersion Behavior of Laser-synthesized silicon carbide nanopowders in ethanol for Electrophoretic Infiltration. Journal of the European Ceramic Society, Vol 32, Issue 14, 3837-3850, 2012. p p y ( ) p [5] A. Coupé, H. Maskrot, E. Buet, A. Renault, P.J. Fontaine, L. Chaffron, Dispersion Behavior of Laser-synthesized silicon carbide nanopowders in ethanol for Electrophoretic Infiltration. Journal of the European Ceramic Society, Vol 32, Issue 14, 3837-3850, 2012. Innovative SiC/SiC Composites for Nuclear Applications posites fo ons VEMBER 2012 NOS Workshop, Mate December 5-7 uder, C. Lor Buet, S Pois ottet, M. Zab of refractory materials for pin cladding of 4t actors /SiC composites : stly driven by GFR fuel objectives (2004-2010) y extended to other applications : SFR & PWR MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France aterial (>> 1000°C) esistance n sparency sistance + mechanical properties + thermal propertie iati act ron osio htn tion R&D Matrix: protects the fiber and displys load transfer Fibre: ensures the mechanical strenght Interphase: bonding between fiber and matrix SIC/SIC COMPOSITE ? MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France transfer 1µm With phase Deflection of the cracks C/SiC is a non brittle ceramic 12, CEA – I s /S C O UC C O e fiber: under irradiation ⇒ ⇒ ⇒ ⇒ u Tyranno SA3 fibers only high temperature ⇒ ⇒ ⇒ ⇒ A3 fibers looks better al conductivity ⇒ ⇒ ⇒ ⇒ A3 fibers looks better MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France A3 fibers looks better Cost ⇒ ⇒ ⇒ ⇒ 3 fiber is cheaper (30%) HNS TSA3 ity ties e interphase: PyC e matrix: SiC CVI TSA TSA is is the the target target! EA – INSTN Saclay, France SIC FOR e fiber: b y es int ma nd Ty 3 e al ss Thermal exchange = High λ Irradiation mecha behavior astight lastic λ SiC is lowered under irradiation (highly lowered at low temperatures) Strain to failure εR > 0,5% MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE low temperatures) ner for s oncept - Deal with it ! - Use of SA3 reinforcement - Process a specific matrix for composites ⇒very long term work - Ok with HNS - No solutions with - Look for high irradiated mecha behavior. ment of a gastight component prepared from HNS SiC/SiC h HNS utions w or hig me SiC/S 2, CEA – INST nt trix - - - irra be ared from er fo ncep ent C C 8 – 2,9 D∼∼∼∼2.3-2.4 ±±± ING : INFLUENCE OF BRAIDING AND GRINDING MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 6 iding 3D braiding With and without grinding ded (machined) composite ginal posite • Properties can be tailored thanks to appropriate braiding • Grinding has no significant effect on C MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 6 • Properties can be tailored thanks to appropriate braiding • Grinding has no significant effect on C ing ed ( om nal osit , RIZATION: WHICH TEMPERATURE LIMITATION? of a thermal treatment (2h in Ar) on mechanical HNS tube MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 7 iC tube is not sensitive to very high temperature in inert atmosphere HNS fiber ance \| PAG ure S fiber WHICH TEMPERATURE LIMIT reatment (2h in Ar) on mechanical HNS tube NOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, Fr t sensitive to very high temperat nert atmosphere HNS OS t s ne M be is n in iC of s erial for Pin cladding: raiding (45°) 2 layers cal behavior is the traction or internal ERIZATION : FATIGUE MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE traction or internal swelling Fatigue tests: 20 -200MPa at 5 Hz No failure after 500 000 cycles! ber 5-7, 2012, CEA – INSTN Saclay, France \| PAG 00MPa at 5 Hz ailure after 500 000 cycles! 5 500 000 sions and tolerances of CMC composites Products External and internal dimensio within 0,01mm tolerance RIZATION : DIMENSIONNING MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PA ernal dimensions: ±0.01 mm city < 0.03mm with mean value of 0.02 mm. city < 0.05mm with mean value of 0.04 mm. 0.05mm with mean value of 0.04 mm htness < 0.02mm with mean value of 0.005 mm tness < 0.04mm with mean value of 0.02 mm hness) < 5µm and Rz (max roughness) < 30µm Very good dimensional accuracies (could be improved for internal part) mposites l and internal dimen hin 0,01mm toleranc 2012, CEA – INSTN Saclay, France e of 0.02 mm. of 0.04 mm. 04 mm ue of 0.005 mm ue of 0.02 mm ghness) < 30µm al accuracies internal part) es Nica CEA – INST ess) < 3 ccurac ternal p olerances of CMC com Externa with MINOS Workshop - December 5-7, 2 nsions: ±0.01 mm mm with mean value mm with mean value th mean value of 0.0 02mm with mean val 4mm with mean valu µm and Rz (max roug y good dimensiona uld be improved for al dime < 0.03 < 0.05 5mm w ss < 0 s < 0.0 ss) < 5 Ve (co ricit 0.0 htn htne ghn nsio Pro SiC/SiC CEA SiC CVD (R&H) CEA SiC/SiC posites ERIZATION: IMPURITIES CONCENTRATION Very few mpurities ities (Fe, S, N, O, H) -Nicalon S fibers MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 10 es (Fe, S, N, O, H) calon S fibers CEA \| 21 Novembre 2012 AGE 10 SiC/SiC CEA SiC CVD (R&H) CEA SiC/SiC mposites TERIZATION: IMPURITIES CONCENTRATION Very few impurities rities (Fe, S, N, O, H) -Nicalon S fibers SiC/SiC CEA SiC PURITIES CONCENTRATIO H) MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, F CEA \| 21 Novemb ) es Nica EA os Process: Process: ss CVI + EPI + PIP Objective : Increase thermal conductivity of SiCf/SiC by lowering porosity Raw material Interphase PyC + SiC Pre-densification EPI + PIP SiC green Matrix T°, P TIVE PROCESSING MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 11 o Cf/ PyC/ SiC nano + SiC Polymer Cf/ PyC/ SiC/ SiC nano + SiC Polymer of a SiC layer on composites ives : Densification and smoothing of SiCf/SiC composites Bubbles: Polymer/resin reactions Raw material ve process could be used for densification of hexagonal avid oral) for which requirements are less harsh sites hex h – INSTN on o ss ha f SiCf/ er 5-7, 201 densi ents a orosity PIP S M ss mm mm mm EA Patent) ING : « SANDWICH » CONCEPT e (CEA/DMN) n CMC Failure limit (σσσσF~300MPa - εεεεF~0,9%) e (CEA/DMN) n CMC Failure limit (σσσσF~300MPa - εεεεF~0,9%) All stages of pr are done in C MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PA nsures tightness (processing in LTMEX) mechanical resistance nd reproducible e of cladding is supposed to be tight up to failure of the pi Internal tube SiC/SiC: liner Ta : External tube SiC/SiC: 0,6 0,8 1 tion (%) tic limit Pa - εεεεE~0,04%) of microcracking tion [%] 0,6 0,8 1 tion (%) tic limit Pa - εεεεE~0,04%) of microcracking tion [%] of p in Saclay, Franc H » CONCEPT All stages o are done i LTMEX) essing e 1 it 0,9%) it 0,9%) 1 ng 1 ng ures mech d rep of c 0,6 on ( on 0,6 on ( on Sandwich Concept – Choice of liner 5E-04 6.0E-04 6.5E-04 7.0E-04 7.5E-04 8.0E-04 8.5E-04 9.0E-04 9.5E-04 1.0E-0 1/T(K-1) ka et al) Ni (Naka et al) Co (Naka et al) a et al) V et Cr (Naka et al) Zr (Naka et al) ka et al) Nb (Naka et al) Ta (Naka et al) numurthy et Schmid-Fetzer) Ni (Bhanumurthy et Schmid-Fetzer) Cr (Bhanumurthy et Schmid-Fetzer) ka et Feng) Mo (Yoon et al) W (Lee et al) ratyan et al) Nb (Joshi et al) Mo (Kharatyan et al) CH » CONCEPT: WHICH LINER? MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 13 ntalum and Niobium are the best candidates for GFR Is it still true for PWR or BWR ? C 1400°C 1200°C 1000°C 900°C 800°C 750°C Ta Nb b AGE 13 0°C 1200° talu Ta ndwich Concept – tightness during tensile test CHARACTERIZATION: PERMEATION 150 200 250 300 350 400 F/So (MPa) pression relative Hélium : 2 bar fin acquisition spectro en continu acquisition uniquement aux palliers MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| 14 Detection limit « sandwich » concept allows to keep tightness up failure of SiC/SiC pin cladding 0 50 100 0 2000 4000 6000 8000 10000 12000 1400 temps (s) acquisition uniquement aux palliers 14 imit ss up 2000 14 MINOS andwic NbC NbC SiC TaC Ta5Si4C Ta5Si3 TaSi2 Ta Précipités Ta2C Nb – 1000h – 1200°C – Sandwich Ta 0h 63h 250h 1200 C ICH CHARACTERIZATION MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 15 250h 560h 1000h encouraging results with Ta ction zones are not symmetric observed with plates) her characterization needed 63h 50h PAGE 50h 60h 00h wich Ta 0 63 25 25 en tio obs er en tio obs er NbC CTERIZATION: IRRADIATION 0 200 400 600 800 1000 1200 0 0.2 0.4 0.6 0.8 Strain (%) Tensile Stress (MPa) unirradiated Sample 1 (3.1 dpa) Sample 2 (3.1 dpa) rageing results have being CVI minicomposite (CROCUS n performed in OSIRIS) XT STEP: MINOS Workshop - December 5-7, 2012, CEA – INSTN Saclay, France \| PAGE 16 0 (sodium, 550°C up to 105-120 dpa SiC) of SiC/SiC composites at such doses ding sandwich specimens) PIE are expected for 2015 XT STEP: should should start start on on december december 19, 2012 19, 2012 rati netw al p ilo wo app
https://openalex.org/W3015944293	https://digital.library.unt.edu/ark:/67531/metadc1639367/m2/1/high_res_d/s10086-020-1853-x.pdf	English	null	Fabrication of densified wood via synergy of chemical pretreatment, hot-pressing and post mechanical fixation	Journal of wood science	2,020	cc-by	7,843	Introductioni methods, softening of wood material before mechanical pressing is required. The process of softening is known as a pretreatment and combined with following mechani- cal pressing, achieving better compressing result. The thermo-mechanical densification [3–5], thermal-hydro- mechanical [6–8], hygro-mechanical steam treatment [9, 10], mechanical-microwave treatment [11], and high- pressure treatment [12, 13] were developed to manufac- ture densified wood, in which the thermal-hydro is the most common and simplest pretreatment method cur- rently. The cell walls of wood would be swelled by water although it cannot penetrate into crystalline region of cellulose. Then, the glass-transition temperature of amor- phous polymers (hemicellulose and lignin) decreases under the hydrothermal condition. As a result, the pre- treated wood allows low compressive force while without Wood densification is one of the effective techniques for improving the strength of low density wood species, which can effectively extend wood product application due to the enhanced density of surfaces or entirety of wood. As early as 1930s, the densified wood was used replacing metal materials in military aircraft in USA and Germany. In recent decades, many approaches were completed for manufacturing densified wood, such as mechanical thermal pressing [1], viscoelastic thermal compression (VTC) [2]. Whatever the compression Correspondence: shijt@njfu.edu.cn 1 College of Materials Science and Engineering, Nanjing Forestry University, Nanjing 210037, China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. Abstract Wood densification can improve the strength of low density wood species and extend wood product applications. To enhance the wood compressive quality, chemical pretreatments for pristine wood have widely been used. Densified Abies wood was fabricated by combining NaOH/Na2SO3 solution treatment, hot-pressing and post mechanical fixa- tion. The appearance, color, chemical composition, and physiology and mechanical properties before and after the densification treatment were examined by the colorimeter, FTIR and mechanical testing machine, respectively. Sur- face color of Abies wood was changed obviously after the densification. The values of brightness L and b* decreased but the value of a* showed a slight increase in the densified wood. FTIR results confirmed that the color changes can be explained by the degradation of hemicellulose and lignin in wood cell walls and migration of extractives during the densification process. Sufficient removal of wood polymers resulted in the average compression ratio of about 80% in the radial direction of the natural wood. The density of densified wood increased with the wood thickness up to 1.227 g cm−1, accounting for a 169% increase compared to that of the pristine wood. Modulus of rupture (MOR) and modulus of elasticity (MOE) in the thickness direction of densified wood also markedly enhanced. Degradation of polymers in wood cell walls also was reconfirmed by the difference of fracture interface. All the results suggested that the densified softwood can be easily fabricated using the proposed method and the new densified softwood can be appropriately used as interior decoration materials. Keywords: Densification, Abies wood, Decorative panel, Hot-pressing, NaOH/Na2SO3 pretreatment Jiangtao Shi1,2* , Junyi Peng1, Qiongtao Huang2, Liping Cai1,3 and Sheldon Q. Shi3 Jiangtao Shi1,2* , Junyi Peng1, Qiongtao Huang2, Liping Cai1,3 and Sheldon Q. Shi3 Shi et al. J Wood Sci (2020) 66:5 https://doi.org/10.1186/s10086-020-1853-x Shi et al. J Wood Sci (2020) 66:5 https://doi.org/10.1186/s10086-020-1853-x Journal of Wood Science Shi et al. J Wood Sci (2020) 66:5 https://doi.org/10.1186/s10086-020-1853-x Journal of Wood Science Open Access Correspondence: shijt@njfu.edu.cn 1 College of Materials Science and Engineering, Nanjing Forestry University, Nanjing 210037, China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. Wood material and densified treatment i Abies spp. wood was cut into blocks with a size of 80 mm × 50 mm (longitudinal × tangential), which had three different thicknesses (in the radial direction) of 17.75, 8.25 and 3.15 mm. The initial average moisture content of wood was 8.76%. After the densified treat- ment, the average thicknesses of the thicker, medium and thinner samples were 3.25, 1.67 and 0.81 mm, respectively. Densified wood was fabricated by combin- ing chemical pretreatment and hot-pressing technology. First, equivalent volumes of 2.5 mol L−1 NaOH and 0.4 mol L−1 Na2SO3 aqueous solution were mixed, which is widely used in the chemical pulping industry. Wood samples were immersed into the prepared mixed solu- tion and heated at 95 °C for 5 h in a water bath. After the wood sank into the solution and the color was darkened, the chemical pre-treated wood was washed successively with 1% HCl and deionized water and kept in water. The secondary step was hot-pressing with a press of DSKE (Wabash, USA) at 25 MPa, 160 °C for 12 h followed by covering wood surface with tinfoil and interrupted heat- ing and hold the pressure for 24 h. All the densified wood samples were dried at 80 °C for 24 h and stored for the following characterization. There were five replicates for each wood thickness. i However, combination of chemical pretreatment and mechanical compression maybe an effective method to solve the recovery in densified wood [15]. Wood poly- mers always degraded and extracted in the chemical pretreatment. Alkaline solution, such as sodium hydrate or potassium hydroxide (10–20%), is the mostly chemi- cal pretreatment method used for the wood densification manufacturing. On the other hand, alkaline solution is normally used in the pulping industry. Under the alka- line condition, cellulose, hemicellulose and lignin all would be swelled or hydrolyzed at different levels. Wood disintegration in cells or cell walls facilitates the maxi- mum compression in hot press. Ammonia is another reagent for pretreatment in the manufacturing of densi- fied wood because it has a relatively high affinity to the polymers in wood cell walls. Although ammonia treat- ment shows no obvious effect on the content of wood polymers, it can easily access crystal region of cellulose and disrupt the lignin–carbohydrate complex. Shi et al. J Wood Sci (2020) 66:5 Page 2 of 9 densified Abies wood via the co-processing of chemical pretreatment, hot-pressing and post mechanical fixation. The color, chemical composition and physical/mechani- cal properties of the wood after and before densification were evaluated. breaking wood cell walls during the next hot-pressing. However, partly elastic deformation is reversible in this densified wood, especially in earlywood. Therefore, the recovery of deformation under humidity condition is an inevitable problem. Previous reports stated that water absorption expansion ratio in the compression direction reached up to 50% if the deformation in densified wood was not fixed [14]. Some methods, such as high-tem- perature steam, resin impregnation, and high-frequency microwave heating, are mostly used for deformation fixa- tion of densified wood in the wood industry. Wood material and densified treatment Moreo- ver, the plasticizing effect of the ammonia treatment is temporary and the deformed wood would be return- ing back to its initial form and the strong pungent smell after the ammonia treatment limits its industrial applica- tion [16, 17]. Later, Song et al. manufactured the high- strength densified wood by coalescent method which combined NaOH/Na2SO3 solution pretreatment and hot press at 100 °C, exhibiting high specific tensile strength of 422.2 MPa cm3 g−1, which is higher than some metals [18]. In recent years, NaOH/Na2SO3 solution system was widely used to degrade hemicellulose and lignin in wood cell walls for preparing transparent wood composites [19], super flexible wood [20] or other functional wood materials [21]. SEM and FTIRh The scanning electron microscopy (SEM, Quanta200, FEI, USA) was employed to observe the microstruc- ture on the transverse section and tangential section of the densified wood. Before SEM observation, control wood and chemical-treated wood samples were oven- dried until the constant weight was obtained and cov- ered by gold sputtering. The observation condition was under vacuum and at an accelerating voltage of 25 kV. Fracture behavior of pristine wood and densified wood was observed after destruction in mechanical testing on Quanta200 at the same condition. The Fourier trans- form infrared spectrum of wood surface was collected using Nicolet 6700 (Thermo Scientific, USA) equipped with Smart orbit using attenuated total reflectance (ATR) model. Every sample was scanned between 400 and 4000 cm−1 at a resolution of 4 cm−1. To quantitatively compare the spectra of differently densified wood with the spectra of pristine wood for evaluating the chemical changes, the average spectra were further normalized based on the high band at nearly 1027 cm−1, assigned to C–O stretching, even this band does not remain invari- able throughout the whole experiments, because both lignin and carbohydrate exhibit this band [22, 23]. dry mass Before treated −dry mass After treated/ dry mass Before treated × 100 . The lengths of all test samples were around 78.5 mm. The densified wood and pristine wood samples were cut into approximately 12.0 mm in width. The modulus of rup- ture (MOR) and modulus of elasticity (MOE) were tested using the mechanical testing machine (UTM4304,Suns, Shenzhen, China). Three-point bending model was per- formed and the transverse loading was carried on the tangential surface of wood at the middle span of the test samples. The span of the test set was 56.0 mm and the loading rate was 5 N min−1. After the mechanical test- ing, the final average moisture contents of pristine wood, thick densified wood, medium densified wood and thin densified wood were 8.58, 7.38, 8.29 and 7.68%, respec- tively. All mechanical data were calculated by built- in software MaterialTest 4.0. Excel (Microsoft, USA) and Origin 8.0 were employed to analyze data and plot figures. Test of chromaticity value Th h CR 5 y The chromameter CR-5 (Konica Minolta, INC, Japan) was employed for chromaticity value test of wood sur- face during the densification treatment. The wood tan- gential section was measured using the chromameter under optional target mask (3 mm diameter) and the D65 light source (Daylight, color temperature of 6504 K). The measurement type was reflectance mode at 10° observer angle and specular component was excluded. Color measurements were completed at nine locations on each sample and mean values were calculated for each wood sample. The color was described using the CIELab system, which was established by International Commis- sion on Illumination in 1976. The CIELab* system con- sists of three perpendicular axes, where L* describes the lightness, a* the chromatic coordinates on the green to red axis and b* the chromatic coordinates on the blue to yellow axis. Five replicates in each wood thickness were completed. Widely planted in North America and Nordic region, Abies wood is one of the vital softwood species used in residential buildings and other construction materials. Compared to hardwood, softwood commonly consists of single cell type, more than 90% of tracheid, result- ing in some special properties, such as the simple wood texture, and lightly color, lower density, which limits it to be applied for interior decoration or wall panels. This work aims to develop a feasible method for fabricating Shi et al. J Wood Sci (2020) 66:5 Page 3 of 9 Determination of physical propertiesh Three different thicknesses of Abies wood (Fig. 1a) were selected for fabricating densified wood by the radial compression loading mode. The wood appearance and color were changed under the synergy of chemical pre- treatment and hot-pressing (Fig. 1). In Fig. 1b, it can be found that the wood was squashed and its color The oven-dry density of all samples was calculated by “dry- mass/dry-volume”. The compression ratio (CR) was calcu- lated as follows: (thickness Before treated −thickness After treated)/ thickness Before treated × 100. (thickness Before treated −thickness After treated)/ thickness Before treated × 100. Fig. 1 Appearance and color changes of pristine wood (PW) and densified wood. a Three pristine wood samples with different thicknesses; b densified wood; c chromatic value of pristine wood and densified wood. DW densified wood Fig. 1 Appearance and color changes of pristine wood (PW) and densified wood. a Three pristine wood samples with different thicknesses; b densified wood; c chromatic value of pristine wood and densified wood. DW densified wood Shi et al. J Wood Sci (2020) 66:5 Page 4 of 9 Page 4 of 9 the trend of wood surface became reddish. In conifer wood, it was shown directly proportional relationship between the extractive content and redness values [27]. This suggested that more extractives moved to the wood surface during the densification process. After compar- ing the colors of three different wood thicknesses, it was found that both L* and b* values of the thickest wood were lower than those in the wood samples with medium and thin thicknesses. With the increase of wood thick- ness, b* value trended toward to decrease. The value of a* showed no noticeable differences. The results suggested that the thickness of pristine wood affected its color after the densification treatment. This might be explained by the difference of chemical reaction efficiency with dif- ferent wood thicknesses. More cell walls in the thickest wood meant more chemical reaction and generation of chromophoric groups, which would be shine through in the hot-pressing process. The total color difference (ΔE) was calculated based on the reference to pristine wood. The following formula was utilized: darkened after the densification. On the densified wood surface, the light and black color alternation was caused by early wood and late wood, which was resulted from differences in the amount and size of wood cell walls. Determination of physical propertiesh The extraction of sodium hydroxide solution from wood matrix resulted in the color changes after the densification. Previous studies reported that the NaOH/Na2SO3 solution system can dissolve the extrac- tives in wood and can also degrade hemicellulose and lignin in wood cell walls [15]. The degradation of hemi- cellulose and lignin was another reason for wood color changes in the densified wood [24]. i Due to the polysaccharide composition of hemicel- lulose, various saccharic acids are gradually generated through the peeling reaction started from the reduc- ing terminal group of polysaccharides in alkaline envi- ronment. Moreover, new chromophoric group may be polymerized by various saccharic acids and extractions [25]. In addition, lignin was also subjected to dearylation and dealkylation reaction with both nucleophile SO3 2− and OH− ions in chemical pretreatment and also pro- duced some new chromophoric groups, such as quinone, epoxide, or lignosulphonates. Another reason for the color changes in wood densification was thermochroma- tism of the chemicals on wood surface. In this work, the 160 °C hot-pressing temperature and aerobic condition affected the migration and oxidation of chromophoric and auxochrome groups. The different colors between early wood and late wood perhaps were caused by the more cell wall polymers for stronger reaction degree. Bekhta et al. also reported that the brightness decreased in the short-term thermo-mechanically densified veneer of various wood species [26]. These mentioned results suggested that the synergistic effect of chemical pretreat- ment, hot-pressing and post mechanical fixation was responsible for color darkening in the densified wood. From the appearance, the densification treatment signifi- cantly improved the decorative value of Abies wood. E = (Lx −L0)2 + (ax −a0)2 + (bx −b0)21/2 , where Lx, ax and bx are values measured in the densified wood and L0, a0 and b0 are values measured in the pris- tine wood. Figure 1c shows that all densified wood pre- sented significantly ΔE, which can be easily visible with naked eyes. It was indicated that the densification in this work influenced wood color seriously. FTIR analysis It was indicated that the polar functional groups conjugated with benzene ring caus- ing some changes. Because of the highly susceptible to nucleophilic reactions between carbonyl C=O in lignin and hemicellulose and SO3 2− and OH− ions in NaOH/ Na2SO3 solution, the disappearance of absorption band at 1650 cm−1 was related to the vanishing of conjugate carbonyl group attached to benzene ring. The sharp band at 1507 cm−1 was attributed to vibration absorp- tion of benzene ring skeletal. In Fig. 2, this band was still present in the densified wood suggesting that there was no chemical reaction of benzene ring in NaOH/ Na2SO3 solution. A certain amount of lignin in wood cell walls benefited to bonding strength in microfibrils. After normalization, the intensity of absorption band at 1507 cm−1 increased more in the densified wood than the pristine wood, indicating the increase of lignin relative content. It was also confirmed by the increased intensity ratio of I1507/I1372 (Table 1). To the contrary, previously studies insisted on lignin would be degraded in NaOH/Na2SO3 solution [18, 30]. This contradictory can be explained by the migration of degraded prod- ucts of hemicellulose and lignin and depositing on the surfaces of the densified wood. The FTIR data col- lected on the densified wood surfaces by ATR mode in this study confirmed this finding as well. In addition, the lignin molecular structure may be changed from vitreous to high-elastic [1] and the changes of band at 1261 cm−1 (C–H of guaiacyl ring in lignin) indicated that lignin unit may be changed. The bands 1425 cm−1 (C–H2 scissoring vibration), 1372 cm−1 (C–H bending vibration) and 897 cm−1 (C1 vibration) can character- ize cellulose and hemicellulose. And previous reports stated that the relative crystallinity of cellulose can be characterized by the band intensity ratio I1372/I2900 and I1427/I897 [31, 32] and it was shown that this ratio was higher in the densified wood than the pristine wood. It can be inferred that the degradation of hemicellulose and partly amorphous cellulose resulted in the recom- bination of cellulose microstructure and finally caused the increase in cellulose crystallinity. Comparatively, the wood thickness might influence wood chemical properties to some extent. FTIR analysis To uncover the changes of wood cell wall polymers dur- ing the densification process, the infrared spectra were Fig. 2 Original infrared spectrum of pristine wood (PW) and densified wood (DW) Fig. 2 Original infrared spectrum of pristine wood (PW) and densified wood (DW) To quantify the changes of wood color in the densifi- cation, chromaticity value on wood surface was meas- ured by the chromameter CR-5. In Fig. 1c, more wood brightness value (L) and b value decreased in the den- sified wood than those in the pristine wood. Decreas- ing of L* means wood color darkening, which may be caused by the degradation of hemicellulose and move- ment of extractives. Kubovský et al. also reported that the decrease of L* value in wood surface was closely related to the degradation of hemicellulose [24]. The decreas- ing of b* value describes the trend of the wood surface towards bluish, which can be explained by the reduction of a tonality component in color and a slight decreas- ing value of color saturation [26]. However, the slightly increased a* value in the densified wood indicated that Fig. 2 Original infrared spectrum of pristine wood (PW) and densified wood (DW) Fig. 2 Original infrared spectrum of pristine wood (PW) and densified wood (DW) Shi et al. J Wood Sci (2020) 66:5 Page 5 of 9 collected and are shown in Fig. 2. The attribution of absorption band was according to the previous publish of Pandey [28]. The bands at 3378 cm−1 and 2900 cm−1 attributed to O–H and C–H stretching vibration in cel- lulose, hemicellulose and lignin. Some characteristic bands were analyzed in untreated wood and densified wood. The absorption band at 1729 cm−1 attributed to C=O stretching vibration in unconjugated ketones, which was a characteristic band of hemicellulose [25, 28]. Disappearance of this band in the densified wood was caused by the deacetylation in hemicellulose, indi- cating that hemicellulose entirely dissolved in NaOH/ Na2SO3 solution. After the densification treatment, the intensity ratio of I1372/I1507 decreased, which sug- gested that the content of holocellulose reduced and also proved the decrease in relative content of hemi- cellulose [29]. The band in range of 1588–1650 cm−1 appeared shoulder absorption peak in the pristine wood but turned into sharp peak only at 1588 cm−1 (C=C stretching vibration on benzene ring) in the den- sified wood spectrum. FTIR analysis The probable reasons which affected the FTIR results were (1) the more massive thick specimens had greater potential for migrating and accumulating some chromatic extractives at the surface than thin specimens and (2) the inside moisture in the thin wood sample was more easily volatilized than the thicker wood in the hot-processing treatment. collected and are shown in Fig. 2. The attribution of absorption band was according to the previous publish of Pandey [28]. The bands at 3378 cm−1 and 2900 cm−1 attributed to O–H and C–H stretching vibration in cel- lulose, hemicellulose and lignin. Some characteristic bands were analyzed in untreated wood and densified wood. The absorption band at 1729 cm−1 attributed to C=O stretching vibration in unconjugated ketones, which was a characteristic band of hemicellulose [25, 28]. Disappearance of this band in the densified wood was caused by the deacetylation in hemicellulose, indi- cating that hemicellulose entirely dissolved in NaOH/ Na2SO3 solution. After the densification treatment, the intensity ratio of I1372/I1507 decreased, which sug- gested that the content of holocellulose reduced and also proved the decrease in relative content of hemi- cellulose [29]. The band in range of 1588–1650 cm−1 appeared shoulder absorption peak in the pristine wood but turned into sharp peak only at 1588 cm−1 (C=C stretching vibration on benzene ring) in the den- sified wood spectrum. It was indicated that the polar functional groups conjugated with benzene ring caus- ing some changes. Because of the highly susceptible to nucleophilic reactions between carbonyl C=O in lignin and hemicellulose and SO3 2− and OH− ions in NaOH/ Na2SO3 solution, the disappearance of absorption band at 1650 cm−1 was related to the vanishing of conjugate carbonyl group attached to benzene ring. The sharp band at 1507 cm−1 was attributed to vibration absorp- tion of benzene ring skeletal. In Fig. 2, this band was still present in the densified wood suggesting that there was no chemical reaction of benzene ring in NaOH/ Na2SO3 solution. A certain amount of lignin in wood cell walls benefited to bonding strength in microfibrils. After normalization, the intensity of absorption band at 1507 cm−1 increased more in the densified wood than the pristine wood, indicating the increase of lignin relative content. It was also confirmed by the increased intensity ratio of I1507/I1372 (Table 1). To the contrary, previously studies insisted on lignin would be degraded in NaOH/Na2SO3 solution [18, 30]. FTIR analysis This contradictory can be explained by the migration of degraded prod- ucts of hemicellulose and lignin and depositing on a and b values indicate the intensity ratio of the absorption band and the maximum absorption band near 1027 cm−1 in that curve Physical and mechanical characterization Physical and mechanical characterization Figure 3 shows the oven-dried densities, compression ratios (CR), MOE and MOR of the wood before and after densification. The wood oven-dry density increased over 1.00 g cm−1 in all densified woods compared to 0.455 g cm−1 of the pristine wood (Fig. 3a). The incre- ment of density depended on wood sample thickness and the highest density was 1.227 g cm−1 (169% higher than the pristine wood) in the thickest wood sample. Moreo- ver, wood density increased from the thin to the thick densified wood sample and CR showed the similar trend. The higher density appeared in the thicker wood sample, and the higher compressibility was obtained. Whereas, the lower density appeared in the thinner wood sam- ple and the lower compressibility was achieved. As well known, many studies investigated various compression Table 1 The band intensity and ratio of pristine wood (PW) and densified wood (DW) a and b values indicate the intensity ratio of the absorption band and the maximum absorption band near 1027 cm−1 in that curve I1372/I1507 I1507/I1372 I1372/I2900 I1427/I897 Ia 1588 Ib 1507 PW 0.40 2.47 0.32 1.11 – 0.16 Thin DW 0.39 2.57 0.43 4.63 0.39 0.29 Medium DW 0.32 3.21 0.49 5.50 0.81 0.39 Thick DW 0.33 3.00 0.37 3.50 0.45 0.33 Table 1 The band intensity and ratio of pristine wood (PW) and densified wood (DW) Shi et al. J Wood Sci (2020) 66:5 Shi et al. J Wood Sci Page 6 of 9 Fig. 3 Physical and mechanical characteristics of pristine wood (PW) and densified wood (DW). CR compression ratio, LWR loss weight ratio, MOR modulus of rupture, MOE modulus of elasticity Fig. 3 Physical and mechanical characteristics of pristine wood (PW) and densified wood (DW). CR compression ratio, LWR loss weight ratio, MOR modulus of rupture, MOE modulus of elasticity ratios of densified wood under different processing con- ditions. It was reported that the compression ratio was generally lower than 30% after the densification with- out any chemical pretreatment [33, 34]. However, in this work, the average compression ratio was approximately 80% in the radial direction of wood. We inferred that the pretreatment with NaOH/NaSO3 solution was one of the reasons accounting for the higher compression ratio in densified wood. Also, the higher compaction ratio can be obtained by other pretreatments, such as saturated steam. Physical and mechanical characterization The maximum MOR is about 228.5 MPa in the thinnest densified wood and increased by 97% than the pristine wood. MOE also increased up to 33.55 GPa in the thinnest densified wood and 346% of that in pristine wood. Overall, the increment of strength showed negatively relation with the wood sample thickness. Cruz et al. reported that the content of hemicellulose in the densified wood showed high nega- tive correlation to MOE and MOR [8], while the positive correlation was shown compared to the density of wood. As well known, wood cell wall is mainly consisted of cel- lulose, hemicellulose and lignin, and the cellulose chain aggregates microfibrils and serves as skeleton as well as hemicellulose and lignin are crosslinked into microfibrils. These three polymers have different contributions to the mechanical properties of wood [39]. Hence, in this work, the synergistic effect of hemicellulose and lignin was degraded or partly degraded during the NaOH/Na2SO3 pretreatment combined with recombination of wood cells in the hot-pressing process, resulting in the increase in density and significant increase in MOR and MOE for the densified wood. Physical and mechanical characterization Kutnar and Kamke treated Douglar-fir wood by pressurized steam before compression and obtained a compaction ratio of 153% using hydrothermal treatment with a pressure of lower than 5 MPa [35]. According to the previous reports, part removal or degradation of amorphous polymers in wood is beneficial to adequate compression [36, 37]. In this work, wood hemicellulose and partly lignin were degraded in the pretreatment with NaOH/NaSO3 solution, which was confirmed by FTIR results, and allowed the wood easier to be com- pressed in the hot-processing treatment. Meanwhile, the much higher density and strength of the wood could be achieved under sufficient compressibility [38]. This prob- ably mainly attributed to synergistic effect of degradation of hemicellulose and lignin in the chemical pretreatment and recombination of wood cells during the hot-pressing process. Abies wood is easily densified and cell recom- bined because of the swelling of cell wall in the alkaline medium environment of NaOH/Na2SO3 solution. From the similar loss weight ratio (about 15%) in the densi- fied wood, it was inferred that the wood samples with three different thicknesses subjected same degradation degrees.h Compared to the pristine wood, both MOR and MOE were increased in the densified wood. The maximum MOR is about 228.5 MPa in the thinnest densified wood and increased by 97% than the pristine wood. MOE also increased up to 33.55 GPa in the thinnest densified wood and 346% of that in pristine wood. Overall, the increment of strength showed negatively relation with the wood sample thickness. Cruz et al. reported that the content of hemicellulose in the densified wood showed high nega- tive correlation to MOE and MOR [8], while the positive correlation was shown compared to the density of wood. As well known, wood cell wall is mainly consisted of cel- lulose, hemicellulose and lignin, and the cellulose chain aggregates microfibrils and serves as skeleton as well as hemicellulose and lignin are crosslinked into microfibrils. These three polymers have different contributions to the mechanical properties of wood [39]. Hence, in this work, the synergistic effect of hemicellulose and lignin was degraded or partly degraded during the NaOH/Na2SO3 pretreatment combined with recombination of wood cells in the hot-pressing process, resulting in the increase in density and significant increase in MOR and MOE for the densified wood. Compared to the pristine wood, both MOR and MOE were increased in the densified wood. Microstructure of densified wood On the other hand, the densified wood showed layered fracture interface (Fig. 4f, g), which can be explained by the longitudinal sliding in tracheid aggregates. The main reason of this fracture behavior was the decreased adhesion between tracheids after the deg- radation of hemicellulose and lignin in middle lamella. Although layered fractures occurred in all the densified wood samples, it was not obvious in the thinnest sample (Fig. 4g). A number of fibers present in fracture surface of the thick densified wood (Fig. 4h) further confirmed excellent mechanical properties to make sure it can be widely used in wood structure building. However, the tracheid lumens on the transverse section of the den- sified wood almost vanished (Fig. 4c). Because of the regular radial arrangement, only cracks were present inside the compressed tracheids in the thickness direc- tion. It was probably caused by the degradation of middle lamella at the loading direction during the hot-pressing. Meanwhile, previous reports suggested that such hydro- thermal conditions generated more microcracks on wood cell walls [25, 40]. Wood tracheids showed a bunch of aggregates on the tangential surface in the densified wood (Fig. 4d), which indicated that wood tracheids subjected to sufficient compression and recombination after the chemical, hot-pressing and mechanical co-pro- cessing. Furthermore, the fracture surfaces of the natu- ral wood and densified wood were also observed by SEM (Fig. 4e–h). The three-point bending method and loading on the thickness direction of wood samples were used. In Fig. 4e, the fracture interface of natural wood shows jagged irregularly fracture and tracheids were pulled out to each other (Fig. 4e). On the other hand, the densified wood showed layered fracture interface (Fig. 4f, g), which can be explained by the longitudinal sliding in tracheid aggregates. The main reason of this fracture behavior was the decreased adhesion between tracheids after the deg- radation of hemicellulose and lignin in middle lamella. Although layered fractures occurred in all the densified wood samples, it was not obvious in the thinnest sample (Fig. 4g). A number of fibers present in fracture surface of the thick densified wood (Fig. 4h) further confirmed the degradation of intercellular layers and secondary cell walls of tracheid. Another reason should be considered was the lateral expansion of different wood thicknesses in the hot-processing treatment. Microstructure of densified wood When all specimens were compressed with the same compression stress, the thin specimens had more lateral restraint than the thick speci- mens due to the friction of the press plates [41]. At last but not least, because of the complicated wood structure, the thicker specimens were more likely to experience the fracture and stress concentration during compres- sion, which may destroy wood structure easily and finally reduced MOR and MOE. There is sn evidence of fracture between tracheids in Fig. 4d, f. The tracheids are clearly displaced out of longitudinal alignment as a result of the Poisson effect. Microstructure of densified wood To testify the changes on microstructure of wood cell walls, SEM was used to observe the transverse and tan- gential surfaces of the wood samples before and after the densification. On the transverse surface, it was shown the honeycomb holes (tracheid lumen) on natural Abies wood tracheids, which were arranged radial-regularly (Fig. 4a). Wood tracheids showed as grooves were inter- val with uniserial wood rays on the tangential surface (Fig. 4b). As well known, Abies wood is one of the soft- wood and its typical regular cell arrangement provides The MOR and MOE in the thickness direction of the densified wood and pristine wood are shown in Fig. 3b. Shi et al. J Wood Sci (2020) 66:5 Shi et al. J Wood Sci Page 7 of 9 Fig. 4 SEM of pristine wood (a cross-section, b tangential section), densified wood (c cross-section, d tangential section), and rupture of various wood (e pristine wood, f thick densified wood, g thin densified wood, h magnifying of thick densified wood). T tracheid, R rays, RD resin duct Fig. 4 SEM of pristine wood (a cross-section, b tangential section), densified wood (c cross-section, d tangential section), and rupture of various wood (e pristine wood, f thick densified wood, g thin densified wood, h magnifying of thick densified wood). T tracheid, R rays, RD resin duct excellent mechanical properties to make sure it can be widely used in wood structure building. However, the tracheid lumens on the transverse section of the den- sified wood almost vanished (Fig. 4c). Because of the regular radial arrangement, only cracks were present inside the compressed tracheids in the thickness direc- tion. It was probably caused by the degradation of middle lamella at the loading direction during the hot-pressing. Meanwhile, previous reports suggested that such hydro- thermal conditions generated more microcracks on wood cell walls [25, 40]. Wood tracheids showed a bunch of aggregates on the tangential surface in the densified wood (Fig. 4d), which indicated that wood tracheids subjected to sufficient compression and recombination after the chemical, hot-pressing and mechanical co-pro- cessing. Furthermore, the fracture surfaces of the natu- ral wood and densified wood were also observed by SEM (Fig. 4e–h). The three-point bending method and loading on the thickness direction of wood samples were used. In Fig. 4e, the fracture interface of natural wood shows jagged irregularly fracture and tracheids were pulled out to each other (Fig. 4e). Conclusions In summary, the densified wood samples with three different thicknesses were fabricated by the combina- tion of chemical pretreatment, hot-pressing and post mechanical treatments. Surface color of Abies wood changed obviously after the densification. Quantitative results indicated that the values of brightness L* and b* decreased but the value of a* showed a slight increase in the densified wood. The FTIR examinations confirmed that the color changes can be explained by the degrada- tion of hemicellulose and lignin in wood cell walls and migration of extractives during the densification process. New chromophoric groups generated from re-polym- erization of wood cell wall degradation product were the other reason for the color changes in the densified Shi et al. J Wood Sci (2020) 66:5 Page 8 of 9 Page 8 of 9 wood. In this work, sufficient removal of wood polymers resulted in the average compression ratio of about 80% in the radial direction of the pristine wood. Furthermore, the increased compression ratio positively related to the wood density. The density of wood increased with wood thickness up to 1.227 g cm−1, counting 169% higher than the pristine wood. Being benefited from the increased density, MOR and MOE in the thickness direction of the densified wood also markedly enhanced. The fracture interface of the densified wood reconfirmed the deg- radation of polymers in wood cell walls. Moreover, the thickness of initial wood sample affected the color, den- sity, chemical groups and mechanical properties in the densified wood. All the mentioned results suggested that the densified softwood can be easily fabricated using the proposed method and the new densified softwood can be considered as an interior decoration material. 4. Wehsener J, Brischke C, Meyer-Veltrup L, Hartig J, Haller P (2018) Physical, mechanical and biological properties of thermos-mechanically densified and thermally modified timber using the Vacu3-process. 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Yu Y, Zhang F, Zhu S, Li H (2017) Effects of high-pressure treatment on Poplar wood: density profile, mechanical properties, strength potential index, and microstructure. BioRes 12(3):6283–6297 The authors declare that they have no competing interests. The authors declare that they have no competing interests. 18. Song J, Chen C, Zhu S, Zhu M, Dai J, Ray U, Li Y, Kuang Y, Li Y, Quispe N, Yao Y, Gong A, Leiste UH, Bruck HA, Zhu JY, Vellore A, Li H, Minus ML, Jia Z, Martini A, Li T, Hu L (2018) Processing bulk natural wood into a high- performance structural material. Nature 554:224–228 Funding The authors like to thanks for finical supporting of the National Key Research & Development Program of China (2017YFD0600204). i 16. Berzins GV, Rocens K (1970) Strength and elasticity of the ammonia treated and compressed birch wood. 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https://openalex.org/W4295996720	https://boletimcn.museu-goeldi.br/bcnaturais/article/view/782/570	English	null	The amphibians of Pará, Brazil	Boletim do Museu Paraense Emílio Goeldi. Ciências Naturais	2,022	cc-by	15,559	Palavras-chave: Lissamphibia. Amazônia. Lista de espécies. Herpetologia. Cassundé, G. F., Sturaro, M. J., Maciel, A. O., Prudente, A. L. C., Sarmento, J. F. M., & Peloso, P. (2022). The amphibians of Pará, Brazil. Boletim do Museu Paraense Emílio Goeldi. Ciências Naturais, 17(2), 445-473. http://doi.org/10.46357/bcnaturais.v17i2.782 Autora para correspondência: Gisele Ferreira Cassundé. Museu Paraense Emílio Goeldi. Programa de Pós-Graduação em Zoologia (UFPA/ MPEG). Setor de Vertebrados. Av. Perimetral, 1901 – Terra Firme. Belém, PA, Brasil. CEP 66077-830 (gi.cassunde@hotmail.com). Recebido em 09/07/2021 Aprovado em 10/02/2022 Responsabilidade editorial: José Nazareno Araújo dos Santos Junior The amphibians of Pará, Brazil Gisele Ferreira CassundéI \| Marcelo José SturaroII \| Adriano Oliveira MacielIII \| Ana Lúcia da Costa PrudenteIV \| João Fabrício Melo SarmentoIV \| Pedro PelosoI, III IUniversidade Federal do Pará. Programa de Pós-Graduação em Zoologia. Belém, Pará, Brasil IIUniversidade Federal de São Paulo. Departamento de Ecologia e Biologia Evolutiva. Diadema, São Paulo, Brasil IIIMuseu Paraense Emílio Goeldi/MCTI. Coordenação de Zoologia. Programa de Capacitação Institucional. Belém, Pará, Brasil IVMuseu Paraense Emílio Goeldi/MCTI. Coordenação de Zoologia. Belém, Pará, Brasil Abstract: Pará is the second largest state in Brazil, with all its territory within the Amazonian rainforest. The state has a wide variety of habitats and bioregions and is home to a large diversity of species. In the race to uncover this diversity and to provide support for future research, we have compiled the first list of amphibian species occurring in Pará, based on the identification of specimens deposited in scientific collections and analysis of primary literature. We recorded 195 species of amphibians, 30 of which are endemic to the state, and five represented new occurrence records. Despite its large territory, high number of endemic species and very intense levels of deforestation, only five species are officially considered threatened with extinction. We discuss reasons as to why so few species are threatened and why this number is likely to increase soon. We also discuss issues related to taxonomy, such as the occurrence of multiple unnamed species that have already been discovered in Pará. Finally, we highlight some opportunities and challenges for future research areas with amphibian diversity and taxonomy in the state of Pará. Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 ARTIGOS CIENTÍFICOS INTRODUCTION et al., 2005). The consensus is that at least nine areas of vertebrate endemism can be easily identified in Amazonia (Silva et al., 2005). Due to its wide territory, geographic placement, and the fact that its territory is divided by three of the largest tributaries of the Amazon River (Tapajós, Xingu and Tocantins), Pará includes portions of five of the nine areas of endemism: Belém, Guyana, Rondônia, Tapajós and Xingu (Figure 1). Therefore, the State’s fauna is extremely rich and has many endemic species. The Amazonian Rainforest (or simply, Amazonia) is distributed over nine countries in South America, with more than half of its area in Brazil (Ab’Saber, 1977). Knowledge about the species diversity, distribution and phylogeny of Amazonian organisms remains, nonetheless, incipient. Several areas of Amazonia have never been sampled and thousands of specimens of its biota have accumulated in scientific collections, fruits of decades of expeditions, have been insufficiently studied (Oren & Albuquerque, 1991; Ávila-Pires et al., 2010; Peloso, 2010; Jenkins et al., 2015; Steege et al., 2016; Prudente et al., 2018). The biodiversity of Pará has been studied since the first scientific expeditions to South America (Papavero et al., 2000; Papavero & Teixeira, 2011, 2013). Among the various explorers who passed through Pará, Spix and Von Martius stand out—they were in Belém in 1819 and described several animal species in Amazonia, including amphibians (Spix, 1824). Additional examples of amphibian species described from material certainly obtained in Pará since the 19th century exist (e.g., Günther, 1859 [1858]; Goeldi, 1907, figure 2), but despite almost two hundred years of documented research with amphibians in the state, the number of species, and their distribution in the Pará is largely unknown. Like most biotic groups, information about Amazonian amphibians is limited, mostly due to a lack of basic studies on species taxonomy and distribution (Ávila-Pires et al., 2007; Stuart et al., 2008; Peloso, 2010; Guerra et al., 2020). Moreover, many Amazonian species have yet to be discovered or are awaiting formal taxonomic recognition (Vacher et al., 2020; Moura & Jetz, 2021). Combined, these factors cause uncertainty regarding species richness in the region, with discrepant estimates of the number of Amazonian amphibians varying from 221 species in older assessments (Caldwell, 1996; Duellman, 1999), to 609 (Mayer et al., 2019), to nearly 900 in more recent studies (Vacher et al., 2020). Keywords: Lissamphibia. Amazonia. Checklist. Herpetology. Resumo: O Pará é o segundo maior estado do Brasil, com todo o seu território dentro da floresta amazônica. O estado possui grande variedade de habitats e bio-regiões e tem, portanto, potencial para ser um dos maiores depositários da biodiversidade de anfíbios da América do Sul. Na corrida para descobrir essa diversidade e fornecer subsídios para pesquisas futuras, compilamos a primeira lista de espécies de anfíbios do Pará, com base na identificação de espécimes depositados em coleções científicas e análise da literatura primária. Registramos 195 espécies de anfíbios, das quais 30 são endêmicas do estado e cinco representaram novos registros de ocorrência. Apesar de seu extenso território, elevado número de espécies endêmicas e níveis muito intensos de desmatamento, apenas cinco espécies são oficialmente consideradas ameaçadas de extinção. Discutimos as razões pelas quais tão poucas espécies estão ameaçadas e as razões pelas quais esse número provavelmente aumentará em breve. Também discutimos questões relacionadas à taxonomia, como, por exemplo, a ocorrência de várias espécies não nomeadas no Pará. Por fim, destacamos algumas oportunidades e desafios para futuras áreas de pesquisa com diversidade e taxonomia de anfíbios no estado do Pará. 445 BY 445 BY 445 The amphibians of Pará, Brazil INTRODUCTION As in the rest of the Amazonia, Pará represents an area where the number of amphibian species described recently Figure 1. Map of northern South America representing the endemic areas of the state of Pará. Pará encompasses five areas of endemism and has a large number of endemic and endangered vertebrate species. Map: Gisele Cassundé (2021). Pará is the second largest Brazilian state, with 1,245,870.798 km² of territorial extension, second only to Amazonas (IBGE, 2019), also in the Amazonian region. Pará has all its territory in Amazonia, but includes several unique types of vegetation, such as savanna enclaves in the northern portion, and areas of Amazonia-Cerrado ecotone in the south, mainly along the border with the state of Tocantins, which confers a wide variety of phytophysiognomies (Goulding et al., 2003). Such characteristics make the State a depository of a unique biodiversity in South America. For example, Amazonia has been subdivided into ‘areas of endemism’ (geographic area that limits the distribution of several species), largely defined based on the co-distribution of terrestrial vertebrates (Silva Figure 1. Map of northern South America representing the endemic areas of the state of Pará. Pará encompasses five areas of endemism and has a large number of endemic and endangered vertebrate species. Map: Gisele Cassundé (2021). 446 446 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Figure 2. Reproduction of original figures of species named over a century ago and for which the type-locality is in Pará. Top: “Hyla (= Boana) multifasciata”, by Albert Günther (Gunther, 1859 [1858]: plate 8, figure D). The type locality was given as “Pará”, later interpreted as probably Belém (Bokermann, 1966). Bottom: “Hyla (=Trachycephalus) resinifictrix”, by Emílio Goeldi (Goeldi, 1907). The type-locality for this species is Missão Santo Antônio do Prata, Maracanã river, Pará, Brazil. published for several locations in the state (e.g., Ávila-Pires & Hoogmoed, 1997; Azevedo-Ramos & Galatti, 2002; Ávila- Pires et al., 2010; Mendes-Pinto & Souza, 2011; Bernardo et al., 2012; Pinheiro et al., 2012; Vaz-Silva et al., 2015; Hoogmoed & Galatti, 2019). Our main objective was to compile the list of amphibian species in Pará. During the process of evaluation and construction of the list, we identified some of the major difficulties associated with the study of amphibians in the Amazonian rainforest. INTRODUCTION We conclude our contribution with an outline and definitions of a few priorities for future studies with amphibians in the State. COLLECTION SPECIMENS FROM MPEG COLLECTION SPECIMENS FROM MPEG (Segalla et al., 2021). From that list, we considered that all species recognized to occur in the Brazilian territory could potentially also occur in Pará. We then searched for each of these potential species in the electronic databases Amphibian Species of the World (Frost, 2022), AmphibiaWeb (AmphibiaWeb, 2022), and IUCN Red List of Threatened Species (IUCN, 2022)—these databases have information based on literature about the distribution of each species. Based on the data from these databases, those with records for Pará were preliminarily included in the list and we then searched for voucher specimens of such species in collections or in the literature. A species was only included if specimens were either analyzed by one of us or mentioned in recent and relevant taxonomic literature. (Segalla et al., 2021). From that list, we considered that all species recognized to occur in the Brazilian territory could potentially also occur in Pará. We then searched for each of these potential species in the electronic databases Amphibian Species of the World (Frost, 2022), AmphibiaWeb (AmphibiaWeb, 2022), and IUCN Red List of Threatened Species (IUCN, 2022)—these databases have information based on literature about the distribution of each species. Based on the data from these databases, those with records for Pará were preliminarily included in the list and we then searched for voucher specimens of such species in collections or in the literature. A species was only included if specimens were either analyzed by one of us or mentioned in recent and relevant taxonomic literature. The first step of the study consisted of identifying specimens deposited in natural history collections. The MPEG collection, the main source of data used here, was officially founded in 1965 by Osvaldo Rodrigues da Cunha, and currently holds over 40,000 specimens of amphibians, most of which collected in Pará (a little over 22,000). The collection holds invaluable material which testifies for the magnificent diversity of amphibians in the State. These holdings have been used to produce studies on the diversity of species in the region for over a century (Goeldi, 1907), and continues to do so in the present. For this reason, we relied heavily on the material listed in the MPEG collection to build the list. A primary list was compiled from the database of the MPEG amphibian collection. An electronic spreadsheet was exported from the MPEG archives including all amphibian entries of the collection. RESULTS We recorded 195 species composing the fauna of amphibians in the state of Pará. This diversity is mostly formed by Anura (178 species), followed by Gymnophiona (15 species), and Caudata (two species). Among these, five species are new occurrence records for the State: Boana diabolica, Boana leucocheila, Pristimantis gutturalis, Pristimantis reichlei and Trachycephalus coriaceus. Thirty species (about 15% of all species included) represent species that only exist in Pará (endemic species). These are: Anura—Adelphobates castaneoticus, Adenomera COLLECTION SPECIMENS FROM MPEG This spreadsheet was filtered to include only specimens collected within the limits of Pará. After filtering, we derived a list of all taxa with occurrences in Pará. After this primary list was created, we checked the actual collection specimens to search for at least one voucher specimen for each species in the list. If a voucher specimen was found and its ID confirmed, the species occurrence was confirmed. However, in many cases, specimens are wrongly identified—in such cases, if no voucher specimen was found at MPEG, the species was not immediately included in the list. In these cases, we searched for records of the species in other collections, or in the primary literature (i.e., taxonomic literature). We immediately included in the list, formally named species for which type material (holotypes or at least one paratype) were obtained in Pará, even if we did not have access to the specimens for confirmation purposes. In the case of works, such as species lists, extensions of species distribution, taxonomic reviews, among others, the record was only included without the analysis of testimony material in some cases (e.g., recent taxonomic reviews, 2005–present; phylogenetic analyses with broad sampling that is sufficient to unambiguously identify a sample). Most of the time, we attempted to analyze the voucher specimens that support the registration to confirm the identification. MATERIAL AND METHODS The List of Amphibians of Pará, presented in this study, was fundamentally based on the analysis of material deposited in the Coleção Herpetológica Osvaldo Rodrigues da Cunha, at the Museu Paraense Emílio Goeldi (MPEG), Belém, Pará, Brazil, and on the literature. However, we also included information on selected specimens deposited in other collections worldwide and analysed by us: AMNH (American Museum of Natural History, New York, EUA); CHUNB (Coleção Herpetológica da Universidade de Brasília, Brasília, Brazil); INPA-H (Coleção de Herpetologia, Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil); LZATM (Laboratório de Zoologia de Altamira, Altamira, Brazil); MNRJ (Museu Nacional, Rio de Janeiro, Rio de Janeiro, Brazil); MCP (Museu de Ciências e Tecnologia da Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil); MZUSP (Museu de Zoologia, Universidade de São Paulo, São Paulo, Brazil); NHMW (Naturhistorisches Museum, Zoologische Abtheilung, Wien, Austria); USNM (Smithsonian, National Museum of Natural History, Washington, EUA). The inclusion of data from the literature was essential, as it allowed us to include species for which we did not find any specimens in the collections we examined. A list of voucher specimens used to confirm the species occurrence in Pará is given in Appendix 1. Figure 2. Reproduction of original figures of species named over a century ago and for which the type-locality is in Pará. Top: “Hyla (= Boana) multifasciata”, by Albert Günther (Gunther, 1859 [1858]: plate 8, figure D). The type locality was given as “Pará”, later interpreted as probably Belém (Bokermann, 1966). Bottom: “Hyla (=Trachycephalus) resinifictrix”, by Emílio Goeldi (Goeldi, 1907). The type-locality for this species is Missão Santo Antônio do Prata, Maracanã river, Pará, Brazil. is high (Maciel et al., 2018; Peloso et al., 2018; Pinheiro et al., 2018; Rojas-Zamora et al., 2018; Carvalho et al., 2019; Kaefer et al., 2019; Moraes et al., 2019; Simões et al., 2019; Carvalho et al., 2020; Oliveira et al., 2020) and current genetic studies indicate a great diversity of species not yet described occurring in the State (Fouquet et al., 2016; Peloso et al., 2018; Carvalho et al., 2020; Jaramillo-Martinez et al., 2020; Vacher et al., 2020; Fouquet et al., 2021b). However, the number of amphibian species in Pará has never been estimated based on a detailed review of biological collection data. Moreover, there is no compiled list of amphibians in Pará, although some punctual inventories have already been 447 447 The amphibians of Pará, Brazil ADDITIONAL COLLECTIONS AND LITERATURE DATA The second stage of the study consisted of confirming or adding species to the list based on the literature, and on data from additional collections (other than MPEG). To verify that our search was nearly exhaustive, we initially investigated the official list of amphibians in Brazil, provided by the Brazilian Society of Herpetology 448 448 448 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Hyalinobatrachium muiraquitan, Pristimantis giorgii, Pristimantis latro, Rhinella magnussoni, Scinax villasboasi, and Trachycephalus helioi; Caudata—Bolitoglossa paraensis and Bolitoglossa tapajonica; and Gymnophiona—Microcaecilia butantan, Microcaecilia trombetas and Rhinatrema uaiuai. The complete list of species currently known for Pará is given below (Table 1). amicorum, Adenomera aurantiaca, Adenomera inopinata, Adenomera martinezi, Adenomera phonotriccus, Adenomera tapajonica, Allobates carajas, Allobates grillicantus, Allobates magnussoni, Allobates masniger, Allobates nunciatus, Allobates tapajos, Amazophrynella bilinguis, Amazophrynella gardai, Amazophrynella minuta, Amazophrynella xinguensis, Chiasmocleis papachibe, Dendropsophus minimus, amicorum, Adenomera aurantiaca, Adenomera inopinata, Adenomera martinezi, Adenomera phonotriccus, Adenomera tapajonica, Allobates carajas, Allobates grillicantus, Allobates magnussoni, Allobates masniger, Allobates nunciatus, Allobates tapajos, Amazophrynella bilinguis, Amazophrynella gardai, Amazophrynella minuta, Amazophrynella xinguensis, Chiasmocleis papachibe, Dendropsophus minimus, Table 1. List of amphibian species from the State of Pará, including a list of taxa currently considered endemic to the State. Information on the conservation status of the species by the IUCN was included considering the following categories: Data Deficient (DD), Least Concern (LC), Near Threatened (NT), Vulnerable (VU), Endangered (EN), Critically Endangered (CR), Not Evaluated (NE). (Continue) Table 1. List of amphibian species from the State of Pará, including a list of taxa currently considered endemic to the State. Information on the conservation status of the species by the IUCN was included considering the following categories: Data Deficient (DD), Least Concern (LC), Near Threatened (NT), Vulnerable (VU), Endangered (EN), Critically Endangered (CR), Not Evaluated (NE). ADDITIONAL COLLECTIONS AND LITERATURE DATA (Continue) IUCN Endemic Taxon Taxon Taxon IUCN Endemic AMPHIBIA Order ANURA Allophrynidae Savage, 1973 Allophryne ruthveni Gaige, 1926 LC Aromobatidae Grant, Frost, Caldwell, Gagliardo, Haddad, Kok, Means, Noonan, Schargel, & Wheeler, 2006 Allobates carajas Simões, Rojas, & Lima, 2019 NE X Allobates crombiei (Morales, 2002) DD Allobates femoralis (Boulenger, 1884) LC Allobates grillicantus Moraes, & Lima, 2021 NE X Allobates magnussoni Lima, Simões, & Kaefer, 2014 NE X Allobates marchesianus (Melin, 1941) LC Allobates masniger (Morales, 2002) DD X Allobates nunciatus Moraes, Pavan, & Lima, 2019 NE X Allobates sumtuosus (Morales, 2002) DD Allobates tapajos Lima, Simões, & Kaefer, 2015 NE X Anomaloglossus stepheni (Martins, 1989) LC Bufonidae Gray, 1825 Amazophrynella bilinguis Kaefer, Rojas-Zamora, Ferrão, Farias, & Lima, 2019 NE X Amazophrynella bokermanni (Izecksohn, 1994) LC Amazophrynella gardai Mângia, Koroiva, & Santana, 2020 NE X Amazophrynella minuta (Melin, 1941) LC X Amazophrynella xinguensis Rojas-Zamora, Fouquet, Ron, Hernández-Ruz, Melo-Sampaio, Chaparro, Vogt, Carvalho, Pinheiro, Ávila, Farias, Gordo, & Hrbek, 2018 NE X Atelopus hoogmoedi Lescure, 1974 NE Atelopus spumarius Cope, 1871 VU Rhaebo guttatus (Schneider, 1799) LC Rhinella castaneotica (Caldwell, 1991) LC Order ANURA Allophrynidae Savage, 1973 Allophryne ruthveni Gaige, 1926 LC Aromobatidae Grant, Frost, Caldwell, Gagliardo, Haddad, Kok, Means, Noonan, Schargel, & Wheeler, 2006 Allobates carajas Simões, Rojas, & Lima, 2019 NE X Allobates crombiei (Morales, 2002) DD Allobates femoralis (Boulenger, 1884) LC Allobates grillicantus Moraes, & Lima, 2021 NE X Allobates magnussoni Lima, Simões, & Kaefer, 2014 NE X Allobates marchesianus (Melin, 1941) LC Allobates masniger (Morales, 2002) DD X Allobates nunciatus Moraes, Pavan, & Lima, 2019 NE X Allobates sumtuosus (Morales, 2002) DD Allobates tapajos Lima, Simões, & Kaefer, 2015 NE X Anomaloglossus stepheni (Martins, 1989) LC Bufonidae Gray, 1825 Amazophrynella bilinguis Kaefer, Rojas-Zamora, Ferrão, Farias, & Lima, 2019 NE X Amazophrynella bokermanni (Izecksohn, 1994) LC Amazophrynella gardai Mângia, Koroiva, & Santana, 2020 NE X Amazophrynella minuta (Melin, 1941) LC X Amazophrynella xinguensis Rojas-Zamora, Fouquet, Ron, Hernández-Ruz, Melo-Sampaio, Chaparro, Vogt, Carvalho, Pinheiro, Ávila, Farias, Gordo, & Hrbek, 2018 NE X Atelopus hoogmoedi Lescure, 1974 NE Atelopus spumarius Cope, 1871 VU Rhaebo guttatus (Schneider, 1799) LC Rhinella castaneotica (Caldwell, 1991) LC 449 449 The amphibians of Pará, Brazil Taxon IUCN Endemic Rhinella dapsilis (Myers & Carvalho, 1945) LC Rhinella diptycha (Cope, 1862) DD Rhinella granulosa (Spix, 1824) LC Rhinella major (Müller & Hellmich, 1936) NE Rhinella magnussoni Lima, Menin, & Araújo, 2007 LC X Rhinella margaritifera (Laurenti, 1768) LC Rhinella marina (Linnaeus, 1758) LC Rhinella mirandaribeiroi (Gallardo, 1965) NE Rhinella ocellata (Günther, 1858) LC Rhinella proboscidea (Spix, 1824) LC Centrolenidae Taylor, 1951 Hyalinobatrachium mondolfii Señaris & Ayarzagüena, 2001 LC Hyalinobatrachium muiraquitan Oliveira & Hernández-Ruz, 2017 NE X Hyalinobatrachium iaspidiense (Ayarzagüena, 1992) DD Ceratophryidae Tschudi, 1838 Ceratophrys cornuta (Linnaeus, 1758) LC Dendrobatidae Cope, 1865 (1850) Adelphobates castaneoticus (Caldwell & Myers, 1990) LC X Adelphobates galactonotus (Steindachner, 1864) LC Adelphobates quinquevittatus (Steindachner, 1864) LC Ameerega braccata (Steindachner, 1864) LC Ameerega flavopicta (Lutz, 1925) LC Ameerega hahneli (Boulenger, 1884) LC Ameerega munduruku Neves, Silva, Akieda, Cabrera, Koroiva, & Santana, 2017 NE Ameerega trivittata (Spix, 1824) LC Dendrobates tinctorius (Cuvier, 1797) LC Ranitomeya amazonica (Schulte, 1999) DD Eleutherodactylidae Lutz, 1954 Adelophryne gutturosa Hoogmoed & Lescure, 1984 LC Phyzelaphryne miriamae Heyer, 1977 LC Hemiphractidae Peters, 1862 Hemiphractus scutatus (Spix, 1824) LC Hylidae Rafinesque, 1815 Boana boans (Linnaeus, 1758) LC Boana caiapo Pinheiro, Cintra, Valdujo, Silva, Martins, Silva, & Garcia, 2018 NE Boana calcarata (Troschel, 1848) LC Boana cinerascens (Spix, 1824) LC Table 1. ADDITIONAL COLLECTIONS AND LITERATURE DATA Taxon 451 451 The amphibians of Pará, Brazil Taxon IUCN Endemic Osteocephalus leprieurii (Duméril & Bibron, 1841) LC Osteocephalus oophagus Jungfer & Schiesari, 1995 LC Osteocephalus taurinus Steindachner, 1862 LC Pseudis paradoxa (Linnaeus, 1758) LC Pseudis tocantins (Linnaeus, 1758) LC Scarthyla goinorum (Bokermann, 1962) LC Scinax boesemani (Goin, 1966) LC Scinax fuscomarginatus (Lutz, 1925) LC Scinax garbei (Miranda-Ribeiro, 1926) LC Scinax nebulosus (Spix, 1824) LC Scinax proboscideus (Brongersma, 1933) LC Scinax rostratus (Peters, 1863) LC Scinax ruber (Laurenti, 1768) LC Scinax villasboasi Brusquetti, Jansen, Barrio-Amorós, Segalla, & Haddad, 2014 NE X Scinax x-signatus (Spix, 1824) LC Sphaenorhynchus lacteus (Daudin, 1800) LC Trachycephalus coriaceus (Peters, 1867) LC Trachycephalus cunauaru Gordo, Toledo, Suárez, Kawashita-Ribeiro, Ávila, Morais, & Nunes, 2013 NE Trachycephalus hadroceps (Duellman & Hoogmoed, 1992) LC Trachycephalus helioi Nunes, Suárez, Gordo, & Pombal, 2013 NE X Trachycephalus resinifictrix (Goeldi, 1907) LC Trachycephalus typhonius (Linnaeus, 1758) LC Leptodactylidae Werner, 1896 (1838) Adenomera amicorum Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Adenomera andreae (Müller, 1923) LC Adenomera aurantiaca Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Adenomera heyeri Boistel, Massary, & Angulo, 2006 LC Adenomera hylaedactyla (Cope, 1868) LC Adenomera inopinata Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Adenomera kayapo Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE Adenomera martinezi (Bokermann, 1956) LC X Adenomera phonotriccus Carvalho, Giaretta, Angulo, Haddad, & Peloso, 2019 NE X Adenomera tapajonica Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Table 1. (Continue) Table 1. ADDITIONAL COLLECTIONS AND LITERATURE DATA (Continue) Table 1. Table 1. 450 450 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Taxon IUCN Endemic Boana courtoisae Fouquet, Marinho, Réjaud, Carvalho, Caminer, Jansen, Rainha, Rodrigues, Werneck, Lima, Hrbek, Giaretta, Venegas, Chávez, & Ron, 2021 NE Boana dentei (Bokermann, 1967) LC Boana diabolica (Fouquet, Martinez, Zeidler, Courtois, Gaucher, Blanc, Lima, Souza, Rodrigues, & Kok, 2016) NE Boana geographica (Spix, 1824) LC Boana icamiaba Peloso, Oliveira, Sturaro, Rodrigues, Lima, Bitar, Wheeler, & Aleixo, 2018 NE Boana lanciformis (Cope, 1871) LC Boana leucocheila (Caramaschi & Niemeyer, 2003) DD Boana multifasciata (Günther, 1859) LC Boana punctata (Schneider, 1799) LC Boana raniceps (Cope, 1862) LC Boana steinbachi (Boulenger, 1905) NE Boana wavrini (Parker, 1936) LC Dendropsophus brevifrons (Duellman & Crump, 1974) LC Dendropsophus cachimbo (Napoli & Caramaschi, 1999) DD Dendropsophus gaucheri (Lescure & Marty, 2000) LC Dendropsophus haraldschultzi (Bokermann, 1962) LC Dendropsophus leucophyllatus (Beireis, 1783) LC Dendropsophus marmoratus (Laurenti, 1768) LC Dendropsophus melanargyreus (Cope, 1887) LC Dendropsophus microcephalus (Cope, 1886) LC Dendropsophus minimus (Ahl, 1933) DD X Dendropsophus minusculus (Rivero, 1971) LC Dendropsophus minutus (Peters, 1872) LC Dendropsophus nanus (Boulenger, 1889) LC Dendropsophus ozzyi Orrico, Peloso, Sturaro, Silva, Neckel-Oliveira, Gordo, Faivovich, & Haddad, 2014 NE Dendropsophus parviceps (Boulenger, 1882) LC Dendropsophus reticulatus (Jiménez de la Espada, 1870) NE Dendropsophus rossalleni (Goin, 1959) LC Dendropsophus sarayacuensis (Shreve, 1935) LC Dendropsophus schubarti (Bokermann, 1963) LC Dendropsophus triangulum (Günther, 1869) LC Dendropsophus walfordi (Bokermann, 1962) LC Dryaderces inframaculata (Boulenger, 1882) DD Lysapsus laevis (Parker, 1935) LC Lysapsus limellum Cope, 1862 LC Osteocephalus cabrerai (Cochran & Goin, 1970) LC Table 1. (Continue) Table 1. ADDITIONAL COLLECTIONS AND LITERATURE DATA Taxon IUCN Endemic Osteocephalus leprieurii (Duméril & Bibron, 1841) LC Osteocephalus oophagus Jungfer & Schiesari, 1995 LC Osteocephalus taurinus Steindachner, 1862 LC Pseudis paradoxa (Linnaeus, 1758) LC Pseudis tocantins (Linnaeus, 1758) LC Scarthyla goinorum (Bokermann, 1962) LC Scinax boesemani (Goin, 1966) LC Scinax fuscomarginatus (Lutz, 1925) LC Scinax garbei (Miranda-Ribeiro, 1926) LC Scinax nebulosus (Spix, 1824) LC Scinax proboscideus (Brongersma, 1933) LC Scinax rostratus (Peters, 1863) LC Scinax ruber (Laurenti, 1768) LC Scinax villasboasi Brusquetti, Jansen, Barrio-Amorós, Segalla, & Haddad, 2014 NE X Scinax x-signatus (Spix, 1824) LC Sphaenorhynchus lacteus (Daudin, 1800) LC Trachycephalus coriaceus (Peters, 1867) LC Trachycephalus cunauaru Gordo, Toledo, Suárez, Kawashita-Ribeiro, Ávila, Morais, & Nunes, 2013 NE Trachycephalus hadroceps (Duellman & Hoogmoed, 1992) LC Trachycephalus helioi Nunes, Suárez, Gordo, & Pombal, 2013 NE X Trachycephalus resinifictrix (Goeldi, 1907) LC Trachycephalus typhonius (Linnaeus, 1758) LC Leptodactylidae Werner, 1896 (1838) Adenomera amicorum Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Adenomera andreae (Müller, 1923) LC Adenomera aurantiaca Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Adenomera heyeri Boistel, Massary, & Angulo, 2006 LC Adenomera hylaedactyla (Cope, 1868) LC Adenomera inopinata Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Adenomera kayapo Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE Adenomera martinezi (Bokermann, 1956) LC X Adenomera phonotriccus Carvalho, Giaretta, Angulo, Haddad, & Peloso, 2019 NE X Adenomera tapajonica Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira, & Haddad, 2021 NE X Table 1. (Continue) Adenomera martinezi (Bokermann, 1956) Adenomera phonotriccus Carvalho, Giaretta, Angulo, Haddad, & Peloso, 2019 452 452 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Table 1. ADDITIONAL COLLECTIONS AND LITERATURE DATA Taxon IUCN Endemic Engystomops freibergi (Donoso-Barros, 1969) LC Hydrolaetare schmidti (Cochran & Goin, 1959) LC Leptodactylus fuscus (Schneider, 1799) LC Leptodactylus intermedius Lutz, 1930 NE Leptodactylus knudseni Heyer, 1972 LC Leptodactylus labyrinthicus (Spix, 1824) LC Leptodactylus leptodactyloides (Andersson, 1945) LC Leptodactylus longirostris Boulenger, 1882 LC Leptodactylus macrosternum Miranda-Ribeiro, 1926 NE Leptodactylus myersi Heyer, 1995 LC Leptodactylus mystaceus (Spix, 1824) LC Leptodactylus paraensis Heyer, 2005 LC Leptodactylus pentadactylus (Laurenti, 1768) LC Leptodactylus petersii (Steindachner, 1864) LC Leptodactylus pustulatus (Peters, 1870) LC Leptodactylus rhodomystax Boulenger, 1884 LC Leptodactylus stenodema Jiménez de la Espada, 1875 LC Physalaemus cuvieri Fitzinger, 1826 LC Physalaemus ephippifer (Steindachner, 1864) LC Lithodytes lineatus (Schneider, 1799) LC Pseudopaludicola boliviana Parker, 1927 LC Pseudopaludicola canga Giaretta & Kokubum, 2003 DD Pseudopaludicola mystacalis (Cope, 1887) LC Microhylidae Günther, 1858 (1843) Chiasmocleis avilapiresae Peloso & Sturaro, 2008 LC Chiasmocleis bassleri Dunn, 1949 LC Chiasmocleis hudsoni Parker, 1940 LC Chiasmocleis papachibe Peloso, Sturaro, Forlani, Gaucher, Motta, & Wheeler, 2014 NE X Chiasmocleis shudikarensis Dunn, 1949 LC Ctenophryne geayi Mocquard, 1904 LC Elachistocleis carvalhoi Caramaschi, 2010 LC Elachistocleis helianneae Caramaschi, 2010 LC Elachistocleis magna Toledo, 2010 NE Elachistocleis surinamensis (Daudin, 1802) LC Hamptophryne boliviana (Parker, 1927) LC Otophryne pyburni Campbell & Clarke, 1998 LC Synapturanus ajuricaba Fouquet, Leblanc, Fabre, Rodrigues, Menin, Courtois, Dewynter, Hölting, Ernst, Peloso, & Kok, 2021 NE Table 1. ADDITIONAL COLLECTIONS AND LITERATURE DATA (Continue) 453 453 453 The amphibians of Pará, Brazil Taxon IUCN Endemic Synapturanus mirandaribeiroi Nelson & Lescure, 1975 LC Odontophrynidae Lynch, 1969 Proceratophrys concavitympanum Giaretta, Bernarde, & Kokubum, 2000 DD Proceratophrys rondonae Prado & Pombal, 2008 NE Phyllomedusidae Günther, 1858 Callimedusa tomopterna (Cope, 1868) LC Cruziohyla craspedopus (Funkhouser, 1957) LC Phyllomedusa bicolor (Boddaert, 1772) LC Phyllomedusa vaillantii Boulenger, 1882 LC Pithecopus hypochondrialis (Daudin, 1800) LC Pipidae Gray, 1825 Pipa arrabali Izecksohn, 1976 LC Pipa pipa (Linnaeus, 1758) LC Pipa snethlageae Müller, 1914 LC Ranidae Batsch, 1796 Lithobates palmipes (Spix, 1824) LC Strabomantidae Hedges, Duellman, & Heinecke, 2008 Pristimantis chiastonotus (Lynch & Hoogmoed, 1977) LC Pristimantis fenestratus (Steindachner, 1864) LC Pristimantis giorgii Oliveira, Alves da Silva, Guimarães, Penhacek, Martínez, Rodrigues, Santana, & Hernández-Ruz, 2020 NE X Pristimantis gutturalis (Hoogmoed, Lynch, & Lescure, 1977) LC Pristimantis latro Oliveira, Rodrigues, Kaefer, Pinto, & Hernández-Ruz, 2017 NE X Pristimantis marmoratus (Boulenger, 1900) LC Pristimantis ockendeni (Boulenger, 1912) LC Pristimantis pictus Oliveira, Alves da Silva, Guimarães, Penhacek, Martínez, Rodrigues, Santana, & Hernández-Ruz, 2020 NE Pristimantis zeuctotylus (Lynch & Hoogmoed, 1977) LC Pristimantis zimmermanae (Heyer & Hardy, 1991) LC Table 1. (Continue) Table 1. Table 1. Order CAUDATA 454 454 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Taxon IUCN Endemic Rhinatrematidae Nussbaum, 1977 Rhinatrema bivittatum (Guérin-Méneville, 1838) LC Rhinatrema gilbertogili Maciel, Sampaio, Hoogmoed, & Schneider, 2018 NE Rhinatrema uaiuai Maciel, Sampaio, Hoogmoed, & Schneider, 2018 NE X Siphonopidae Bonaparte, 1850 Brasilotyphlus guarantanus Maciel, Mott, & Hoogmoed, 2009 NE Microcaecilia butantan Wilkinson, Antoniazzi, & Jared, 2015 NE X Microcaecilia marvaleewakeae Maciel & Hoogmoed, 2013 NE Microcaecilia rochai Maciel & Hoogmoed, 2011 NE Microcaecilia trombetas Maciel & Hoogmoed, 2011 NE X Siphonops annulatus (Mikan, 1820) LC Typhlonectidae Taylor, 1968 Atretochoana eiselti (Taylor, 1968) DD Nectocaecilia petersii (Boulenger, 1882) LC Potomotyphlus kaupii (Berthold, 1859) LC Typhlonectes compressicauda (Duméril & Bibron, 1841) LC Table 1. (Conclusion) Table 1. and molecular analyses will change the taxonomic status of these populations and in the future a new Microcaecilia will compose the list of amphibians in the state of Pará (A. O. M., personal communication, 2022). In addition to the species included in the present list, others are candidates to be included in the list in the future—these taxa do occur in Pará (based on literature records): at least six species from the Boana semilineata group (one from the Boana geographica complex and five from the Boana semilineata complex) (Fouquet et al., 2016; Peloso et al., 2018), and two species of Synapturanus (Fouquet et al., 2021a) have already been confirmed to be new species (some of which already being described, PLVP, personal observation; Fouquet et al., 2021b). The species Ameerega picta guayanensis, currently considered a subspecies of A. picta (often, but unofficially, regarded as a synonym) may represent a valid taxon. Specimens from MPEG (MPEG 2803, MPEG 2805, MPEG 2807, MPEG 2815–2816, MPEG 2828, MPEG 17495–17503), obtained in the northern part of Pará, are compatible with diagnostic characters of this taxon. However, a more detailed taxonomic study is needed to confirm the status of this taxon. A recent study with Atelopus suggests that an unnamed species of the genus occurs in southern Pará (the Tapajós river) (Jorge et al., 2020), but this is pending a more robust analysis of the taxonomic status of these populations. Finally, we did not include Chiasmocleis jimi Caramaschi & Cruz, 2001 in our list—a species currently included and listed as valid in both Segalla et al. (2021) and Frost (2022), and for which paratypes are available at MPEG. Order CAUDATA The species was named based on specimens from Amazonas and Pará (type-locality Humaitá, Amazonas, Brazil). Peloso et al. (2014) suggested that both species are not unambiguously An undergoing study on the Microcaecilia from Pará will probably provide additions of new caecilians. Maciel & Hoogmoed (2011a) denominated all populations of this genus from south of the Amazon River, and east to the Tapajós river as Microcaecilia taylori Nussbaum & Hoogmoedi, 1979 based on external morphology. New undergoing morphological 455 455 The amphibians of Pará, Brazil states of Mato Grosso (Aripuanã and Apiacás) and Rondônia (Nova Carolina), Brazil. Pansonato et al. (2011) confirmed its occurrence in Mato Grosso and expanded the range within that State. We confirm the presence of the species in Pará from specimens from the municipalities of Juruti and Itaituba (MPEG 14271; MPEG 27239–27244; MPEG 22393–22395; MPEG 22210–22211; MPEG 22347– 22349; MPEG 33538; MPEG 37062). Most specimens we correctly identified as Boana leucocheila and in some cases as B. lanciforms. Despite most specimens being correctly labeled in the collection, the occurrence in Pará was never published and remained unavailable in the literature. differentiated based on a detailed analysis of morphology and advertisement call from throughout the range of both species (including the analyses of both types). De Sá et al. (2019) reinstated C. jimi as a valid species, based on their interpretation of genetic data. However, the clades labeled as C. jimi and C. hudsoni by De Sá et al. (2019) did not include the type-locality of C. jimi, whereas they form sister clades for which the distributions apparently overlap. Furthermore, many of the specimens included in the genetic study of De Sá et al. (2019) were examined (for morphology) by Peloso et al. (2014), including specimens in both clades. De Sá et al. (2019) did not provide any new evidence that could be used to diagnose both taxa. Given that the lineages labeled C. jimi and C. hudsoni by De Sá et al. (2019) form a clade that is diagnoseable based on morphological and acoustic characters, we suggest these two taxa should not be treated as separate species until more convincing evidence is available. Trachycephalus coriaceus Trachycephalus coriaceus is widely distributed in Amazonia, but interestingly, no record of the species existed for Pará. The species was originally described for Suriname, with vague information about its type locality (Peters, 1867). Order CAUDATA However, over the years its distribution broadened to include French Guiana, Guyana, Upper Amazon Basin in Colombia, Ecuador, Peru, Bolivia, and downstream into Rondônia and Amazonas, near Manaus, Brazil (Frost, 2022). In Brazil, there are known records from the state of Acre (Souza, 2009), Amazonas (Zimmerman & Rodrigues, 1990) and, more recently, Rondônia (Meneghelli et al., 2017). The species occurrence for Pará was confirmed for the municipalities of Marabá (MPEG 35500) and Parauapebas (MPEG 41293). Boana diabolica Boana diabolica Boana diabolica is known to occur in most of French Guiana and in the state of Amapá, Brazil (Fouquet et al., 2016). The record of the species for Pará was anticipated, since some points of occurrence, defined by Fouquet et al. (2016), are close to the border between Amapá and Pará. A single specimen from Pará was found in MPEG (MPEG 33920), obtained in the municipality of Almeirim. This specimen was previously included in the genetic analysis of Peloso et al. (2018)—therein tentatively assigned to B. diabolica. After a detailed examination of that specimen, we concluded it is a member of that species and confirm the occurrence of B. diabolica in Pará. Pristimantis gutturalis Pristimantis gutturalis was described by Hoogmoed et al. (1977) and listed as occurring in French Guiana. Later, its distribution was expanded to Suriname and Brazil (Amapá) (Ouboter & Jairam, 2012; Frost, 2022). Here, we expand the distribution of the species to Pará (MPEG 21395–21396, MPEG 27873–27874) from Porto Trombetas, municipality of Oriximiná. Boana leucocheila Boana leucocheila was described and named by Caramaschi & Niemeyer (2003), who suggested a distribution in the 456 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 published lists and when compared to numbers given in Toledo & Batista (2012), followed by São Paulo, Minas Gerais and Rio de Janeiro. However, comparing the number of species from different regions can be relatively uninformative, especially if we consider the different methodologies applied, intrinsic differences between the regions, differences in the size of the areas, disparate sampling efforts, and especially the number of taxonomists and taxonomic work published within these regions (Duellman & Thomas, 1996; Martins & Oliveira, 1998). Pará covers a large territorial area with several areas that are little studied or have never been sampled at all, whereas the states of São Paulo and Rio de Janeiro, for example, have been much better studied historically (Guerra et al., 2020). These factors certainly contribute to São Paulo and Rio de Janeiro having a better understanding of its biodiversity, resulting in relatively large lists of amphibians, and further highlighting the urgent need for additional work with amphibians in Pará (see also Guerra et al., 2020). Pristimantis reichlei was described and named by Padial & De La Riva (2009), as occurring for Bolivia and Peru. The first record of the species for Brazil was in 2010, for the municipality of Senador Guiomard, Acre (Melo-Sampaio & Souza, 2010). After ten years, no other records for the country have been published. During the analysis of MPEG specimens, originally identified as P. peruvianus in the collection database, we concluded that the specimens are actually P. reichlei, collected in the municipalities of Vitória do Xingu and Itaituba (MPEG 36826–36827, MPEG 39015–39019). DISCUSSION Few Brazilian states have recent (i.e., from 2000 to present) lists of amphibian species occurring within their political limits, namely: Rio Grande do Sul—86 species (Machado & Maltchik, 2007); Espírito Santo—133 spp. (Almeida et al., 2011); São Paulo—236 spp. (Rossa-Feres et al., 2011); Piauí—55 spp. (Roberto et al., 2013); Alagoas—74 spp. (Almeida et al., 2016); Ceará—57 spp. (Roberto & Loebmann, 2016); Rio de Janeiro—201 spp. (Dorigo et al., 2018); Mato Grosso do Sul—97 spp. (Souza et al., 2017); Tocantins—90 spp. (Silva et al., 2020). A list for Goiás reported 39 species (Santos et al., 2014), but a recent publication that jointly reports the richness in Goiás and Distrito Federal included 114 species (Vaz-Silva et al., 2020). In addition to these carefully elaborated lists, Toledo & Batista (2012) provided estimates of number of species for all States, based on IUCN’s and amphibian collection’s data—their data show numbers that are similar to the individually published lists, but also provide estimates for states where official lists have never been published. In Toledo & Batista (2012) the greatest species richness was found in Amazonas (212 spp.), Minas Gerais (259 spp.), São Paulo (241 spp.), and Rio de Janeiro (205 spp.)—the authors estimated a total of 148 species for Pará, considerably less than what we report herein. O d h h P á i i h h Considering the size of the state of Pará (larger than Colombia), we consider that the amphibian fauna in this region has so far been poorly studied. Although several sites across Pará have been studied and inventoried for amphibian diversity, only a few local/regional lists have been published—discussing these is beyond the scope of this work, but some are worth mentioning. Crump (1971) studied amphibian diversity in the vicinities of Belém and reported the occurrence of at least 41 species (37 frogs, 3 caecilians, one salamander) in the region. Her study detailed habitat use, abundance, and several other ecological aspects for most species, and is a fairly complete account of the amphibian fauna in the capital of Pará. A later study reported 55 species over the same general area (Galatti et al., 2007), and more recently, a study focused on Parque Estadual do Utinga (PEUT, a relatively large and well-preserved forest fragment within Belém) reported a list comprising of 50 amphibian species (Ávila-Pires et al., 2018). DISCUSSION Surprisingly, these are the only detailed studies of amphibian diversity published for Belém, Our data shows that Pará is among states with the largest number of amphibian species registered among 457 The amphibians of Pará, Brazil The amphibians of Pará, Brazil ON THE QUALITY OF THE SPECIES LIST the second largest city of the Brazilian Amazonia. These studies are further discussed below (see section “Endemic and Threatened Species”). Data on the geographical and temporal distribution of organisms are often obtained from direct information from databases of zoological collections, and, more recently, citizen science (Dickinson et al., 2010; Callaghan et al., 2019). Very often, in the case of natural history collections, data from the registration books are made available worldwide, and are used for further studies without careful reviews of original entry identifications (Goodwin et al., 2015). Some examples involving information obtained from online databases without a careful assessment of the quality of the information can be seen in Feeley & Silman (2011), Escalante et al. (2013), and Meyer et al. (2015). Additional studies reported over the amphibian fauna from broad geographical areas (based on short-term surveys), or narrow areas that have been more densely sampled and studies over time. Examples of the first is a series of expeditions conducted to the northern part of Pará (Ávila-Pires et al., 2010)—north of the Amazonas River (part of the Guiana Shield). Over the course of seven expeditions to different localities, a team of researchers mostly from Museu Paraense Emílio Goeldi reported a total of 80 amphibian species in the region, with the number of species per site varying from 21 to 36 (Ávila-Pires et al., 2010). In another example, Vaz-Silva et al. (2015), compiled data on species richness over a large area known as Volta Grande do Xingu (in the Xingu River)—109 species of amphibians were recorded for this region, a diversity considerably higher compared to the other inventories carried out in Pará (representing over 52% of all species found in the state). At least one other region has been relatively well studied and have relatively detailed accounts about amphibian diversity published—Floresta Nacional de Carajás where species richness has been reported to be as high as 71 species (Neckel-Oliveira et al., 2012; Pinheiro et al., 2012). DISCUSSION Although the metadata from collection databases is extremely important for meta-analysis in studies on biodiversity, the practice is not without problems and criticism (Goodwin, et al., 2015; Peloso, 2010; Vollmar et al., 2010). Erroneous data recorded in collections and museums are the result of a chain of problems that can accumulate over time. The mistaken and outdated identifications in regional, national or global databases consequently provide erroneous information about species diversity and distribution patterns. In an assessment of the accuracy of identifying plants deposited in scientific collections worldwide, Goodwin et al. (2015) mention that more than half of the studied specimens were incorrectly identified. Although the study was restricted to only a few groups of plants, the authors mention the expectation that the pattern will be the same for most taxonomic groups. To generate a better understanding of the geographical distribution of species richness in Pará, further studies are urgently needed. This will be a hard task to accomplish, as many areas have not been sufficiently sampled, and specimens collected sporadically over the area of the State are spread across multiple collections (often misidentified). This is aggravated by the fact that many places where inventories had been carried out have not seen these lists published or were published in the so-called grey literature (e.g., annals of meetings, technical reports, websites without a permanent link). Noticeable places with large amounts of material available at the MPEG collection but without lists published are, for example, the upper and middle Tapajós River regions, the upper Xingu River, and the Marajó island. In the case of our list, a preliminary study involving only specimens from a frog family deposited in the MPEG collection also revealed a reasonable rate of identification errors (Ferreira et al., 2016). The list of Microhylidae based on raw (uncorrected) data from MPEG led to a total of 15 species, but after re-identification of the material in the collection, the list was reduced to only 13 species. It is noteworthy, however, that despite the similar numbers, the corrected list is considerably different from the original list, with only nine species common to both lists (Table 2: see also Ferreira et al., 2016). 458 458 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. DISCUSSION 2022 Among the factors pointed out by Goodwin et al. (2015) and Ferreira et al. (2016) as probable causes of the high rate of misidentifications in the collections are the lack of recent systematic review works for most groups (which causes new errors to be incorporated into the collections), and the lack of constant review of the identification of the material incorporated in the collections (which means that errors inserted in the databases are not corrected despite sufficient taxonomic information to do so). It is clear from these two examples, how important taxonomic revision work and periodic revision of the material deposited in the collections are. For example, the correct re-identification of several specimens of Microhylidae from MPEG was only possible because two of the largest genera of Microhylidae have undergone relatively recent systematic reviews (Caramaschi, 2010; Peloso et al., 2014). Table 2. List of taxa of the Microhylidae family registered for the state of Pará obtained from the raw data from the MPEG collection database (Original List), and after reviewing the specimens of the collection (Refined List). Table adapted from Ferreira et al. (2016). Taxon Original List Refined List Justification Chiasmocleis avilapiresae Present Confirmed - Chiasmocleis bassleri Present Confirmed - Chiasmocleis hudsoni Present Confirmed - Chiasmocleis papachibe Absent Added Species recently described and endemic to Pará (Peloso et al., 2014) Chiasmocleis shudikarensis Present Confirmed - Chiasmocleis ventrimaculata Present Removed Species with no occurrence in Eastern Amazonia. Specimens reidentified as C. bassleri, C. papachibe Ctenophryne geayi Present Confirmed - Elachistocleis bicolor Present Removed Specimens reidentified as E. carvalhoi, E. helianneae, E. surinamensis Elachistocleis carvalhoi Present Confirmed - Elachistocleis helianneae Absent Added Several specimens in the collection previously identified as E. bicolor, E. ovalis Elachistocleis magna Absent Added Several specimens in the collection previously identified as E. ovalis Elachistocleis pearsei Present Removed Specimens reidentified as E. carvalhoi Elachistocleis surinamensis Absent Added Specimens reidentified as E. carvalhoi Hamptophryne boliviana Present Confirmed - Otophryne pyburni Present Confirmed - Synapturanus mirandaribeiroi Present Confirmed - Synapturanus salseri Present Removed Specimens reidentified as S. mirandaribeiroi or as unnamed species of Synapturanus Chiasmocleis jimi Present Removed Junior synonym of C. hudsoni (Peloso et al., 2014). See text for details Elachistocleis ovalis Present Removed Nominal species is not valid (Caramaschi, 2010). Specimens reidentified as E. carvalhoi, E. helianneae, E. DISCUSSION Systematic and recent reviews by some groups allowed for the easy and accurate re-identification of most of the analyzed material. For example, due to the relative low diversity and the existence of recent reviews for several taxa in Gymnophiona and Caudata (Maciel & Hoogmoed, 2011b; Brcko et al., 2013, respectively) the identification of the material of these groups was greatly facilitated. Likewise, recent work with some Anura groups has also been of great value (examples in Caramaschi, 2010; Caminer & Ron, 2014; Caminer et al., 2017; Carvalho et al., 2020; Peloso et al., 2014). Finally, recent genetic studies have also helped to identify some MPEG specimens with a high degree of reliability (Castroviejo- Fisher et al., 2011; Jungfer et al., 2013; Fouquet et al., 2014, 2016, 2021b; Peloso et al., 2018). For several other groups, however, the lack of recent review work and known taxonomic problems made it extremely difficult to identify the available material. Consequently, the degree of reliability in the identifications is much higher for some groups than for others. Some species were not possible to identify to the species level, due to confusion in their diagnosis or due to the presence of a set of similar species indistinguishable morphologically. For example, Hyalinobatrachium has, in theory, three species occurrying in Pará: H. mondolfi, H. muiraquitan and H. iaspidiense (Oliveira & Hernández-Ruz, 2017). However, in the literature (Castroviejo-Fisher et al., 2011) H. mondolfi and H. iaspidiense are indistinguishable when based solely on morphological characters. The overlap of characters between H. mondolfi, H. iaspidiense, H. taylori and H. tricolor hindered the diversity sampling of the Centrolenidae family in Pará. It is possible that some of the specimens identified as H. iaspidiense refer to H. tricolor. The same occurs with H. mondolfi, where some of the specimens may represent records of H. taylori. Many species of amphibians are difficult to identify based solely on the evaluation of morphological characters—many important characters are lost or altered once the animals are preserved, while other characters are subjective. Moreover, some species cannot be distinguished at all based on morphology alone, and one must rely on alternative sources of data, such as bioacoustics, genetics, or behavior. Peloso (2010) reinforced that morphological, molecular and bioacoustic data, when used in an integrated manner, are extremely effective tools for the identification of amphibians in the Brazilian Amazonia. DISCUSSION magna mily registered for the state of Pará obtained from the raw data from the MPEG collection database ecimens of the collection (Refined List). Table adapted from Ferreira et al. (2016). 459 459 459 The amphibians of Pará, Brazil TAXONOMY, A GREAT CHALLENGE In the present study, the problem was once again detected, although not quantified. Systematic and recent reviews by some groups allowed for the easy and accurate re-identification of most of the analyzed material. For example, due to the relative low diversity and the existence of recent reviews for several taxa in Gymnophiona and Caudata (Maciel & Hoogmoed, 2011b; Brcko et al., 2013, respectively) the identification of the material of these groups was greatly facilitated. Likewise, recent work with some Anura groups has also been of great value (examples in Caramaschi, 2010; Caminer & Ron, 2014; Caminer et al., 2017; Carvalho et al., 2020; Peloso et al., 2014). Finally, recent genetic studies have also helped to identify some MPEG specimens with a high degree of reliability (Castroviejo- Fisher et al., 2011; Jungfer et al., 2013; Fouquet et al., 2014, 2016, 2021b; Peloso et al., 2018). For several other groups, however, the lack of recent review work and known taxonomic problems made it extremely difficult to identify the available material. Consequently, the degree of reliability in the identifications is much higher for some groups than for others. Some species were not possible to identify to the species level, due to confusion in their diagnosis or due to the presence of a set of similar species indistinguishable morphologically. For example, Hyalinobatrachium has, in theory, three species occurrying in Pará: H. mondolfi, H. muiraquitan and H. iaspidiense (Oliveira & Hernández-Ruz, 2017). However, in the literature (Castroviejo-Fisher et al., 2011) H. mondolfi and H. iaspidiense are indistinguishable when based solely on morphological characters. The overlap of characters between H. mondolfi, H. iaspidiense, H. taylori and H. tricolor hindered the diversity sampling of the Centrolenidae family in Pará. It is possible that some of the specimens identified as H. iaspidiense refer to H. tricolor. The same occurs with H. mondolfi, where some of the specimens may represent records of H. taylori. Moreover, several specimens in several collections (including MPEG) remain unidentified. In the present study, the problem was once again detected, although not quantified. DISCUSSION Several are the examples where integrative taxonomy helped in the discovery and characterization of Amazonian species, including species from Pará (Padial et al., 2010; Jansen et al., 2011; Simões et al., 2013; Caminer & Ron, 2014; Peloso et al., 2014, 2018; Fouquet et al., 2016, 2021a; Carvalho et al., 2020). Despite recent advances and the efforts of several researchers working directly in systematic studies of Amazonian amphibians, an enormous number of species still await formal description. Several lineages have already been formally recognized as species, but not described and named, are still awaiting validation by taxonomists. As examples, we highlight some lineages recognized as independent evolutionary units (species) through genetic studies, which were never named (Fouquet et al., 2012, 2014, 2016, 2021b; Funk et al., 2012; Jungfer et al., 2013; Caminer & Ron, 2014; Caminer et al., 2017; Peloso et al., 2018). Among these species not described, some occur in Pará, as shown by our analysis of specimens of collection and the provenance of the samples used in those studies. The discovery of these taxonomic holes highlights the need to renew and intensify efforts to discover and catalog amphibians in the Amazon region, especially in Pará, where the destruction and fragmentation of the forest is extremely higher compared to other regions. Finally, it is easy to point out several examples of groups that need more systematic studies. Many groups with difficult identification and low genetic sampling need more 460 460 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 levels of deforestation, very few species of Pará are officially considered threatened with extinction. The current official Brazilian red list (“Lista Nacional Oficial de Espécies da Fauna Ameaçadas de Extinção”, MMA, 2014) includes 42 species of amphibians, of which only one is known from Pará: the salamander Bolitoglossa paraensis (Vulnerable) (Figure 4). The last update for the list of threatened species of Pará was published by the state government in 2007 (“Lista de Espécies da Flora e da Fauna Ameaçadas no Estado do Pará”, Governo do Estado do Pará, 2007) and includes three species: the salamander Bolitoglossa paraensis, and two frogs, Rhinella ocellata and Pseudopaludicola canga (Figure 4). The IUCN Red list includes Atelopus spumarius as threatened with extinction, and our list include this species. attention. ENDEMIC AND THREATENED SPECIES The distribution of organisms in the world is not random, resulting in the tendency for biodiversity to aggregate on specific areas (Sigrist & Carvalho, 2008). The concentration of organisms with restricted distribution, generated by historical factors, define the areas of endemism (Harold & Mooi, 1994; Morrone, 1994; Linder, 2001). These areas are important for providing support to postulate hypotheses about the history of the biotas and for harboring unique species (Cracraft, 1988, 1994; Morrone, 1994; Morrone & Crisci, 1995; Silva et al., 2004). Why so few species threatened with extinction, given the large diversity, a considerable number of endemic species, and major threats to the environment? Peloso (2010) discussed some of the reasons why, and many of which also likely apply here. Among the most important factors impeding correct assessment of threats and number threatened species is taxonomy, and considerable deficiencies in data related to geographical distributions. For example, Peloso (2010) mentioned the problems related to taxonomy and conservation in Adenomera. At the time, diversity of the group was vastly underestimated, and two species were considered widespread in the Amazonia (A. andreae and A. hylaedactyla), despite evidence that they might represent distinct species—both considered as Least Concern (LC). Peloso (2010) then posed some questions: Are all known, but undescribed, species in the complexes also of Least Concern? Are those widespread entities? Time has shown that these concerns were warranted. Fouquet et al. (2014) based on genetic data showed that both A. andreae and A. hylaedactyla represented complexes of many species, some with considerable narrow ranges. Since then, ten new species of Adenomera had been named, mostly from Amazonia, and several of them from Pará (Carvalho et al., 2019, 2020; Frost 2022). Among these at least one, A. phonotriccus, has an extremely narrow geographic range, We recorded 30 species endemics to Pará (Table 1; Figure 3). Although many species can represent actual endemism, it is also possible that some will be found in neighboring states and countries. This is especially true for recently named species. For example, several of the endemic species were only named in the last five years, with the majority between the years 2019 and 2022 (e.g., Adenomera amicorum, Adenomera aurantiaca, Adenomera inopinata, Adenomera phonotriccus, Adenomera tapajonica, Allobates carajas, Allobates nunciatus, Allobates tapajos, Amazophrynella bilinguis, Amazophrynella xinguensis, Hyalinobatrachium muiraquitan, Microcaecilia butantan, Pristimantis giorgii, Pristimantis latro, and Rhinatrema uaiuai). DISCUSSION Always problematic are the specimens of the genera Allobates, Amazophrynella, Atelopus, Bolitoglossa, Dendropsophus, Leptodactylus, Microcaecilia, Pristimantis, Rhinella (especially those of the Rhinella margaritifera group), and Scinax. Despite some major improvement in some of these groups, a lot remains to be done—all have in common the fact that they include several species that are morphologically similar, but genetically and biologically distinct (i.e., cryptic species). ENDEMIC AND THREATENED SPECIES The sole fact that these species are endemic to the state of Pará suggests that additional conservation attention is warranted to these species. Despite its large territory, great diversity (almost 200 species), high levels of endemism, and very intense 461 461 The amphibians of Pará, Brazil The amphibians of Pará, Brazil gure 3. Example of amphibian species endemic to the state of Pará: A) Amazophrynella xinguensis; B) Rhinella magnussoni; C) Pristima ro; D) Adelphobates castaneoticus; E) Adenomera phonotriccus; F) Chiasmocleis papachibe; G) Bolitoglossa tapajonica; H) Rhinotrema uai photos by PLVP, except (H), by T.C. Ávila-Pires. Figure 3. Example of amphibian species endemic to the state of Pará: A) Amazophrynella xinguensis; B) Rhinella magnussoni; C) Pristimantis latro; D) Adelphobates castaneoticus; E) Adenomera phonotriccus; F) Chiasmocleis papachibe; G) Bolitoglossa tapajonica; H) Rhinotrema uaiuai. All photos by PLVP, except (H), by T.C. Ávila-Pires. 462 462 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 and occur within a devastated portion of Pará’s Amazonian forest (Carvalho et al., 2019). It is likely that A. phonotriccus is threatened with extinction, although an official assessment by IUCN is pending. This conclusion was only possible due to the concentrated effort the resolve the taxonomic status of these likely endangered populations (Carvalho et al., 2019, 2020). It is likely that more species relate to the example above—narrow range, threatened species being treated as widespread, Least Concern taxa. We urge that stakeholders invest heavily in initiatives that accelerate taxonomy and the collection/publication of distribution and natural history data on Amazonian biodiversity. Figure 4. Endangered species of amphibians from Pará: A) Pará’s Lungless Salamander (Bolitoglossa paraensis). The only species from Pará included in the Brazilian redlist (“Lista nacional oficial de espécies da fauna ameaçadas de extinção”: MMA, 2014). Photo from Santa Bárbara do Pará, Pará, Brazil; B) Pseudopaludicola canga, from Carajás; and C) Rhinella ocellata, from Serra do Cachimbo. Both P. canga and R. ocellata were included in the state’s red list (“Lista de espécies da flora e da fauna ameaçadas no estado do Pará”: Governo do Estado do Pará, 2007). Figure 4. Endangered species of amphibians from Pará: A) Pará’s Lungless Salamander (Bolitoglossa paraensis). The only species from Pará included in the Brazilian redlist (“Lista nacional oficial de espécies da fauna ameaçadas de extinção”: MMA, 2014). REFERENCES Great challenges lie ahead on our path to a decent understanding of amphibian diversity in Amazonia. Our study highlighted a high species richness of amphibians in Pará—a total of 195 species registered, of which five represent new records, and 30 are considered endemic to the region. This is the largest species richness among all Brazilian states with published or estimated species lists, after São Paulo and Rio de Janeiro, respectively. The high diversity is not surprising, giving the size of the state and the diversity of habitats present therein. 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South American Journal of Herpetology, 13(2), 170–182. https://doi.org/10.2994/ sajh-d-17-00040.1 Silva, L. A., Carvalho, P. S., Pereira, E. A., Fadel, R. M., Dantas, S. P., Brandão, R. A., & Santana, D. J. (2020). Richness, diversity patterns, and taxonomic notes of amphibians from the tocantins state. Biota Neotropica, 20(1), e20190838. https:// doi.org/10.1590/1676-0611-BN-2019-0838 Simões, P. I., Kaefer, I. L., Farias, I. P., & Lima, A. P. (2013). An integrative appraisal of the diagnosis and distribution of Allobates sumtuosus (Morales, 2002) (Anura, Aromobatidae). Zootaxa, 3746(3), 401-421. https://doi.org/10.11646/ zootaxa.3746.3.1 Prudente, A. L. C., Sarmento, J. F. M., Ávila-Pires, T. C. S., Maschio, G. F., & Sturaro, M. J. (2018). How much do we know about the diversity of Squamata (Reptilia) in the most degraded region of Amazonia? South American Journal of Herpetology, 13(2), 117-130. https://doi.org/10.2994/SAJH-D-17-00009.1 Roberto, I. ACKNOWLEDGEMENTS J., Ribeiro, S. C., & Loebmann, D. (2013). Anfíbios do estado do Piauí, Nordeste do Brasil: Um ensaio preliminar. Biota Neotropica, 13(1), 322–330. https://doi.org/10.1590/ S1676-06032013000100031 Simões, P. I., Rojas, D., & Lima, A. P. (2019). A name for the nurse-frog (Allobates, Aromobatidae) of Floresta Nacional de Carajás, Eastern Brazilian Amazonia. Zootaxa, 4550(1), 71–100. https://doi.org/10.11646/zootaxa.4550.1.3 468 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 Souza, M. B. (2009). Anfíbios: Reserva Extrativista do Alto Juruá e Parque Nacional da Serra do Divisor, Acre (Série Pesquisa e Monitoramento Participativo em Áreas de Conservação Gerenciadas por Populações Tradicionais, Vol. 2). Instituto de Filosofia e Ciências Humanas. Valsecchi, J., Marmontel, M., Franco, C. L. B., Cavalcante, D. P., Cobra, I. V. D., Lima, I. J., . . . Monteiro, V. (2017). Atualização e composição da lista – novas espécies de vertebrados e plantas na Amazônia 2014-2015. Iniciativa Amazônia Viva da Rede WWF/WWF-Brasil/Instituto de Desenvolvimento Sustentável Mamirauá. Souza, F. L., Prado, C. P. A., Sugai, J. L. M. M., Ferreira, V. L., Aoki, C., Landgref-Filho, P., . . . Duleba, S. (2017). Amphibian diversity of Mato Grosso do Sul State, Brazil. Iheringia, Série Zoologia, 107(suppl.), e2017152. https://doi.org/10.1590/1678- 4766e2017152 Vaz-Silva, W., Oliveira, R., Gonzaga, A., Pinto, K., Poli, F., Bilce, T., . . . Pinheiro, R. (2015). Contributions to the knowledge of amphibians and reptiles from Volta Grande do Xingu, northern Brazil. Brazilian Journal of Biology, 75(3), 205–218. https://doi. org/10.1590/1519-6984.00814bm Spix, J. B. (1824). Animalia Nova Sive Species NovaeTestudinum et Ranarum Quas in Itinere perBrasiliam Annis MDCCCXVII- MCCCCXX Jussu etAuspidis Maximiliani Joseph I. Bavarie Regis. Monachii, S. Hübschmanni. Vaz-Silva, W., Maciel, N., Nomura, F., Morais, A., Guerra-Batista, V., Lopes, D., . . . Bastos, R. (2020). Guia de identificação das espécies de anfíbios (Anura e Gymnophiona) do estado de Goiás e do Distrito Federal, Brasil Central. Sociedade Brasileira de Zoologia. https://doi.org/10.7476/9786587590011 Steege, H., Vaessen, R. W., Cárdenas-López, D., Sabatier, D., Antonelli, A., De Oliveira, S. M., . . . Salomão, R. P. (2016). The discovery of the Amazonian tree flora with an updated checklist of all known tree taxa. Scietific Reports, 6, 29549. https://doi.org/10.1038/srep29549 Vollmar, A., Macklin, J. A., & Ford, L. S. (2010). Natural history specimen digitization: challenges and concerns. Biodiversity Informatics, 7(2), 93-112. https://doi.org/10.17161/ bi.v7i2.3992 Stuart, S., Hoffmann, M., Chanson, J., Cox, N., Berridge, R., Ramani, P., & Young, B. (2008). Threatened amphibians of the World. ACKNOWLEDGEMENTS Lynx Edicions, IUCN, Conservation International. World Wide Fund for Nature (WWF). (2010). Amazon alive! A decade of discovery 1999-2009. https://wwf.panda.org/wwf_ news/?200056/AmazonAliveAdecadeofdiscovery1999--2009 Toledo, L. F., & Batista, R. F. (2012). Integrative study of Brazilian Anurans: geographic distribution, size, environment, taxonomy, and conservation. Biotropica, 44(6), 785–792. https://doi.org/10.1111/j.1744-7429.2012.00866.x Zimmerman, B. L., & Rodrigues, M. T. (1990). Frogs, snakes, and lizards of the INPA/WWF reserves near Manaus, Brazil. In A. H. Gentry (Eds.), Four Neotropical rainforests (pp. 426–454). Yale University Press. Vacher, J. P., Chave, J., Ficetola, F. G., Sommeria-Klein, G., Tao, S., Thébaud, C., . . . Fouquet, A. (2020). Large-scale DNA-based survey of frogs in Amazonia suggests a vast underestimation of species richness and endemism. Journal of Biogeography, 47(8), 1781–1791. https://doi.org/10.1111/jbi.13847 AUTHORS’ CONTRIBUTIONS G. F. Cassundé contributed to formal analysis, conceptualization, data curation, investigation, validation, and writing (original draft); M. J. Sturaro to data curation, investigation, and writing (review and editing); A. O. Maciel to data curation, investigation, and writing (review and editing); A. L. C. Prudente to data curation, resources, and writing (review and editing); J. F. M. Sarmento to data curation, and writing (review and editing); and P. Peloso to project administration, funding acquisition, conceptualization, data curation, supervision, and writing (original draft). AUTHORS’ CONTRIBUTIONS G. F. Cassundé contributed to formal analysis, conceptualization, data curation, investigation, validation, and writing (original draft); M. J. Sturaro to data curation, investigation, and writing (review and editing); A. O. Maciel to data curation, investigation, and writing (review and editing); A. L. C. Prudente to data curation, resources, and writing (review and editing); J. F. M. Sarmento to data curation, and writing (review and editing); and P. Peloso to project administration, funding acquisition, conceptualization, data curation, supervision, and writing (original draft). 469 469 469 469 The amphibians of Pará, Brazil Appendix 1. Voucher of testimony specimens used to generate the list of amphibians in the State of Pará. Appendix 1. Voucher of testimony specimens used to generate the list of amphibians in the State of Pará Order Anura Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. Order Anura Order Anura Adelophryne gutturosa: BM 1983.1139; LACM 44277; LACM 44278; Adelphobates castaneoticus: MPEG 10329; MPEG 10581; MPEG 22000; MPEG 25186; Adelphobates galactonotus: MPEG 2921-2924; Adelphobates quinquevittatus: MPEG 6691; MPEG 6709; MPEG 6947; Adenomera amicorum: INPA-H 40490–40499; INPA-H 40501–40510; Adenomera andreae: MPEG 30747; Adenomera aurantiaca: INPA-H 40518–40521; Adenomera heyeri: MPEG 30095–30101; Adenomera hylaedactyla: MPEG 30903; Adenomera inopinata: INPA-H 40517; Adenomera kayapo: CFBH 43885; MPEG 41619–41620; Adenomera martinezi: CHUNB 40218; CHUNB 40220; Adenomera phonotriccus: MPEG 41155–41156; Adenomera tapajonica: INPA-H 40515–40516; Allobates carajas: INPA-H 38635; INPA-H 38642; INPA-H 38643; Allobates crombiei: MPEG 37859; MPEG 37860; Allobates grillicantus: MPEG 43038–43050; Allobates femoralis: MPEG 29444–29445; MPEG 33686–33687; MPEG 33799; MPEG 33800–33882; Allobates magnussoni: MPEG 30888–30891; MPEG 33623-33627; Allobates marchesianus: MPEG 16193; MPEG 18187; MPEG 18193; MPEG 18201–18202; MPEG 18206; Allobates masniger: USNM 303585–303587; MZUSP 69166; MZUSP 69167; Allobates nunciatus: INPA-H 40305; INPA-H 40307; INPA-H 40482; INPA-H 40486; Allobates sumtuosus: USNM 303591– 303593; MZUSP 69157; MZUSP 69158–69160; Allobates tapajos: MPEG 3198–31801; MPEG 35854–35855; Allophryne ruthveni: MPEG 2972; MPEG 3751; MPEG 10083; MPEG 30390; Amazophrynella bilinguis: INPA-H 39780; INPA-H 39782; INPA-H 39784; Amazophrynella bokermanni: MPEG 24656; MPEG 24669; MPEG 27191; MPEG 27201; MPEG 27204; MPEG 27208; Amazophrynella gardai: ZUFMS-AMP12821–12828; Amazophrynella minuta: MPEG 23228; MPEG 23234; MPEG 23239; MPEG 23243; Amazophrynella xinguensis: INPA–H 35471; INPA–H 35482, INPA–H 35493; INPA–H 35472; Ameerega braccata: MPEG 712; MPEG 2860; MPEG 2864; MPEG 3207; MPEG 3266; MPEG 3268; MPEG 3313; Ameerega flavopicta: MPEG 28442–28443; Ameerega hahneli: MPEG 25076–25083; MPEG 25373; MPEG 29425; MPEG 30291; Ameerega munduruku: ZUFMS-AMP 03762; ZUFMS-AMP 03747–03749; Ameerega trivittata: MPEG 30687–30691; Anomaloglossus stefeni: MPEG 17175; MPEG 15185–15186; Atelopus hoogmoedi: MPEG 25071–25075; MPEG 30988–30998; Atelopus spumarius: MPEG 22606; MPEG 34479; Boana boans: MPEG 21969–21971; MPEG 21973; MPEG 33461–33462; Boana caiapo: MZUSP 138987–139009; Boana calcarata: MPEG 25634; MPEG 25657; MPEG 30468; MPEG 38866–38870; Boana cinerascens: MPEG 35593–35595; MPEG 25638–25643; Boana courtoisae: MPEG 30336; MPEG 30341–30342; Boana dentei: MPEG 30299–30300; Boana diabolica: MPEG 33920; Boana geographica: MPEG 1316–1317; MPEG 8162; Boana icamiaba: MNRJ 89836– 89839, MNRJ 90997–91000; MPEG 27245–27249; MPEG 40100–40110; Boana lanciforms: MPEG 17118–17122; MPEG 37451–37453; Boana leucocheila: MPEG 14271; MPEG 22210–22211; MPEG 22347–22349; MPEG 22393– 22395; MPEG 27239–27244; MPEG 33538; MPEG 37062; Boana multifasciata: MPEG 31637–31638, MPEG 33324, MPEG 39182, MPEG 39185; Boana punctata: MPEG 8813; MPEG 761; MPEG 37414; MPEG 32313; MPEG 19804; Boana raniceps: MPEG 21264–21268; MPEG 11722–11727; MPEG 8308; MPEG 6387; MPEG 6337; MPEG 6347; MPEG 21158; Boana steinbachi: MPEG 39405; MPEG 39532; MPEG 39534–39535; MPEG 25661; MPEG 29648– 25650; MPEG 36753; Boana wavrini: MPEG 11718–11721; MPEG 5923; MPEG 27247; MPEG 37061; Callimedusa tomopterna: MPEG 4330–4334; MPEG 11157; MPEG 11159-11162; MPEG 31978; Ceratophrys cornuta: MPEG 15625; MPEG 1525; MPEG 28987–28988; Chiasmocleis avilapiresae: MPEG 23276–23280; MPEG 23287–23293; Chiasmocleis bassleri: MPEG 18574; MPEG 28326; Chiasmocleis hudsoni: MPEG 14172; MPEG 32696–32704; 470 Bol. Order Anura 2022 Chiasmocleis papachibe: MPEG 30683; MPEG 27788; Chiasmocleis shudikarensis: MPEG 6603; MPEG 30396–30404; Cruziohyla craspedopus: MPEG 38389; Ctenophryne geayi: MPEG 11400–11401; Dendrobates tinctorius: MPEG 27875; 27877; MPEG 19965; Dendropsophus brevifrons: MPEG 25558–25561; MPEG 14367–14368; MPEG 25516; MPEG 11647–11657; Dendropsophus cachimbo: MZUSP 21912; MNRJ 17298–17299; MZUSP 21911, 21913–21918, 21920– 21926; Dendropsophus gaucheri: MPEG 38719; Dendropsophus haraldschultzi: MPEG 33289–33290; Dendropsophus leucophyllatus: MPEG 8808; MPEG 19008–19023; Dendropsophus marmoratus: MPEG 20515; MPEG 27459; MPEG 27461; MPEG 12076–12077; Dendropsophus melanargyreus: MPEG 31466–31483; MPEG 18945–18954; Dendropsophus microcephalus: MPEG 35553; MPEG 35544; MPEG 35555; MPEG 15104; MPEG 27546; Dendropsophus minimus: NHMW 19436; Dendropsophus minusculus: MPEG 7560; MPEG 16428–16444; Dendropsophus minutus: MPEG 16481–16489; MPEG 31700–31704; MPEG 30910–30920; Dendropsophus nanus: MPEG 21635; 21642; MPEG 21659; MPEG 21647; MPEG 21634; Dendropsophus ozzyi: MPEG 27263–27279; Dendropsophus parviceps: MPEG 38719; Dendropsophus reticulatus: MPEG 19565; MPEG 16025; Dendropsophus rossalleni: MPEG 16365; MPEG 20050–20052; Dendropsophus sarayacuensis: MPEG 61340; Dendropsophus schubarti: MPEG 25537–25541; Dendropsophus triangulum: MPEG 32310–32311; MPEG 15976; Dendropsophus walfordi: MPEG 17104–17115; Dryaderces inframaculata: MPEG 35725–35728; Elachistocleis carvalhoi: MPEG 3200; MPEG 3258; MPEG 3260; Elachistocleis helianneae: MPEG 1767; MPEG 8929–8931; MPEG 29415–29420; MPEG 6875; MPEG 6934; MPEG 6939; MPEG 20566; MPEG 21423–21479; MPEG 35720–35724; MPEG 6929–6931; MPEG 5992; MPEG 6353; Elachistocleis magna: MPEG 23785; MPEG 31705–31709; MPEG 34579–34581; Elachistocleis surinamensis: MPEG 28130–28132; MPEG 28444; MPEG 28445; MPEG 35710–35713; MPEG 35866; Engystomops freibergi: MPEG 40244–40246; Hamptophryne boliviana: MPEG 24705–24710; MPEG 30488–30491; Hemiphractus scutatus: INPA-H 38116–38118; Hyalinobatrachium iaspidiense: MPEG 30871; MPEG 38896; MPEG 38899; Hyalinobatrachium mondolfii: MPEG 38906; MPEG 30872; MPEG 38898; Hyalinobatrachium muiraquitan: LZA 841; LZA 842; LZA 843; LZA 844; Hydrolaetare schmidti: MPEG 8790; MPEG 18330–18333; Leptodactylus fuscus: MPEG 31713–31719; Leptodactylus intermedius: CFBH 39668– 39669; Leptodactylus knudseni: MPEG 17589; MPEG 32314–32326; Leptodactylus labyrinthicus: MZUSP 21734; MPEG 5784; MPEG; 14197; MPEG 14207; MPEG 14320; MPEG 14326; Leptodactylus leptodactyloides: MPEG 11516; MPEG 11537; MPEG 14404–14406; Leptodactylus longirostris: MPEG 30831–30848; MPEG 33282-33285; Leptodactylus macrosternum: CHUNB 31189; Leptodactylus myersi: MPEG 30758–30765; MPEG 19744–19746; Leptodactylus mystaceus: MPEG 29113–29114; MPEG 34048–34052; Leptodactylus paraenses: MZUSP 69318; MPEG 25764–25770; MPEG 33319; MPEG 32588–32590; Leptodactylus pentadactylus: MPEG 25771–25774; MPEG 25781–25783; Leptodactylus petersii: CFBH 16742; CFBH 167423; Leptodactylus pustulatus: MPEG 35832–35834; Leptodactylus rhodomystax: MPEG 25871–25876; Leptodactylus stenodema: MPEG 20643; MPEG 20648; MPEG 21403–21405; MPEG 29423–29424; Lithobates palmipes: MPEG 29451–29452; Lithodytes lineatus: MPEG 25707– 25709; MPEG 30849–30869; Lysapsus laevis: MPEG 33751–33754; MPEG 20015–20021; MPEG 33631–33632; MPEG 33277–33281; Lysapsus limellum: MPEG 19805–19809; Osteocephalus cabrerai: MPEG 27212–27222; Osteocephalus leprieurii: MPEG 38937; MPEG 38942; MPEG 27900; MPEG 37614–37617; Osteocephalus oophagus: MPEG 38134; MPEG 38130–38131; Osteocephalus taurinus: MPEG 40258; MPEG 22205–22207; MPEG 29991–29996; Otophryne pyburni: MPEG 17605; Phyllomedusa bicolor: MPEG 38121–38123; MPEG 39890; Phyllomedusa vaillantii: MPEG 12121–12128; MPEG 36583; MPEG 36605; MPEG 29009; 29391; Physalaemus cuvieri: MPEG 19759–19760; MPEG 30882–30887; Physalaemus ephippifer: MPEG 6125–-6128; MPEG 34624–34626; Phyzelaphryne miriamae: 471 471 471 The amphibians of Pará, Brazil USNM 239363; Pipa arrabali: MPEG 25155–25158; MPEG 27915; MPEG 31305; Pipa pipa: MPEG 8791–8800; MPEG 25935; MPEG 32486; Pipa snethlageae: MPEG 16939; MPEG 23275; Pithecopus hypochondrialis: MPEG 20631; MPEG 40218–40222; MPEG 9301–9324; Potamotyphlus kaupii: MPEG 7345; Pristimantis chiastonotus: MPEG 32222; MPEG 32224; MPEG 32227; MPEG 32229; Pristimantis fenestratus: MPEG 33477–33480; Pristimantis giorgii: LZAG 1381–1389; MPEG 21145–21147; MPEG 34838–33847; MPEG 35610–35613; Pristimantis gutturalis: MPEG 21395; MPEG 21396; MPEG 27873; MPEG 27873; Pristimantis latro: LZATM 0063; LZATM 139; LZATM 197; LZATM 467; LZATM 739; LZATM 747; MPEG 26050; MPEG 26059; MPEG 31415–31416; Pristimantis marmoratus: MPEG 30085; MPEG 30088; Pristimantis ockendeni: MZUSP (field number BM153); Pristimantis pictus: ZUFMS–AMP 8540–8543; Pristimantis zeuctotylus: MPEG 29684–29692; MPEG 30892–30899; Proceratophrys concavitympanum: MPEG 40377; MPEG 40379; Proceratophrys rondonae: MPEG 33451; MPEG 22517; Pseudis paradoxa: MPEG 18328; Pseudis tocantins: MPEG 34803–34805; MPEG 35827–35829; MPEG 28111; MPEG 38102; Pseudopaludicola boliviana: UFMT 15981; UFMT 16176; UFMT 16177; UFMT 16178; UFMT 16181; Pseudopaludicola canga: ZUEC 6088; ZUEC 6274; ZUEC 6275; Pseudopaludicola mystacalis: AAG-UFU 6262; Ranitomeya amazonica: MPEG 22742; MPEG 19706; MPEG 24602–24608; MPEG 17417; Rhaebo guttatus: MPEG 30693; MPEG 3422; MPEG 39567–39570; Rhinella castaneotica: MZUSP 67162; Rhinella dapsilis: CFBHT 12072; Rhinella diptycha: MPEG 39197; Rhinella granulosa: MPEG 21983– 21988; Rhinella magnussoni: INPA-H 19527–195310; Rhinella major: MPEG 31779– 31780; MPEG 31782; MPEG 31790–MPEG 31792; Rhinella margaritifera: MPEG 30766–30790; Rhinella marina: MPEG 39194–39196; MPEG 39198– 39199; Rhinella mirandaribeiroi: MPEG 31757–31760; Rhinella ocellata: CHUNB 40244; Rhinella proboscidea: MPEG 33829–33831; Scarthyla goinorum: CZPB-AA 949–950; Scinax boesemani: MPEG 16565–16577; MPEG 33675–33677; MPEG 22356–22357; Scinax fuscomarginatus: MPEG 31325–31359; Scinax garbei: MPEG 30301– 30304; MPEG 31827; MPEG 36831–36832; Scinax nebulosus: MPEG 37622–37624; MPEG 31631–31634; MPEG 31636; MPEG 24993–25503; Scinax proboscideus: MPEG 29668; Scinax rostratus: MPEG 28060–28061; Scinax ruber: MPEG 28100; MPEG 31608–31610; MPEG 37427–37428; MPEG 6350; Scinax villasboasi: CHUNB 34498−34500; CHUNB 34502; CHUNB 34505−34510; CHUNB 40156–40157, CHUNB 40159−40161; Scinax x-signatus: MPEG 16506–16507; MPEG 6008; MPEG 6012–6014; MPEG 6017–6018; Sphaenorhynchus lacteus: MPEG 36842; MPEG 3106; MPEG 739; MPEG 33709; MPEG 6136–3138; MPEG 6185; MPEG 6187; Synapturanus ajuricaba: MPEG 29453– 29454; MPEG 29456–MPEG 29458; INPA-H 28519; INPA-H 35751; INPA-H 38464; Synapturanus mirandaribeiroi: MPEG 19962–19964; MPEG 20007–20011; Trachycephalus coriaceus: MPEG 35500; 41293; Trachycephalus cunauaru: MZUSP 71146; Trachycephalus helioi: MPEG 32558; MPEG 39200; MPEG 20507; Trachycephalus hadroceps: MPEG 43224;Trachycephalus resinifictrix: MPEG 15507; MPEG 8650; MPEG 16961; MPEG 14710; Trachycephalus typhonius: MPEG 38068; MPEG 34057; MPEG 20873. Order Anura Order Caudata Bolitoglossa paraensis: MPEG 31682; Bolitoglossa tapajonica: MPEG 22176. Bolitoglossa paraensis: MPEG 31682; Bolitoglossa tapajonica: MPEG 22176. Order Gymnophiona Atretochoana eiselti: MPEG 33621; Brasilotyphlus guarantanus: MPEG 22170; Caecilia gracilis: MPEG 8368; MPEG 9848; MPEG 15857; Caecilia tentaculata: MPEG 22068–22073; MPEG 22811; Microcaecilia butantan: MZUSP 143389; Microcaecilia marvaleewakeae: MPEG 21896; Microcaecilia rochai: MPEG 14596–14597; Microcaecilia trombetas: 472 472 472 Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 MPEG 26476; Nectocaecilia petersii: UFOPA-H 1231; Potamotyphlus kaupii: MPEG 7345; Rhinatrema bivittatum: MPEG 23548–23549; Rhinatrema gilbertogili: MPEG 16975; MPEG 17435; MPEG 19966–19967; Rhinatrema uaiuai: MPEG 26477; Siphonops annulatus: MPEG 33734; Typhlonectes compressicauda: MPEG 7337–7339; MPEG 7355–7361; MPEG 7363–7374. Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 MPEG 26476; Nectocaecilia petersii: UFOPA-H 1231; Potamotyphlus kaupii: MPEG 7345; Rhinatrema bivittatum: MPEG 23548–23549; Rhinatrema gilbertogili: MPEG 16975; MPEG 17435; MPEG 19966–19967; Rhinatrema uaiuai: MPEG 26477; Siphonops annulatus: MPEG 33734; Typhlonectes compressicauda: MPEG 7337–7339; MPEG 7355–7361; MPEG 7363–7374. Bol. Mus. Para. Emílio Goeldi. Cienc. Nat., Belém, v. 17, n. 2, p. 445-473, maio-ago. 2022 MPEG 26476; Nectocaecilia petersii: UFOPA-H 1231; Potamotyphlus kaupii: MPEG 7345; Rhinatrema bivittatum: MPEG 23548–23549; Rhinatrema gilbertogili: MPEG 16975; MPEG 17435; MPEG 19966–19967; Rhinatrema uaiuai: MPEG 26477; Siphonops annulatus: MPEG 33734; Typhlonectes compressicauda: MPEG 7337–7339; MPEG 7355–7361; MPEG 7363–7374. 473 473 473
https://openalex.org/W4387119693	https://www.fracturae.com/index.php/fis/article/download/4428/3886	English	null	The behavior of reinforced lightweight concrete beams with initial cracks	Frattura ed integrità strutturale	2,023	cc-by	7,571	Received: 09.07.2023 Accepted: 14.09.2023 Online first: 18.09.2023 Published: 01.10.2023 Received: 09.07.2023 Accepted: 14.09.2023 Online first: 18.09.2023 Published: 01.10.2023 Copyright: © 2023 This is an open access article under the terms of the CC-BY 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. KEYWORDS. Lightweight concrete, Rehabilitation, Carbon fiber, U-wrapping, CFRP. M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 ABSTRACT. This research examines the performance of reinforced lightweight concrete beams subjected to several degrees of damage (50%, 60%, 70%, and 100%). It can use a sheet made of Carbon Fiber Reinforced Polymer (CFRP) to reinforce. The Full U-wrapping rehabilitation method was tested in the presented experimental program. In this method, CFRP sheets are attached to the bottom only and the side and bottom of the beam section. Experiments proved that the service load (Ps) increases by 7.06 % from a damage level of 50 % to 70 %, rises by 1.21 % from a damage level of 60 % to 70 %, and falls by 3.07 % from a damage level of 100 %. The result also rose for the fortified sample by 11.99%. Increases of 42.67 %, 33.07 %, and 23.73 % in the stiffness ratio (k) were observed at damage intensities of 50, 60, and 70 %, respectively. Damage at lower severity levels is increasing at a faster rate. The ductility of the restored LWC beams is more excellent than the control, as with the stiffness. Damage levels of 50%, 60%, and 70% saw increased ductility of 35.60, 34.92, and 34.69 %, respectively. Citation: Hasan, M. Q., Abdulridha, A. J.,The behavior of reinforced lightweight concrete beams with initial cracks, Frattura ed Integrità Strutturale, 66 (2023) 297-310. Citation: Hasan, M. Q., Abdulridha, A. J.,The behavior of reinforced lightweight concrete beams with initial cracks, Frattura ed Integrità Strutturale, 66 (2023) 297-310. The behavior of reinforced lightweight concrete beams with initial cracks Mustafa Q. Hasan, Abdulkhalik J. Abdulridha Department of Civil Engineering, College of Engineering, Al-Nahrain University, Jadriya, Baghdad, Iraq mustafa88qusay@gmail.com Abdulkhalik.J.AbdulRidha@nahrainuniv.edu.iq, https://orcid.org/0000-0001-6403-2325 Mustafa Q. Hasan, Abdulkhalik J. Abdulridha Department of Civil Engineering, College of Engineering, Al-Nahrain University, Jadriya, Baghdad, Iraq mustafa88qusay@gmail.com Abdulkhalik.J.AbdulRidha@nahrainuniv.edu.iq, https://orcid.org/0000-0001-6403-2325 INTRODUCTION made from natural materials like pumice, diatomite, and volcanic ash or manufactured materials like perlite, extended schist, clay, slate, and sintered powdered fuel ash (PFA). Lightweight aggregate concrete (LWAC) replaces natural aggregates with manufactured rocks that are less dense but still strong [11-15]. This type of concrete has scientific and commercial benefits, and the environment may also benefit from using LWAC. The mechanical properties of LWAC are determined by mix design (water-to-cement ratio, quality, and aggregates’ characteristics), the preformed foam's quality, and the type of foaming agent [16-19]. FRP can be added to the mix to make concrete or bricks more durable. A significant concern when dealing with FRP [20] is that the reinforcement can separate from the attached material. Changes in body damage substantially affect how smoothing works [21], but only until damage at the interface exceeds that at the body. g ] y g y For many reinforced concrete building systems, reducing the dead load is essential for relieving stress on the supporting structures and cutting costs. Insulation from heat and sound is a common requirement for many newer structures [22]. The relatively high water-to-cement (W/C) ratios were once the norm in the concrete industry. The hybrid variety's resilience to both drought and pests is exceptional. Nonetheless, there are usual difficulties, as shown by shrinking at cracking [23], despite these benefits. While chemical shrinkage occurs in both high and low W/C mixes, low W/C mixes are more prone to self-drying out because of holes and capillary pressure, which produces fractures [23-25]. LWC (lightweight concrete) might solve many of these problems. If the weight placed on an RC beam is more significant than it can support, it will crack. Although unlikely, this can occur, especially if the building's framework is experiencing significant stress [26]. This study looks at the feasibility of employing FRP designs to secure LWC beams. Light Weight Aggregate (LWA) has a reduced density of mass compared to Normal Weight Aggregate (NWA) [27]. Adopting LWA for internal hardening in RC applications may increase the service life of the entire structure by reducing early-age stresses and shrinkage. Researchers have shown that introducing LWA increases moisture, increases mechanical strength across a broader range, and reduces heat conductivity [28]. After drilling, the pressure inside the capillary holes will drop because the cement paste will hydrate and dry out independently, allowing water from the LWA's perforations to soak into the cement. INTRODUCTION It is essential to supply enough water inside the LWA—paste system and guarantee that it is distributed uniformly for proper paste integration. The concept's central defining feature controls water flow inside the grid: the distance between LWA particles. According to the statistics reported in this paper, increasing the LWA substitution ratio might dramatically improve water distribution [22]. Lightweight Expanded Clay Aggregate (LECA) is a kind of LWA produced by heating clay to 1200 C in a rotating kiln. Due to the decompression of the gases upon boiling, thousands of bubbles make up the bulk of this mixture. As a result of these gases, clay tends to expand, taking on a honeycomb pattern [29]. Most rotating kilns have a round form because of the way they produce heat. This chemical will be used as LWA in the synthesis of LWC in this experimental research. FRP use in this capacity has become prominent in recent years. The superiority of some materials over more common ones like steel makes this abundantly clear. Utilizing three primary types of FRPs for RC beam strengthening and repair are studied. CFRP is an early leader in this field. The second variety is a composite reinforced with aramid and glass fibers. CFRP is the best material for strengthening and repairing RC beams. g g g p g When components of a building are upgraded either before or after they begin to fail, this process is known as a retrofit. Restoration, however, covers how a damaged building can be fixed. Recent articles published in peer-reviewed journals in this discipline have dabbled in various formats and mediums. Pilot programs are frequently used to test these methods. People are always making an effort in the pursuit of better results. An essential part of recovery is pinpointing what went wrong. This factor may come into play while deciding what to do next. For these reasons, many scholars maintain that this topic still merits study. Due to the minimal likelihood of repeating border conditions, this is a serious concern. As a result, the strategy taken may be affected by factors such as technology, economics, and even society [30-32]. INTRODUCTION here is a growing interest in using fiber-reinforced polymer FRP technology to update RC construction. FRP has been widely used for repairing and replacing worn-out and broken structures. Changes in CFRP thickness have a significant effect on the ultimate load-bearing capability. Longitudinal soffit-bonded CFRP strips are used to reinforce shear-weak RC beams so that they are better able to bear lateral service loads, as opposed to the more common transversely aligned or wrapped CFRP strips, used in multi-girder bridge decks. [1-4]. Using fewer resources in construction yields savings in both energy and money [5-7]. LWA's output includes a material known as Lightweight Aggregate Concrete (LWAC). T i fT ( ) The compressive strength of LWAC (concrete with a weight per cubic meter of less than 1800 kilograms) is greater than 18 MPa [8-10]. Lightweight Aggregate Concrete (LWAC) is most often used to increase the insulating properties of a structure, particularly in terms of heat and sound. Traditional aggregates are replaced by lightweight aggregates (LWA), which may be The compressive strength of LWAC (concrete with a weight per cubic meter of less than 1800 kilograms) is greater than 18 MPa [8-10]. Lightweight Aggregate Concrete (LWAC) is most often used to increase the insulating properties of a structure, particularly in terms of heat and sound. Traditional aggregates are replaced by lightweight aggregates (LWA), which may be 297 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 made from natural materials like pumice, diatomite, and volcanic ash or manufactured materials like perlite, extended schist, clay, slate, and sintered powdered fuel ash (PFA). Lightweight aggregate concrete (LWAC) replaces natural aggregates with manufactured rocks that are less dense but still strong [11-15]. This type of concrete has scientific and commercial benefits, and the environment may also benefit from using LWAC. The mechanical properties of LWAC are determined by mix design (water-to-cement ratio, quality, and aggregates’ characteristics), the preformed foam's quality, and the type of foaming agent [16-19]. FRP can be added to the mix to make concrete or bricks more durable. A significant concern when dealing with FRP [20] is that the reinforcement can separate from the attached material. Changes in body damage substantially affect how smoothing works [21], but only until damage at the interface exceeds that at the body. OUTLINE OF THE EXPERIMENTAL SECTION d hT he research investigates what happens to beams made of reinforced lightweight concrete when they sustain 50%, 60%, 70%, or 100% damage. Use the CFRP sheet to strengthen. Five reinforced lightweight concrete beams span 1600mm center to center and 1800mm total length. The dimensions of the beam section are a total height of 300mm and the width of 200mm. The 2 Ø 8mm top reinforcement and 3 Ø 12mm bottom steel bars were used as primary reinforcement. Additionally, Ø 10 mm @ 120mm were used as stirrups, illustrated in Fig. 1. Tab. 1 shows the beam specimen descriptions. T The first digit of the specimen number indicates whether the sample was strengthened (S) or repaired (R). The second number (F), means that the Full wrapping method is used over the whole section height. The severity of the damage (in terms of ultimate load) is indicated by the last digit. Tabs. 2 and 3 show the chemical and physical parameters of the regular Portland cement utilized in this experiment, and they conform to the Iraqi standard specification (I.Q.S No.5-2019). We've 298 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 been using natural sand with a maximum particle size of 4.75 millimeters. As shown in Tab. 4, it satisfies the grading requirements of the Iraqi standard specification (I.Q.S No.45-1984). Tab. 5 shows that the maximum diameter of the lightweight coarse aggregate LECA employed in this investigation is 12 mm. This material replaced the coarse aggregate in the LWC. Iraqi standards (I.Q.S. No.45-1984) were used to conduct the necessary testing on this sand. the LWC. Iraqi standards (I.Q.S. No.45-1984) were used to conduct the necessary testing on this sand. Figure 1: Specimen dimensions of the present study Sec. (A-A). Specimen designation Description Control Referential specimen without CFRP SF-100 Strengthened beam with full wrapping CFRP sheets. RF-50 Repaired beam with full U-wrapping CFRP sheets and subjected to 50% of damage. RF-60 Repaired beam with full U-wrapping CFRP sheets and subjected to 60% of damage. RF-70 Repaired beam with full U-wrapping CFRP sheets and subjected to 70% of damage. Table 1: Beam specimens’ descriptions. Oxide Percentage by weight% Limit of IQS No. OUTLINE OF THE EXPERIMENTAL SECTION 5/ 2019 CaO 60.37 - SiO2 20.74 - Al2O3 5.78 - Fe2O3 3.48 - MgO 1.91 < 5.0% SO3 1.85 ≤ 2.8% for C3A ≥ 3.5% Loss on Ignition (L.O.I) 2.44 < 4.0% Lime Saturation Factor (L.S.F) 0.92 0.66 - 1.02 Insoluble residue (I.R) 0.19 < 1.5 % Main compound (Bouge equation) % by weight of cement - Limit of IQS No. 5/ 2019 C3S 41.7 - C2S 27.4 - C3A 10.2 - C4AF 9.9 - Table 2: Chemical analysis of cement. Physical properties Test result Limit of IQS No. 5/ 2019 Fineness, Blaine, gm/cm2 319 >230 Setting Time: Initial, min Setting Time: Final, min 95 410 ≥45 min ≤10hrs Table 3: Physical analysis of cement. Figure 1: Specimen dimensions of the present study Sec. (A-A). Specimen designation Description Control Referential specimen without CFRP SF-100 Strengthened beam with full wrapping CFRP sheets. RF-50 Repaired beam with full U-wrapping CFRP sheets and subjected to 50% of damage. RF-60 Repaired beam with full U-wrapping CFRP sheets and subjected to 60% of damage. RF-70 Repaired beam with full U-wrapping CFRP sheets and subjected to 70% of damage. Table 1: Beam specimens’ descriptions. Oxide Percentage by weight% Limit of IQS No. 5/ 2019 CaO 60.37 - SiO2 20.74 - Al2O3 5.78 - Fe2O3 3.48 - MgO 1.91 < 5.0% SO3 1.85 ≤ 2.8% for C3A ≥ 3.5% Loss on Ignition (L.O.I) 2.44 < 4.0% Lime Saturation Factor (L.S.F) 0.92 0.66 - 1.02 Insoluble residue (I.R) 0.19 < 1.5 % Main compound (Bouge equation) % by weight of cement - Limit of IQS No. 5/ 2019 C3S 41.7 - C2S 27.4 - C3A 10.2 - C4AF 9.9 - Table 2: Chemical analysis of cement. Physical properties Test result Limit of IQS No. 5/ 2019 Fineness, Blaine, gm/cm2 319 >230 Setting Time: Initial, min 95 ≥45 min Figure 1: Specimen dimensions of the present study Sec. (A-A). Figure 1: Specimen dimensions of the present study Sec. (A-A). Table 3: Physical analysis of cement. 299 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 M. Q. OUTLINE OF THE EXPERIMENTAL SECTION Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 Diameter of Sieve (mm) Percent of Passing % 10 100 4.75 99 2.36 96 1.18 90 0.60 84 0.300 50 0.150 12 Table 4: Sand sieve analyses. Diameter of Sieve (mm) Percent of Passing % 75 100 63 100 37.5 100 20 97 14 38 10 30 5 0 2.36 0 Table 5: LECA sieve analyses. Table 5: LECA sieve analyses. The high-range water-reducing additive PC600 (superplasticizer) was used. It has a specific gravity of 1.22, and its properties, meet the requirements of both BS 5075 and ASTM C494. Mixing Water Concrete was mixed and hardened using Baghdad's tap water. This study kept this water clear and pure as much as possible. Reinforcing deformed bars used throughout the present study are 8mm and 10mm, and 12mm in diameter. The reinforcing steel testing results of such bars are listed in Tab. 6. Such tests, are done according to American Testing Standard Measurements (ASTM) A615. These tests were done at the University of Al-Nahrain concrete laboratory. CFRP SikaWrap-300 C unidirectional, woven, carbon-fiber fabrics have been used to reinforce the beams externally. Tab. 7 shows the CFRP laminates' mechanical properties. The bonding material for the appropriate adhesive material for CFRP sheets is Sikadur-330. There's a white paste (the Resin) and a grey paste (the Hardener) that make up the two halves. According to the manufacturer, the ideal mix ratio is (4:1). Tab. 8 shows some of the characteristics of the used adhesive material. Nominal diameter mm Yield stress MPa Yield strain Ultimate strength MPa Ultimate strain Elongation % 8 489.26 0.00244 629.26 0.00282 15.46 10 501.93 0.00253 635.09 0.00301 13.58 12 505.12 0.00268 646.96 0.00357 12.77 Table 6: Tension tests results for steel bars within this study. Properties SikaWrap-300 C Density (g/cm3) 1.8 Thickness (mm) 0.17 Tensile Strength (MPa) 3900 Modulus of Elasticity (GPa) 230 width (mm) 300 Table 7: The technical properties of CFRP. Properties SikaWrap-300 C Density (kg/L) 1.30 (mixed component A+B mixed) (+23°C) Modulus of Elasticity in flexure (GPa) 3.8 (7 days, +23°C) Modulus of Elasticity in tension (GPa) 4.8 (7 days, +23°C) Tensile Strength (MPa) 30 (7 days, +23°C) Elongation at Break (%) 0.88 (7 days at +23°C) Mixing ratio 4 : 1 Table 8: Properties of the used bonding materials. CONCRETE MIX DESIGN he mix-design of the LWC that existed in this study is presented in Tab. (9). This mix reflected 1700 kg/m3 of density. However, the required compression strength and slump were achieved after several trial mixtures. Material Cement (kg/m3) Sand (kg/m3) LECA (kg/m3) Water (kg/m3) Admixture (Liter/m3) Quantity 480 600 500 144 5 T he mix-design of the LWC that existed in this study is presented in Tab. (9). This mix reflected 1700 kg/m3 of density. However, the required compression strength and slump were achieved after several trial mixtures. T he mix-design of the LWC that existed in this study is presented in Tab. (9). This mix reflected 1700 kg/m3 of density. However, the required compression strength and slump were achieved after several trial mixtures. Material Cement (kg/m3) Sand (kg/m3) LECA (kg/m3) Water (kg/m3) Admixture (Liter/m3) Quantity 480 600 500 144 5 Table 9: Concrete mixture proportions. T T OUTLINE OF THE EXPERIMENTAL SECTION Nominal diameter mm Yield stress MPa Yield strain Ultimate strength MPa Ultimate strain Elongation % 8 489.26 0.00244 629.26 0.00282 15.46 10 501.93 0.00253 635.09 0.00301 13.58 12 505.12 0.00268 646.96 0.00357 12.77 Table 6: Tension tests results for steel bars within this study. Properties SikaWrap-300 C Density (g/cm3) 1.8 Thickness (mm) 0.17 Tensile Strength (MPa) 3900 Modulus of Elasticity (GPa) 230 width (mm) 300 Table 7: The technical properties of CFRP. Properties SikaWrap-300 C Density (kg/L) 1.30 (mixed component A+B mixed) (+23°C) Modulus of Elasticity in flexure (GPa) 3.8 (7 days, +23°C) Modulus of Elasticity in tension (GPa) 4.8 (7 days, +23°C) Tensile Strength (MPa) 30 (7 days, +23°C) Elongation at Break (%) 0.88 (7 days at +23°C) Mixing ratio 4 : 1 Table 8: Properties of the used bonding materials. Table 8: Properties of the used bonding materials. 300 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 THE REHABILITATION TECHNIQUE he rehabilitation technique was implemented and examined in the presented experimental program called Full U- wrapping. In this technique, CFRP sheets are installed to cover the section bottom and the full-height of its sides, as shown in Fig. 2. This technique consists of four specimens; the first is RF-50, an LWC beam that was repaired with full-height U-wrapping CFRP sheets and subjected to 50% of damage, as shown in Fig. 3. The second is RF-60, an LWC beam that was repaired with full-height U-wrapping CFRP sheets and subjected to 60% of damage, as shown in Fig. 4. The third is RF-70, an LWC beam that was repaired with full-height U-wrapping CFRP sheets and subjected to 70% of damage, as shown in Fig. 5. The last specimen is SF-100, a strengthened LWC beam by full-height U-wrapping CFRP sheets, as shown in Fig. 6. The structural behavior of the specimen is compared with a control specimen (Referential specimen without CFRP). T T without CFRP). Figure 2: Full-height U-wrapping. Figure 3: Full-height U-wrapping CFRP sheets and subjected to 50% of the damage. Figure 4: Full-height U-wrapping CFRP sheets and subjected to 60% of the damage. Figure 2: Full-height U-wrapping. Figure 2: Full-height U-wrapping. Figure 3: Full-height U-wrapping CFRP sheets and subjected to 50% of the damage. Figure 2: Full-height U-wrapping. Figure 2: Full-height U-wrapping. Figure 3: Full-height U-wrapping CFRP sheets and subjected to 50% of the damage. Figure 3: Full-height U-wrapping CFRP sheets and subjected to 50% of the damage. Figure 4: Full-height U-wrapping CFRP sheets and subjected to 60% of the damage. Figure 4: Full-height U-wrapping CFRP sheets and subjected to 60% of the damage. 301 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 Figure 5: Full-height U-wrapping CFRP sheets and subjected to 70% of the damage. Figure 6: Full-height U-wrapping CFRP sheets and subjected to 100% of the damage. Figure 5: Full-height U-wrapping CFRP sheets and subjected to 70% of the damage. Figure 6: Full-height U-wrapping CFRP sheets and subjected to 100% of the damage. Loading Capacity T g p y Ab. 10 and Fig. 10 shows the size effect degree of damage to the Ps and Pu of LWC beams rehabilitated by full- height U-wrapping CFRP sheets. Changing the degree of damage from 50% to 70% increased the Ps by 7.06% and 1.21% for 50% and 60%, respectively, while it decreased by 3.07 for 100%. In addition, such value increased the strengthened specimen by 11.99%. Service load Ps levels decreased as damage severity increased for LWC beams rehabilitated with full-height U-wrapped CFRP sheets. Using such a technique recovers this value for 50% and 60%, but when this degree is extended to 70%, Ps is lower than the control reading. This can be attributed to the consequent considerable mechanical performance of the CFRP – Epoxy composite that can compensate for the loss in structural rigidity-for Pu, increasing the degree of damage from 50% to 70% decrease this value. However, implementing full-height U-wrapping CFRP sheets can recover this value increasingly (as damage decreased) by 11.47%, 6.31%, and 0.57% for 50%, 60%, and 70% degrees of damage, respectively. Ps value for the strengthened specimen is more than the referential reading by 14.81% due to the consequent gain in structural rigidity. For, Pcr levels reported 40.08 kN and 78.56 kN, which corresponds to an excellency of 96% for the strengthened beam. Further research is needed to investigate the degree of the relation between the degree of damage and the relevant Pcr, Ps, and Pu. T gT Increasing in ultimate load Pu % Ultimate load Pu (kN) Increasing in service load Ps % Service load Ps (kN) Beam Specimen - 197.99 - 168.47 Control 11.47 220.70 7.06 180.34 RF - 50 6.31 210.48 1.21 170.51 RF - 60 0.57 199.12 -3.07 163.30 RF - 70 14.81 227.32 11.99 188.67 SF - 100 Table 10: The Ps and Pu for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. Table 10: The Ps and Pu for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. REHABILITATION PROCEDURE F irst, as shown in Fig. 7, the per-failed specimens were mechanically ground to a satisfactory smoothness using appropriate grinding commercial sheets. After that, an air compressor was used to clean the beams, preventing dust from settling on the specimens. To produce the adhesive material for the CFRP installation, KUT BOND Epoxy was mixed using a laboratory spatula. Figs. 8 and 9 demonstrate how these sheets were placed and established using an appropriate roller to prevent trapped air. F Figure 7: Mechanical Grinding. Figure 8: KUT BOND Epoxy. Figure 7: Mechanical Grinding. Figure 7: Mechanical Grinding. Figure 7: Mechanical Grinding. Figure 8: KUT BOND Epoxy. Figure 8: KUT BOND Epoxy. Figure 8: KUT BOND Epoxy. 302 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 Figure 9: Installing CFRP. Figure 9: Installing CFRP. DEFLECTION Ab. 11 and Fig. 11 demonstrate the extent of damage remedied by full-height U-wrapping CFRP sheets on LWC beams. There is a drop of 24.97 %, 23.95 %, and 21.66 %, respectively, when the damage is increased from 50 to 60 to 70 %, compared to the control. The same three groups had decreases of 6.98%, 8.75%, and 9.35%, respectively. Good mechanical characteristics of CFRP- Epoxy composite (stiffness) in the early response phases led to a reduction in all existing damage degrees in this investigation. Due to the different degrees of crushing the concrete pieces before restoration, the levels also reduced as the degree of damage proceeded. The reinforced beam has a lower weight (31.47% lower) and a higher strength (3.11%) than the control beam (no early crushing in concrete cracking before attaching CFRP sheets). Load-deflection plots for the Control, RF-50, RF-60, RF-70, and SF-100 configurations, are shown in Fig. 12. Phase I (Elastic) of the control specimen lasts until the first cracking limit is reached. By its conclusion, the fissures will be visible. T Ab. 11 and Fig. 11 demonstrate the extent of damage remedied by full-height U-wrapping CFRP sheets on LWC beams. There is a drop of 24.97 %, 23.95 %, and 21.66 %, respectively, when the damage is increased from 50 to 60 to 70 %, compared to the control. The same three groups had decreases of 6.98%, 8.75%, and 9.35%, respectively. Good mechanical characteristics of CFRP- Epoxy composite (stiffness) in the early response phases led to a reduction in all existing damage degrees in this investigation. Due to the different degrees of crushing the concrete pieces before restoration, the levels also reduced as the degree of damage proceeded. The reinforced beam has a lower weight (31.47% lower) and a higher strength (3.11%) than the control beam (no early crushing in concrete cracking before attaching CFRP sheets). Load-deflection plots for the Control, RF-50, RF-60, RF-70, and SF-100 configurations, are shown in Fig. 12. Phase I (Elastic) of the control specimen lasts until the first cracking limit is reached. By its conclusion, the fissures will be visible. T G dT 303 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 Phase II (Phase II) occurs after the initial cracking load when the cracks increase in number and breadth. This continues till the Ps. DEFLECTION Figure 12: Load deflection response for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. DEFLECTION Once this threshold was passed, the last step (step III) created wider-cracks. Phase I and II inflection points are also easily identifiable, as is Ps, and this holds over a wide range of damage intensities. Phase III is extended for the reinforced specimen in comparison to the control. M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 Phase II (Phase II) occurs after the initial cracking load when the cracks increase in number and breadth. This continues till the Ps. Once this threshold was passed, the last step (step III) created wider-cracks. Phase I and II inflection points are also easily identifiable, as is Ps, and this holds over a wide range of damage intensities. Phase III is extended for the reinforced specimen in comparison to the control. Phase II (Phase II) occurs after the initial cracking load when the cracks increase in number and breadth. This continues till the Ps. Once this threshold was passed, the last step (step III) created wider-cracks. Phase I and II inflection points are also easily identifiable, as is Ps, and this holds over a wide range of damage intensities. Phase III is extended for the reinforced specimen in comparison to the control. Figure 10: The Ps and Pu for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. Figure 10: The Ps and Pu for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. Changing in ultimate displacement m  % Ultimate displacement m  (mm) Decreasing in Service displacement s  % Service displacement sP (mm) Beam Specimen - 38.27 - 25.39 Control 3.11 39.46 -31.47 17.4 SF - 100 -6.98 35.6 -24.97 19.05 RF - 50 -8.75 34.92 -23.95 19.31 RF - 60 -9.35 34.69 -21.66 19.89 RF - 70 Table 11: The s  and m  for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. Figure 11: The s  and m  for LWC beams rehabilitated. Figure 11: The s  and m  for LWC beams rehabilitated. Figure 11: The s  and m  for LWC beams rehabilitated. 304 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 Figure 12: Load deflection response for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. FAILURE MODE ig. 13 shows the failure mode visual observation of the control specimen. The normal tension failure mode (flexure failure) was observed for the control specimen. In addition, crack propagation was always expected, and no cracks had reached the extreme compression fiber. Fig. 14 shows the failure mode of the beam specimen (SF-100). It is clear that the cracks propagated increasingly as the degree of damage progressed the RF-50, RF-60, and RF-70, as shown in Fig. 15. After rehabilitation, the mode of failure was transformed to crushing failure for all the degrees of damage proposed. This form of failure was also observed in the strengthened specimen. F F Figure 13: The mode of failure for the beam specimen (Control). Figure 14: The mode of failure beam specimen (SF – 100). Control SF – 100 Figure 13: The mode of failure for the beam specimen (Control). Control Figure 13: The mode of failure for the beam specimen (Control). Figure 14: The mode of failure beam specimen (SF – 100). SF – 100 Figure 14: The mode of failure beam specimen (SF – 100). 305 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 306 Figure 15: The mode of failure for the full-height U-wrapping CFRP sheets rehabilitation technique RF – 50 (Before) RF – 50 (After) RF – 60 (Before) RF – 60 (After) RF – 70 (Before) RF – 70 (After) Figure 15: The mode of failure for the full-height U-wrapping CFRP sheets rehabilitation technique 306 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 STIFFNESS RATIO (K) Figure 16: The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping CFRP sheets. Increasing in stiffness ratio k % Stiffness ratio (k) kN/mm Service load Ps (kN) Service displacement s  (mm) Beam specimen - 6.64 168.47 25.39 Control 42.67 9.47 180.34 19.05 RF - 50 33.07 8.83 170.51 19.31 RF - 60 23.73 8.21 163.30 19.89 RF - 70 63.42 10.84 188.67 17.4 SF - 100 Table 12: The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping CFRP sheets. ( ) y g pp g Figure 16: The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping CFRP sheets. Figure 16: The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping CFRP sheets STIFFNESS RATIO (K) hroughout the current study, the stiffness behavior of LWC rehabilitated beams is characterized by th ratio (k) as shown in Eq. 1: T hroughout the current study, the stiffness behavior of LWC rehabilitated beams is characterized by the stiffness ratio (k) as shown in Eq. 1: T hroughout the current study, the stiffness behavior of LWC rehabilitated beams is characterized by the stiffness ratio (k) as shown in Eq. 1: sP k (1) T T s s P k  (1) where k= Stiffness Ratio (kN/mm), sP = Service load (kN), and s = Service deflection (mm). Tab. 12 and Fig. 16 show the degree of damage effect to k of LWC beams rehabilitated by full-height U-wrapping CFRP sheets. Compared to the control specimen, the stiffness of the rehabilitated LWC beams by full-height U-wrapping CFRP sheets is more than the referential reading. For degrees of damage of 50%, 60%, and 70%, k levels were increased by 42.67%, 33.07%, and 23.73%, respectively. The rate of increase is more in the low levels of degree of damage. This can be attributed to the high levels of Ps and low and the consequent levels due to the reason discussed in the previous section. For the strengthened beam, the stiffness is also more than the control (63.42%) and the rehabilitated beams. Another effort should be devoted to exploring the degree of relation between the degree of damage and the related k. Increasing in stiffness ratio k % Stiffness ratio (k) kN/mm Service load Ps (kN) Service displacement s  (mm) Beam specimen - 6.64 168.47 25.39 Control 42.67 9.47 180.34 19.05 RF - 50 33.07 8.83 170.51 19.31 RF - 60 23.73 8.21 163.30 19.89 RF - 70 63.42 10.84 188.67 17.4 SF - 100 Table 12: The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping CFRP sheets. Figure 16: The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping CFRP sheets. DUCTILITY FACTOR (D) Increasing in stiffness ratio k % Stiffness ratio (k) kN/mm Service load Ps (kN) Service displacement s  (mm) Beam specimen - 6.64 168.47 25.39 Control 42.67 9.47 180.34 19.05 RF - 50 33.07 8.83 170.51 19.31 RF - 60 23.73 8.21 163.30 19.89 RF - 70 63.42 10.84 188.67 17.4 SF - 100 Table 12: The stiffness ratio (k) for LWC control and rehabilitated beams by full-height U-wrapping CFRP sheets. uring this study, the ductility behavior of LWC rehabilitated beams is represented by the stiffness ratio (d) as shown in Eq. 2: D DUCTILITY FACTOR (D) uring this study, the ductility behavior of LWC rehabilitated beams is represented by the stiffness ratio (d) as shown in Eq. 2: m s d   (2) D uring this study, the ductility behavior of LWC rehabilitated beams is represented by the stiffness ratio (d) as shown in Eq. 2: m d   (2) D uring this study, the ductility behavior of LWC rehabilitated beams is represented by the stiffness ratio (d) as shown in Eq. 2: D D (2) 307 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 where d= Ductility factor, m = Service load (kN) and s = Service deflection (mm). Tab. 13 and Fig. 17 show the degrees of damage effect to d of LWC beams rehabilitated by full-height U-wrapping CFRP sheets. As in the stiffness, the ductility of the rehabilitated LWC beams by full-height U-wrapping CFRP sheets is more than the control. For degrees of damage of 50%, 60% and 70%, d levels were increased by 35.60%, 34.92% and 34.69%, respectively. As in the stiffness behavior, the rate of d increase is more when the degree of damage was low due to the s and m  levels. The strengthened specimen has a high level compared to the control (50.46%). Again, this happened due to the inherent low level of s and high level of m . More research on this topic is needed to be undertaken about correlating d to the degree of damage for any CFRP rehabilitation technique. Increasing in Ductility Factor d % Ductility Factor (d) Maximum Deflection (mm) Service Deflection (mm) Beam specimen - 1.51 38.27 25.39 Control 23.98 1.87 35.60 19.05 RF - 50 19.98 1.81 34.92 19.31 RF - 60 15.70 1.75 34.69 19.89 RF - 70 50.46 2.27 39.46 17.4 SF - 100 Table 13: The ductility factor (d) for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. The ductility factor (d) for LWC beams rehabilitated by full-height U-wrapping CFRP sheets. Figure 17: The ductility factor (d) for LWC control beams rehabilitated by full-height U-wrapping CFRP sheets. Figure 17: The ductility factor (d) for LWC control beams rehabilitated by full-height U-wrapping CFRP sheets. Figure 17: The ductility factor (d) for LWC control beams rehabilitated by full-height U-wrapping CFRP sheets. CONCLUSION The rate of escalation is the greatest for moderate and minor damage levels. g g 7. Rehabilitated LWC beams with complete U-wrapped CFRP sheets have superior ductility than the originals, just as they do in stiffness. y 8. The levels of deflection increased by 35.60 %, 34.92 %, and 34.69% for damage levels of 50 %, 60 %, and 70 %, respectively. 8. The levels of deflection increased by 35.60 %, 34.92 %, and 34.69% for damage levels of 50 %, 60 %, and 70 %, respectively. p y 9. Restoring the original structural behavior features of LWC beams is possible with full U CFRP wrapping. 10 Th i l d d i i f h LWC b di i i h i h i i d i i 9. Restoring the original structural behavior features of LWC beams is possible with full U CFRP wrapping. service load and carrying capacity of the LWC beams diminish with increasing deterioration. 11. The ductility and stiffness of LWC beams are affected by the severity of the damage they have sustained. 11. The ductility and stiffness of LWC beams are affected by the severity of the damage they have sustained. 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CONCLUSION he following conclusions can be drawn: T he following conclusions can be drawn: 1. Using full-height U-CFRP wrapping can recover the inherent structural behavior characteristics of LWC beams. T T g 1. Using full-height U-CFRP wrapping can recover the inherent structural beha beams. T beams. 2. Increasing the degree of damage in the LWC beams decreases the performance characterization parameter such as service load and load carrying capacity for the proposed techniques T beams. 2. Increasing the degree of damage in the LWC beams decreases the performance characterization parameter such as service load and load carrying capacity for the proposed techniques. 3. Increasing the degree of damage decreases the relevant ductility and stiffness of LWC beams for all the proposed techniques when the proposed technique is used for strengthening, the flexural performance was increased. 308 M. Q. Hasan et alii, Frattura ed Integrità Strutturale, 66 (2023) 297-310; DOI: 10.3221/IGF-ESIS.66.18 4. The service load (Ps) increased by 7.06 % at a damage level of 50 % versus 60 % and 1.2 % at 70 %, while it decreased by 3.07 % at a damage level of 100 %. The value also contributed to an 11.99% boost in strength in the improved specimen. 4. The service load (Ps) increased by 7.06 % at a damage level of 50 % versus 60 % and 1.2 % at 70 %, while it decreased by 3.07 % at a damage level of 100 %. The value also contributed to an 11.99% boost in strength in the improved specimen. p p 5. Service deflection decreased across the board due to the stiffness of the CFRP-Epoxy composite in the early stages of response, with increases in stiffness ratio (k) of 42.67 %, 33.07 %, and 23.7 for damage levels of 50 %, 60 %, and 70 %, respectively. p p 5. 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https://openalex.org/W4362014600	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_A_Susceptibility_Locus_for_Papillary_Thyroid_Carcinoma_on_Chromosome_8q24/22377552/1/files/39822882.pdf	English	null	Supplementary Figure 2 from A Susceptibility Locus for Papillary Thyroid Carcinoma on Chromosome 8q24	null	2,023	cc-by	327	11 12 20 13 21 * * 26 14 18 24 15 25 19 1. Simplified pedigrees of 25 PTC families. Members affected with PTC are rcles or squares. Hatched circles or squares denote those with benign thyroid disease. Two individuals with anaplastic thyroid cancer in family 21 are indicated with an duals shown here were included in all the linkage analyses described in the text. 11 12 20 13 21 * * 26 14 18 24 15 25 19 1. Simplified pedigrees of 25 PTC families. Members affected with PTC are rcles or squares. Hatched circles or squares denote those with benign thyroid disease. Two individuals with anaplastic thyroid cancer in family 21 are indicated with an duals shown here were included in all the linkage analyses described in the text. 21 26 4 15 8 cted with PTC are with benign thyroid disease. are indicated with an escribed in the text. th benign thyroid disease. re indicated with an scribed in the text. 11 12 20 13 * * 1 18 24 25 19 mplified pedigrees of 25 PTC families. Members affe or squares. Hatched circles or squares denote those w o individuals with anaplastic thyroid cancer in family 21 a shown here were included in all the linkage analyses d 6 20 13 * * 7 amilies. Members affe quares denote those w d cancer in family 21 the linkage analyses d 11 5 12 20 13 * * 18 23 24 25 19 7 e 1. Simplified pedigrees of 25 PTC families. Members aff circles or squares. Hatched circles or squares denote those d. Two individuals with anaplastic thyroid cancer in family 21 viduals shown here were included in all the linkage analyses 5 12 24 25 19 pedigrees of 25 PTC fa es. Hatched circles or sq als with anaplastic thyroid ere were included in all t 5 12 19 11 18 1. Simplified rcles or squa Two individu duals shown h
W4285586792.txt	https://www.researchsquare.com/article/rs-1310829/latest.pdf	en		Effect of uniaxial strain on characteristic frequency of scaled SiGe HBT with embedded stress raiser	Journal of computational electronics	2,022	cc-by	5,759	Effect of Uniaxial Strain On Characteristic Frequency of Scaled SiGe HBT With Embedded Stress Raiser Senlin Kong Chongqing University of Posts and Telecommunications Guanyu Wang (  sigebicmos@foxmail.com ) Chongqing University of Posts and Telecommunications Chunyu Zhou Yanshan University Jianhao Wen Chongqing University of Posts and Telecommunications Peng Ling Chongqing University of Posts and Telecommunications Yingcong Xiang Chongqing University of Posts and Telecommunications Research Article Keywords: SiGe HBT, uniaxial stress, stress raiser, cut-off frequency Posted Date: February 14th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1310829/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Springer Nature 2021 LATEX template Effect of Uniaxial Strain on Characteristic Frequency of Scaled SiGe HBT with Embedded Stress Raiser Senlin Kong1 , Guanyu Wang1, Chunyu Zhou2† , Jianhao Wen1 , Peng Ling1 and Yingcong Xiang1 1 College of Electronic Engineering, Chongqing University of Posts and Telecommunications, 400065, Chongqing, China. 2 Key Laboratory for Microstructural Material Physics of Hebei Province, School of Science, Yanshan University, Qinhuangdao, 066004, Hebei, China. Corresponding author(s). E-mail(s): sigebicmos@foxmail.com; Contributing authors: zhoucy@ysu.edu.cn; † These authors contributed equally to this work. Abstract In order to further improve the performance of scaled silicon-germanium (SiGe) heterojunction bipolar transistor (HBT) and consider the compatibility with mature CMOS process, a novel SiGe HBT is designed by introducing the embedded Si1−y Gey stress raiser into the collector. In the proposed HBT structure, the collector region is subjected to additional uniaxial stress, to enhance the characteristic frequency. The effect of embedded Si1−y Gey stress raiser on the frequency performance with different Ge mole fractions is simulated and analyzed by employing SILVACOr TCAD tools. The simulation results show that the high frequency performance of the device can be significantly improved by applying additional uniaxial stress in the collector. At y = 0.3, the current gain of the device is increased by approximately 6% compared to the case where no stress is applied to the collector region (y = 0). Taking the uniform SiGe base case with the Ge fraction of 0.25 as an example, by adjusting the Ge fraction of the stress raiser, the peak values of f T and f max reach about 507.7 GHz and 730.7 GHz, respectively. Compared with the traditional SiGe HBT without any additional stress in the collector region, f T and f max are respectively increased by 29.1% and 71.5%. When y = 0.1, the proposed device has the best frequency characteristics due to the peak value of the f T ×f max product. Keywords: SiGe HBT, uniaxial stress, stress raiser, cut-off frequency 1 Introduction As integrated circuits (ICs) continue to move towards smaller process nodes, the cut-off frequency (f T ) and maximum oscillation frequency (f max ) of silicon-based devices gradually enter the terahertz (THz) band. Compared with traditional III-V semiconductor devices, silicon-based 1 high-frequency devices have the advantages of low cost, easy mass production, and compatibility with developed CMOS process, then gradually attract the attention of countries around the world [1]. As the core device in RF ICs, SiGe HBT takes advantage of energy band engineering and is fully compatible with mature silicon processes. The “DOTFIVE” and “DOTSEVEN” initiatives Springer Nature 2021 LATEX template were proposed by STMicroelectronics, Infineon, and other research institutes [2, 3]. Its goal is to develop SiGe HBT devices that can be used in communication and remote sensing with (f T ) up to 0.5 ∼ 0.7THz, and give full play to the great potential and technical advantages of SiGe HBT in reference circuits and system applications. The International Semiconductor Technology Roadmap (ITRS) predicts that the f T /f max of HBT will reach 570/610 GHz by 2020 [4]. Currently, the frequency performance of SiGe HBTs is mostly improved at the expense of high process costs, and the frequency performance of SiGe HBTs is improved by continuously optimizing the process flow and reducing the process node to reduce the lateral size of the device [5–7]. On the other hand, as the feature size of ICs becomes smaller and smaller, the isometric scaling technique faces more and more severe challenges. Silicon-based strain technology can effectively improve the mobility of carriers, thus improving the performance of devices, and has become an important mature technology and development direction of high-frequency/highperformance semiconductor devices and ICs [8]. Strain effect now has been introduced into SiGe HBT to improve device performance [9–12]. STMicroelectronics uses stacked metal interconnect wire structure near HBT based on BiCMOS-9W process [9]. The uniaxial stress is introduced into the device by the different thermal expansion coefficients of each metal layer. f T and f max increased by 21% and 12%, respectively. Ref. [10] proposed a new strain Si HBT device structure that uses a relaxed SiGe virtual substrate as the collector region and grows biaxial strain Si (SSi) on it as the emitter region. Compared with the traditional SiGe HBT, the current gain is increased by 11%. It should be noticed that the self-heating effect of virtual SiGe substrate is very significant, which cannot meet the requirement of ultra-high frequency [11]. In Ref. [12], external SiGe stress film is used to apply uniaxial stress to the base region of conventional bulk Si BJT, and TCAD simulation is carried out based on the fluid dynamics (HD) model. The simulation results showed that the f T and f max are only increased by about 5% and 3% respectively, and the improvement effect of a characteristic frequency is very limited. Ref. [13] investigated the effect of global stress on the high-frequency characteristics by full-band MonteCarlo simulation, but the way of applying the additional uniaxial stress is not discussed in detail. In view of the above, the device structure proposed in this work employs the “Embedded SiGe source/drain Technology” which commonly used in the uniaxial strain Si PMOS transistors. In this work. the Si1−y Gey stress raiser is implanted in the collector region of the device, and the uniaxial stress is introduced into the collector region, which is convenient for BiCMOS integration with the strained-Si CMOS process. The influence of Ge mole fraction y in the embedded Si1−y Gey stress raiser on f T and f max was further investigated and simulated analysis was conducted to reveal the physical causes for the influence of additional uniaxial stress in the collector region on characteristic frequency, to provide some potential possibilities for the design of silicon-based terahertz ICs in the future. 2 Device Structure The structure of the NPN SiGe HBT device with additional uniaxial stress in the collector region designed in this work is shown in Fig. 1. In this structure, the base region and emitter region have the same width. The parasitic effect between Si cap layer and SiGe base layer can be reduced and the frequency characteristics of the device can be improved. The emitter region width is reduced to 100 nm to effectively reduce the intrinsic resistance of the base region. Embedded Si1−y Gey stress raiser at both sides of the collector region introduces uniaxial strain, which improves the mobility of carriers, effectively reduces the square resistance of the collector region and the transmission time of carriers in the collector region, reduces the effective collector region width and BC junction capacitance, and improves the frequency characteristics of the device. The Si cap layer is subjected to uniaxial tensile stress and the resulting interfacial barrier in the emitter region effectively reduces the reverse current injected into the emitter region from the base region, thus improving the current gain of the device. In addition, for SiGe HBT fabricated by traditional bipolar process, the BE junction area is usually smaller than the BC junction area, and the current gain is limited by the larger BC junction area. Springer Nature 2021 LATEX template 3 For the structure proposed here, two P+ diffusion regions are implanted below the SiGe base region as the contact part of the external base region, therefore, the current gain can be further improved. For the SiGe base layer with composite strain, the lattice constant of the collector region is further reduced by uniaxial stress compared with that of the conventional biaxial strain. To be consistent with the longitudinal strain tensor εk,Si in Eq. (2), the base region of the composite strain level and strain tensor εk,SiGe is equivalent to: εk,SiGe = (1 + εk,Si ) Fig. 1 Device structure with embedded Si1−y Gey stress raiser Since the lattice constant of SiGe is greater than that of Si, the embedded Si1−y Gey stress raiser generates a longitudinal uniaxial compressive stress σ xx in the collector region. The Si lattice in the collector region is further compressed and the lattice constant decreases. The Si1−x Gex base region is superimposed with uniaxial compressive stress on top of the conventional biaxial compressive stress to form the composite strain base region. The Si cap in the emitter region is also affected by the uniaxial tensile stress σ xx . Considering the requirement of integration with uniaxial strain Si PMOS transistors, uniaxial compressive stress is usually applied along the [110] direction of (001) substrate, and the Si cap and the substrate collector region should have the same strain tensor εSi [13].  0 1 , 4 s44 σxx 1 2 (s11 + s12 )σxx (1) s 11 , s 12 and s 44 are elastic compliance coefficients, the longitudinal strain tensor is:  s12 σxx εSi =  0 0 0 1 (s + s12 )σxx 11 2 1 s σxx 44 4 εk,Si 1 = (s11 + s12 )σxx . 2 a(x) . 1 + 12 (s11 + s12 )σxx (4) For uniaxial compressive stress, σ xx < 0. For uniaxial tensile stress, σ xx > 0. It can be seen that a ∗ (x ) > a(x ) from the above, due to the uniaxial compressive stress introduced in the collector region. This corresponds to the fact that the additional uniaxial stress in the collector region has the effect of increasing the value of the Ge fraction x in the base region caused by the composite strain while maintaining the same level of strain as in the conventional biaxially strained Si1−x Gex base region, the bandgap of the base region is spontaneously reduced and the intrinsic carrier concentration is increased, resulting the bandgap of the Si cap-layer is also reduced and its intrinsic carrier concentration is increased. The characteristic frequency of SiGe HBT mainly refers to the cut-off frequency f T . This work only considers the frequency characteristics under the condition of a small signal and ignores the large injection effect. According to the smallsignal model, the expressions of f T and f max are [14]: fT = 2π[τF + 1 . + CBC ) + (RE + RC )CBC ] (5) r fT = . (6) 8πRB CBC kT qIC (CBE fmax (2) (3) Where a 0 and a(x ) are the lattice constants of relaxed Si and relaxed Si1−x Gex , respectively. If the longitudinal strain tensor of the biaxial strain SiGe base region is consistent with that in Eq. (3), the equivalent lattice constant a ∗ (x ) of the composite strain base region can be expressed as: a∗ (x) = 3 Estimation of Strain Mechanism a0 − 1, a(x) Springer Nature 2021 LATEX template k T/qI C is the BE junction dynamic resistance, I C is the BC junction electron current, C BC is the capacitance of the BC junction, C BE the BE junction capacitance, R E and R C represent the emitter resistance and collector resistance, respectively. τ BC is the forward transit time. Under the condition of small signal, the transit time can be approximately expressed as [15]: τF ≈ W2E W2B 1 WE + )+ . ( β SpE 2DpE 2DnB (7) In order to reflect the effect of stress on the characteristic frequency, this work only considers the Ge fractions of the base layer as a constant, and uniform doping in the base region. In Eq. (7), W E and W B are the longitudinal width of the emitter region, and the longitudinal width of the base region, respectively. β the current gain, D pE , and D nE are minority carrier diffusion coefficients in the emitter region and the base region respectively, S pE represents the holes recombination velocity at the contact interface of the emitter region. electrons injected from the emitter region to the base region, the transport process is not affected by the oxide-like layer, and it is “transparent” for electrons [16]. χh acts as a barrier to the holes injected from the base region, and the holes pass through the oxide-like layer in the form of quantum tunneling. V j is the bias voltage falling on the Si/SiGe BE junction and meets V BE = V j + ∆V n . If the holes in the emitter region comply with the Boltzmann distribution, the holes tunneling current can be written as: JP = JPT (8) q∆Vp − Efp1 4qπm∗h (kT)2 PT exp(− ). 3 h kT √ h is Planck constant, P T,Si ≈ exp(-d χh ) is the holes tunneling probability [17]. m ∗h is the effective mass of holes. The thickness of the silicon cap layer is very thin and is uniformly doped with the concentration is N D . χh increases ∆E V,SSi due to the upward shift of the valence band under the action of stress, and the unneling probability becomes: = PT,SSi ≈ exp(−d q χh + ∆E V,SSi ). (9) Neglecting the recombination in the strained Si cap layer, the hole tunneling current can be rewritten as: s qn2i,SSi PT,SSi qVj kT JP = exp( ) ∗ ND 2πmh kT (10) qn2i,Si qVBE exp( ), = GE,SSi kT Fig. 2 Energy-band of poly-Si/oxide-like layer/strained Si system As shown in Fig. 2, the thickness of the polysilicon emitter is W 1 , and an extremely thin “oxide-like” layer SiOr is formed at the interface with the strained Si layer, the barrier heights of electrons and holes are χe , and χh . The barrier height of electron χe is generally very small. For n i,Si and n i,SSi are the intrinsic carrier concentrations of relaxed and strain Si cap layers, respectively. Due to the uniaxial stress applied on the Si cap layer, the bottom of the conduction band E C decreases. Under the same V BE conditions, ∆V n increases ∆E C,SSi /q, the actual BE junction voltage drop decreases accordingly ∆V j = ∆E C,SSi /q. For the base region of uniform doping and uniform Ge fraction, the Gummel Number of the emitter region, ie. G E,SSi and the collector current density J C are as follows: GE,SSi = q∆Vn + ∆EC,SSi ND n2i,Si ), (11) exp( 2 SpE ni,SSi kT Springer Nature 2021 LATEX template 5 BE qexp( qVkT ) J C ≈ R WB NB [ 0 D∗ (n∗ )2 dx] (12) q(ni,SiGe ) qVBE exp( ), = GB,SSi kT p S pE = P T,SSi kT/2πm∗h is the recombination velocity at the interface of the emitter region. The additional uniaxial stress on the substrate leads to the increase of the intrinsic carrier concentration n ∗i,SiGe in the base region, and the minority carrier diffusion coefficient D ∗B ∝ µnB = (1 + 3x )µn0 [18] in the base layer also increases, µn0 is electron mobility of relaxed Si. It can be seen that the uniaxial stress increases the base Ge fraction x equivalently, thus increases, but ∆E C,SSi will also reduce J C , so the value of J C is mainly determined by Ge fraction y in Si1−y Gey stress raiser. The G B,SSi of the base region can be derived directly from Eq. (12): B i,SiGe 2 q∆Vn + ∆EC,SSi (ni,SiGe )2 WB NB exp( ). ∗ (n∗i,SiGe )2 DB kT (13) DC current gain is defined as β = G E,SSi /G B,SSi [19], from Eqs. (12) and (13), we have the following relationship: GB,SSi = β=( ∗ n2i,Si (n∗i,SiGe )2 DB 1 ND )( 2 ) ( ). 2 SpE ni,SSi (ni,SiGe ) WB NB (14) The uniaxial compressive stress in the collector region will narrow the forbidden bandgap of the collector region. Therefore, the built-in potential V bi in the depletion region of the BC junction decreases and C BC increases, but J C is also affected by the stress. If the fraction y of the stress raiser is very small, the effect of stress on C BC is also very small, then the effect of stress on C BC is negligible [13]. In addition, considering that the stress of the embedded Si1−y Gey stress raiser in this work increases with increasing values of y, dislocations can be introduced within the collector region. The dislocations in Si and Ge are mainly prismatic ones that can form suspension chains, act as donor or acceptor, and have a compensating effect on the mobile carriers in the collector region. Also dislocations can be considered as scattering centers and can affect the mobility and resistivity of carriers, which will also affect the frequency characteristics of the device under greater stress. 4 Simulation Results and Analysis According to the device structure shown in Fig. 1, the parameters of each active region are given in Table 1. The product of R C and C BC in Eq. (5) represents the charge and discharge time of the BC depletion capacitor, which has a significant effect on the frequency of the SiGe HBT. There is a large amount of mobile charge in this region. The width of that depletion region is related to J C . Under the condition of small injection or signal, if only the effect of V BE on J C is considered, C BC can be approximated as: d JC [(qNC − )WBC ] dVCB vS JC dWBC ) . ≈ (qNC − vS dVCB CBC = (15) v S is the electron saturation velocity in the collector region, W BC is the width of the depletion region of the BC junction, and it is also related to J C: p WBC = 2εSi (Vbi + VCB )/(qNC − JC /vS ). (16) Fig. 3 Simulated result of longitudinal stress distribution in the collector Fig. 3 shows the trend of uniaxial additional stress in the collector region along the electron transport direction for different values of the Ge fraction y in the stress raiser. It is obvious that Springer Nature 2021 LATEX template Table 1 Structure and technology parameters for simulations Region Material Thickness(nm) Width(nm) Doping(cm−3 ) Ge fraction(%) N+ Emitter N Emitter cap P+ Base Stress raiser N− Collector N+ Collector P+ Substrate Polysilicon Strain Si Strain Si1−x Gex Strain Si1−y Gey Strain Si/Relaxed Si Relaxed Si Relaxed Si 100 20 30 150 300 200 100 80 80 80 92 280 340 340 3×1019 2.5×1018 2.5×1018 0 1×1017 1×1020 1×1015 0 0 25 0-30 0 0 0 + the stress raiser can effectively introduce uniaxial stress into the collector region. With the increase of y, the additional uniaxial stress generated by the stress raisers increases significantly. Fig. 4 shows the calculated variation of the current gain for different Ge fractions of stress raiser. When y is between 0 and 5%, the current gain decreases with the increasing stress. After that, it increases with the increase of the y, but the increase is very small. The reasons for this can be analyzed according to Eq. (14): y between 0 and 5% corresponds to very small stress (< 0.1 GPa). With such a small uniaxial compressive stress, the equivalent Ge fraction increase in the Si1−x Gex base region is negligible. Along the [110] orientation of (100) substrate, the change of bandgap in the Si cap layer ∆E g,SSi , and the change of valence band edge ∆E V,SSi is also very small. And the barrier height of the oxide-like layer to hole χh is usually about 1 eV [16]. So the tunneling probability P T in Eq. (14) p has little effect, which is approximately β ∝ m∗h . The calculation results of the variation of the average holes effective mass of valence band show that, when the stress is in the range of 0 ∼ 0.2 GPa, decreases greatly, resulting in β being slightly reduced. When the stress is greater than 0.2 GPa, the magnitude of bandgap shift increases with increasing stress. As a result, the accumulation of holes at the interface layer decreases significantly under higher stresses and the height of the holes barrier χh becomes a major factor in the continued increase in β. In general, the first and second terms to the right of the equal sign in Eq. (14) decrease as the stress increases, while the remaining terms increase as the stress increases. When the Ge fraction y is small, term 1 leads to a reduction in β. When y is increased, the magnitude of uniaxial stress is also increased, then terms 3 and 4 mentioned above become the main factors that make β increase. Fig. 4 Variation of DC current gain with Ge fraction y in the stress raiser It should be noted that various factors are affecting the characteristic frequency, and the theoretical analysis in the previous section is intended to provide a relevant physical basis for analyzing the effect of uniaxial stresses applied in the collector region on the frequency performance of the device. Combining the simulation results allows for an in-depth analysis of the physical mechanism affecting the frequency characteristics, rather than establishing a detailed physical model. The distribution of the applied uniaxial stress σ xx along the x -direction of the collector is not uniform. By fitting the stress σ xx distribution curve of Fig. 3, the average stress magnitude over the length of the transverse collector in the range 0 ∼ L can be calculated as: Springer Nature 2021 LATEX template 7 < σxx 1 >= L Z L σxx (x)dx. (17) 0 By substituting the calculation results of Eq. (17) into Eq. (4), the Ge fraction of the composite strained base region under additional uniaxial stress conditions can be obtained according to the relationship between the Si1−x Gex lattice constant and the equivalent Ge fraction x ∗ . The intrinsic carrier concentration of the base region and the diffusion coefficient can be then calculated, and the intrinsic carrier concentration of the strained Si cap layer can be obtained, as detailed in Ref. [20]. A comparison of the theoretical results calculated according to Eq. (14) with the simulation results in Fig. 4 reveals that the DC current gain is consistent with the variation pattern of the Ge fraction y, which can illustrate the correctness of the previous theoretical analysis. The reasons for the difference between the two are: (1) The thickness of the strained silicon capping layer is extremely thin, therefore, for the convenience of the analysis, it is approximated that the recombination of minority carriers in this layer and the effect of the thickness of the layer is ignored. (2) The interfacial state and the recombination velocity S pE in the interface between strained Si cap layer and oxide-like layer depends on different process conditions, and more detailed physical modeling is dependent on the specific fabrication process. (3) Building a more accurate physical model of current gain also requires solving complex differential equations to calculate the distribution functions of the minority carriers and the magnitudes of various microscopic currents in the emitter region [16], which are beyond the scope of this paper. Fig. 5 shows the variation curve of collector current J C with BE junction voltage V BE and stress. It can be seen that the uniaxial additional stress applied to the collector region does increase J C . From Eq. (12), this is mainly due to the increase of the minority carriers’ diffusion coefficient and the equivalent x in the base region, but there is no change in the order of magnitude. This is due to the fact that the effective bias voltage V j falling on the Si/SiGe emitter junction decreases as the variation ∆E C,SSi in the conduction band of the strained Si cap layer increases. In general, J C still increases with increasing y. As y Fig. 5 Variation of collector current with Ge fraction in the stress raiser increases, the recombination between stress raiser and collector region is enhanced by the increased prismatic dislocations, so the variation of J C with y is not obvious. In addition, the theoretical calculation of the collector current according to Eqs. (12) and (13) are also given in Fig. 5, which exhibit a good agreement with the simulation results. This is because the electron current J C is dependent on the energy band variation rather than the height of the oxide-like layer barrier and interface state [16]. According to the definitions of f T and f max , the value of characteristic frequency at a certain collector current I C can be obtained for the variation curves of the common-emitter current gain H 21 and the unidirectional transmission power gain U under small-signal conditions when both parameters are reduced to 0 dB. For example, the curves of H 21 and U with frequency f were analyzed by AC simulation at different V BE conditions for y = 0.1, as shown in Fig. 6 and Fig. 7. By extracting the data from the curves, the frequency characteristic for different Ge fractions y can be obtained, as shown in Fig. 8 and Fig. 9. The cut-off frequency f T and maximum oscillation frequency f max are significantly increased compared to the case without stress raiser (y = 0), by about 29.1% and 71.5% respectively. The peak values of f T and f max reached 507.7 GHz and 730.7 GHz for y = 0.15 and y = 0.1, respectively, entering the “half-THz” band. According to the analysis presented above, the favorable factors Springer Nature 2021 LATEX template Fig. 6 Variation of small-signal current gain H 21 with frequency f Fig. 7 Variation of unilateral power gain U with frequency f leading to an increase in the characteristic frequency as the additional uniaxial stress increases are the increase of current gain, collector current, and interfacial recombination velocity. The additional uniaxial stress equivalently increases the Ge fraction of the base region, as illustrated in section 3, leading to an increased minority carrier diffusion coefficients and a simultaneous decrease in the resistance of in the working base region. The additional stress increases the carrier mobility and reduces the series resistance of the emitter and collector regions. It can also be seen from Fig. 8 that f T increases as y increases from 0 to 0.15 but decreases when y exceeds 0.15. Similarly, f max increases as y Fig. 8 Variation of the cut-off frequency f T with Ge fraction y increases from 0 to 0.1 but decreases when y beyonds 0.1. Referring to Eqs. (5), (14) and (15), it can be concluded that the reason for this situation is mainly the effect of prismatic dislocations in the collector region on the product R C ×C BC . For one thing, the dislocation compensates for the reduction of the free charge (J C /qv S ) in the collector region, leading to an increase in barrier capacitance C BC . Secondly, the lattice distortion caused by the dislocation becomes the scattering center of the carrier, and in the low-doped collector region, its ionization as the dominant center has an anisotropic scattering effect on the carrier, reducing the electron mobility, thus increasing the product R C ×C BC in Eq. (5), resulting in a drop in f T . Fig. 9 depicts the variation trend of f max with collector current and Ge fraction y. From Eq. (6), the trend is similar to that of f T . At higherlevel stresses, C BC increases, so both f T and f max increase and then decrease as y increases. It should be noted that, however, the increase and decrease in f max are greater than the change in f T . In addition to the influence of C BC , the base resistance R B is also another dominant role. Qualitatively, this is most likely due to the thin base region, where the dislocations at the Si/SiGe interface act as a deep energy level, enhancing the recombination of the base region and reducing the concentration of holes in the base region. At the same time the dislocation scattering also hurts the mobility of the base region majority carriers. The concentration compensation and scattering effect Springer Nature 2021 LATEX template 9 5 Conclusion Fig. 9 Variation of the maximum oscillation frequency f max with Ge fraction y of dislocation on the base region majority carriers leads to the increase of R B more than the increase of f T , thus worsening the f max . Fig. 10 Variation of f T ×f max with Ge fraction y For more visual analysis of the frequency characteristics, Fig. 10 shows the variation of f T and f max peaks for different Ge fractions y. It is clear from this that the Ge fractions corresponding to the two peaks are different, and it is not straightforward to obtain the optimal value of the Ge fraction for the design of device to achieve the best frequency performance. For this purpose, the Ge fraction can be determined by determining the peak of the product f T ×f max . It is clear from the graph that f T ×f max peaks at y = 0.1 and that the overall performance is optimal for the frequency characteristics. In this work, a device structure for a scaled SiGe HBT with an embedded Si1−y Gey stress raiser in the collector region is designed. Under additional stress, the theoretical analysis shows that the improvement of the frequency characteristics is mainly due to the change of the strained Si/SiGe band structure, as well as the improvement of the physical parameters such as the DC current gain, the interface recombination rate in the emitter region and the R C ×C BC product, while the effect of the dislocation scattering caused by the stress on the relevant physical parameters is also taken into account. The simulation results show that the introduction of additional uniaxial compressive stresses in the collector region can indeed significantly improve the frequency performance of the device, and the Ge fraction y can be flexibly adjusted to achieve different degrees of enhancement of the characteristic frequency. In particular, the cut-off frequency f T reaches a maximum at y = 0.15, which is close to the Ge fraction of the SiGe source-drain in a strained Si PMOS in a 90 nm process. Moreover, the device structure designed in this work is similar to that of PMOS and is therefore easily integrated with strained Si CMOS processes, resulting in a BiCMOS device structure with a smaller size or layout region and better performance. The research in this work can provide a theoretical basis for the design of future silicon-based Terahertz Devices and ICs, and has some potential application value. In addition, this work only investigates the physical parameters of the device that affect the characteristic frequency, and it is believed that on this basis, by further optimizing the process structure and parameters of the device, even better frequency characteristics can be obtained. Acknowledgments. 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An equivalent heterojunction-like model for polysilicon emitter bipolar transistor. Solid-State Electron, 2003, 47(10):1719-1727. [17] Zekry A, Shaker A, Ossaimee M, et al. A comprehensive semi-analytical model of the polysilicon emitter contact in bipolar transistors. Journal of Computational Electronics, 2017, 17: 246-255. [18] Chang S T, Liu C W, Lu S C. Base transit time of graded-base Si/SiGe HBTs considering recombination lifetime and velocity saturation. Solid-State Electron, 2004, 48(2):207215. [19] Voinigescu S P. High-frequency integrated circuits. Cambridge: Cambridge University Press, 2013. [20] S. Dhar, E. Ungersbock, H. Kosina, T. Grasser and S. Selberherr, ”Electron Springer Nature 2021 LATEX template 11 Mobility Model for h110i Stressed Silicon Including Strain-Dependent Mass,” in IEEE Transactions on Nanotechnology, vol. 6, no. 1, pp. 97-100, Jan. 2007, doi: 10.1109/TNANO.2006.888533.
https://openalex.org/W4379091906	https://zenodo.org/records/7995729/files/5.23.pdf	Bulgarian	null	ИНДИВИДУАЛ ЁНДАШУВ АСОСИДА ТАЪЛИМ СИФАТИНИ ОШИРИШ ИМКОНИЯТЛАРИ	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,090	ИНДИВИДУАЛ ЁНДАШУВ АСОСИДА ТАЪЛИМ СИФАТИНИ ОШИРИШ ИМКОНИЯТЛАРИ Омонова Мухлиса Дўстназар қизи Аннотация. Мазкур мақола индивидуал таълим технологияси, индивидуал ёндашув ва шу орқали таълим-тарбия жараёнининг ташкил этилиши, таълим сифатини оширишда индивидуал таълим-технологияларининг имкониятлари ҳақида маълумотлар моҳияти ёритиб берилган. Калит сўзлар: таълим, инновацион таълим, индивидуал таълим, технология, индивидуал ёндашув, таълим сифати. Дунёда жамиятнинг ҳар томонлама ривожланиши таълим мазмуни ва сифати билан боғлиқ эканлиги ўз исботини топиб бормоқдаТаълимнинг бугунги кундаги вазифаси соҳага қўйилган давлат сиёсатини амалга ошириш, кадрлар тайёрлаш бўйича давлат талабларни сифатли бажариш, ўқув адабиётларининг замонавий авлодини яратиш, ўқитувчи- мураббийларнинг методикасини узлуксиз такомиллаштириш, ўқувчиларга шахсга йўналтирилган таълим технологиялари асосида дарс бериш каби педагогиканинг методологик аҳамиятга эга муаммолари билан бевосита боғлиқ. Ушбу муаммоларнинг ҳал қилиниши таълим мазмунига қўйилаётган давлат талабининг сифатли бажарилишини таъминлайди, таълим беришнинг асосий концептуал масалаларининг ижобий ечимига сабаб бўлади. Бу эса фан методологияси ва методологик тадқиқи билан боғлиқ жараёнлардир. Бугунги кун педагогикасида таълимни шахсга йўналтирилган, компетенциявий, акмеологик, эвристик технологиялар асосида ташкил этиш амалий фаолиятнинг самарали шаклларидан бири сифатида қаралмоқда. Мазкур замонавий ёндашувлар педагогикада методологик аҳамиятга эга мазмун ва шакл ҳодисалари бўлиб, уларда ўқувчи шахсини ҳар томонлама такомиллаштириш, уларга индивидуал ёндашиш, назарий билимларнинг амалий аҳамиятини ошириш, ўқувчиларда муайян компетентликни шакллантириш методлари ва уларнинг иқтидорларини юзага чиқариш билан боғлиқ муҳим масалаларга ўзига хос инновацион ёндашиш назарда тутилади. Таълимнинг узлуксиз равишда ривожланиб бориши дарс машғулотлари жараёнида қўлланилаётган янги педагогик технологияларни самарали татбиқ этиш шаклида кечмоқда. Янги педагогик технологияларни жорий этишдан мақсад таълим жараёни самарадорлигини оширишдан иборат. Шахсга йўналтирилган таълим технологияси ўқувчи шахсини таълим жараёнининг марказига қўйиш, унинг қобилиятини ривожлантириш учун зарур бўлган барча қулайликлар, шарт-шароитлар яратишдан иборат. Мазкур технологияда таълим олувчи таълимнинг кенг имкониятларга эга субъекти сифатида намоён бўлади. Ўқитувчининг ўқувчига муносабати шахсга йўналтирилган таълим технологиясида асосий омиллардан бири сифатида қаралади. Ушбу технологияни лойиҳалаштиришда таълим олувчи шахсининг руҳий, жисмоний ва ёш хусусиятлари, унинг фанга тайёрлик даражасининг ҳисобга олиниши муҳим аҳамият касб этади. Таълим сифати ва самарадорлигини ошириш педагогика фанининг бош масалаларидан бири сифатида қараб келинган. Фандаги ёндашувлар, технологиялар, 823 RAQAMLI TEXNOLOGIYALAR DAVRIDA TILLARNI INTENSIV OʻQITISHNING PSIXOLOGIK- PEDAGOGIK JIHATLARI RESPUBLIKA ILMIY AMALIY ANJUMANI RESPUBLIKA ILMIY-AMALIY ANJUMANI 2023-yil 2-iyun RAQAMLI TEXNOLOGIYALAR DAVRIDA TILLARNI INTENSIV OʻQITISHNING PSIXOLOGIK- PEDAGOGIK JIHATLARI RESPUBLIKA ILMIY-AMALIY ANJUMANI 2023-yil 2-iyun талабаларнинг руҳий ҳолати, эмоциялари, ўз шахсий қадриятларини ҳурмат қилиш; талабаларнинг таълим олиш стратегиясига хос бўлган ўқув кўникмаларини мақсадли шакллантириш; таълим жараёнида профессор-ўқитувчи ва талаба вазифаларини қайта тақсимлаш: профессор-ўқитувчининг етакчилик ролини чеклаш, унга ёрдамчи, маслаҳатчи сифатида қараш. Демак, индивидуал таълимда талабаларнинг мавжуд имкониятлари ва салоҳиятидан келиб чиқиб ўқитиш жараёни ташкил қилинади, уларга максимал даражада ишончи билдирилади. Индивидуал таълим технологиясига асосланган дарс машғулотларида фойдаланиладиган дидактик материалнинг мақсади ўқув дастурини ишлаб чиқиш, талабаларга керакли билим, кўникма ва малакаларни ўргатишдан иборат. Индивидуал таълим технологиясида дидактик материалнинг турлари ўқув матнлари, топшириқ карточкалари, дидактик тест кабилардан иборат. Вазифалар мавзу бўйича, мураккаблик даражаси бўйича, фойдаланиш мақсади бўйича кўп даражали, табақалаштирилган, индивидуал ёндашув, талаба ўқув фаолиятининг етакчи турини (когнитив, коммуникатив, ижодий) ҳисобга олган ҳолда ишлаб чиқилади. Бу ёндашувнинг марказида билим, кўникма, малакаларни ўзлаштиришда эришилганлик даражасини баҳолаш қобилияти туради. Профессор-ўқитувчи талабалар ўртасида карточкаларни тарқатади, уларнинг билиш хусусиятлари ва имкониятларини билиб олади, нафақат билим олиш даражасини аниқлайди, балки ҳар бир талабанинг шахсий хусусиятларини ҳисобга олади, фаолиятнинг шакл ва усулларини танлаш орқали унинг ривожланиши учун энг муносиб шароитни яратади. Ўқитиш методлари таълим технологияси ва ёндашувига мос ҳолда танланиши мақсадга мувофиқ. Акс ҳолда танланган метод кутилган самарани бермайди. Шу нуқтаи назардан, тадқиқот ишимизда индивидуал таълим технологиясига мос методлар мажмуи аниқлаштирилди. Бу ўсувчи-ўзгарувчи кўрсаткич бўлиб, ҳар бир профессор-ўқитувчининг индивидуал педагогик манерасидан келиб чиққан ҳолда танланади. Ўқитиш методлари таълим технологияси ва ёндашувига мос ҳолда танланиши мақсадга мувофиқ. Акс ҳолда танланган метод кутилган самарани бермайди. Шу нуқтаи назардан, тадқиқот ишимизда индивидуал таълим технологиясига мос методлар мажмуи аниқлаштирилди. Бу ўсувчи-ўзгарувчи кўрсаткич бўлиб, ҳар бир профессор-ўқитувчининг индивидуал педагогик манерасидан келиб чиққан ҳолда танланади. RESPUBLIKA ILMIY-AMALIY ANJUMANI 2023-yil 2-iyun методикалар, шакллар ва воситаларнинг муваффақияти, пировардида, таълим сифатини оширишга қўшган ҳиссаси билан баҳоланади. Шундай экан, таълим сифати бу педагогиканинг фундаментал ва субстанционал тадқиқот масаласи бўлиб, таълимнинг бошқа компонентлари унга нисбатан восита сифатида қаралади. Бугунги кун таълимида табақалаштириш, ихтисослаштириш билан боғлиқ ёндашувли жараёнлар, хусусийлаштириш билан боғлиқ мулкий муносабатлар шаклланмоқда. Хусусан, бу жараён умумий ўрта таълимда умумтаълим, ихтисослаштирилган, ижод ва Президент мактаблари шаклида амалга оширилмоқда. Таълимда нисбатан янги технологик ёндашувлар сифатида шахсга йўналтирилган, компетенциявий, эвристик, акмеологик ва индивидуал ёндашувларни келтириш мумкин. Тадқиқотчи М.Қурбонова индивидуал таълим технологиясини таълим олувчи шахсининг қадр-қимматини кўтаришга хизмат қилишини таъминлаган ҳолда, унинг ўзига бўлган ишончини, билишга бўлган қизиқиши оширишга, олган билимларини кундалик ҳаётда қўллай олиш кўникмасини шакллантиришга хизмат қилишини таъкидлайди. Дарҳақиқат, индивидуал таълим технологияси таълим олувчи шахсига қулай имкониятлар яратиш, таълимнинг бош мақсади – таълим олувчи шахсини ҳар томонлама уйғун камол топишини таъминлашга қаратилган ёндашув сифатида қаралмоқда. Индивидуал таълимда талаба шахси таълим жараёнининг марказига қўйилади ва таълим жараёнининг барча компонентлари унинг таълим ва тарбия олишига йўналтирилади. Аслида ҳам, таълим жараёнининг устувор мақсади таълим бериш экан, барча имконият ва воситаларнинг талаба шахсинининг шахсий ва касбий имкониятларини оширишга, билим, малака ва кўникмаларини компетенция даражасига олиб чиқишга йўналтириш таълимнинг сифат ва самарадорлигини оширишга хизмат қилади. Анъанавий таълимда таълимнинг бошқа компонентларига, хусусан, ўқитиш методикасини кучайтириш, воситаларни такомиллаштириш, шаклларни ўзгартириш, технологияларни такомиллаштириш, ўқувчи ва ёрдамчи адабиётларни замонавий талабларга мослаштириш каби жиҳатларга кенг эътибор қаратилди. Аммо таълим олувчи шахсининг талаблари, эҳтиёжлари, имкониятлари етарли даражада эътиборга олинмаганлиги боис юқоридаги таълим компонентлари ўз самарасини етарли даражада намоён қила олмади. Индивидуал таълим технологияси таълим жараёнини ташкил қилиш ва бошқаришдаги мазкур камчиликни бартараф этишга қаратилгани билан аҳамиятлидир. Яъни воситаларнинг такомиллаштирилиши ўша воситалар хизмат қилиши лозим бўлган таълим олуви шахсининг талаб ва эҳтиёжлари, майллари етарли даражада эътиборга олинмагани сабабли ўз вазифасини етарли даражада бажара олмади. Шу нуқтаи назардан индивидуал таълим технологияси Чирчиқ давлат педагогика университетининг асосий стратегик илмий тадқиқот йўналишларидан бири сифатида ўрганилмоқда. Унга кўра, педагогик ва психологик таълимда индивидуал ёндашувнинг хусусиятлари қуйидагилардан иборат: талабаларнинг таълим жараёнидаги мустақиллигини таъминлаш, улар учун мақбул бўлган ўқитиш методларидан фойдаланиш; талабаларнинг мавжуд билим, малака, кўникма ва компетенцияларига, тажрибасига ишонч билдириш; талабаларнинг мавжуд билим, малака, кўникма ва компетенцияларига, тажрибасига ишонч билдириш; талабаларнинг ижтимоий ҳолати, турмуш тарзи ва имкониятларини ҳисобга олган ҳолда ўзини намоён қилишига шароит яратиш; 1. Muxаmеdоv G‘.I., Xо‘jаmqulоv U.N., Klаstеr tа’limgа nisbаtаn innоvаtsiоn yоndаshuv sifаtidа // Tа’lim, fаn vа innоvаtsiyа, 2020/2-sоn. 2. Usаrоv J.Е. Muxаmеdоv G.I. Uzluksiz pеdаgоgik tа’lim klаstеri shаrоitidа kоmpеtеntlik tаlаblаri vа tа’lim sаmаrаdоrligi imkоniyаtlаri // Uzluksiz tа’lim, Mаxsus sоn, 2020. 3. Қурбонова М. Индивидуал таълим – инновацион ўқитиш усули // Academic Research in Educational Sciences. Volume 4/ Issue 3/2023/ Фойдаланилган адабиётлар 1. Muxаmеdоv G‘.I., Xо‘jаmqulоv U.N., Klаstеr tа’limgа nisbаtаn innоvаtsiоn yоndаshuv sifаtidа // Tа’lim, fаn vа innоvаtsiyа, 2020/2-sоn. 2. Usаrоv J.Е. Muxаmеdоv G.I. Uzluksiz pеdаgоgik tа’lim klаstеri shаrоitidа kоmpеtеntlik tаlаblаri vа tа’lim sаmаrаdоrligi imkоniyаtlаri // Uzluksiz tа’lim, Mаxsus sоn, 2020. 3. Қурбонова М. Индивидуал таълим – инновацион ўқитиш усули // Academic Research in Educational Sciences. Volume 4/ Issue 3/2023/ 825
https://openalex.org/W4313188899	http://jurnal.pasca.untad.ac.id/index.php/jstt/article/download/369/238	Indonesian	null	PENGARUH PERUBAHAN TITIK LEMBEK ASPAL AKIBAT PENAMBAHAN VIATOP66 TERHADAP KARAKTERISTIK CAMPURAN HRS-WC	Jurnal Sains dan Teknologi Tadulako	2,020	cc-by-sa	5,791	__________________________________________________________________________ PENGARUH PERUBAHAN TITIK LEMBEK ASPAL AKIBAT PENAMBAHAN VIATOP66 TERHADAP KARAKTERISTIK CAMPURAN HRS-WC Reza Maulana Anshory¹, Mashuri², dan Jurair Patunrangi³ 1,2,3Jurusan Teknik Sipil, Fakultas Teknik, Universitas Tadulako Jl.Soekarno-Hatta Km. 9 Palu, Sulawesi Tengah. Email: Rezamaulanaanshory@gmail.com Reza Maulana Anshory¹, Mashuri², dan Jurair Patunrangi³ 1,2,3Jurusan Teknik Sipil, Fakultas Teknik, Universitas Tadulako Jl.Soekarno-Hatta Km. 9 Palu, Sulawesi Tengah. Email: Rezamaulanaanshory@gmail.com 630) Jurnal Sains dan Teknologi Tadulako 630) Jurnal Sains dan Teknologi Tadulako (p-issn: 2089-8630) Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 Abstrak Peningkatan nilai Titik lembek aspal juga mempengaruhi karakteristik dari campuran HRS-WC. Tujuan penelitian ini untuk mengetahui pengaruh perubahan Titik lembek aspal akibat penambahan Viatop66 terhadap karakteristik perkerasan aspal beton lapisan Aus (HRS-WC). Penambahan variasi kadar viatop66 dalam aspal adalah 0,0% ; 1,0%; 2,0%; 3,0%; 4,0%; dan 5,0% terhadap berat aspal. Penentuan Kadar Aspal Optimum (KAO) campuran HRS-WC menggunakan Metode Marshall. Hasil penelitian mendapatkan bahwa peningkatan nilai Titik lembek aspal akibat penambahan viatop66 ke dalam aspal dapat meningkatkan nilai kepadatan dan VFB campuran HRS-WC dengan persentase peningkatan tertinggi 0,67% untuk kepadatan dan 4,06% untuk VFB campuran HRS-WC pada nilai Titik Lembek Aspal 52,48 °C, menurunkan nilai VIM dan VMA campuran HRS-WC dengan persentase penurunan 11,92% untuk VIM dan 3,24% untuk VMA campuran HRS-WC pada nilai Titik Lembek Aspal 52,48 °C, menurunkan nilai flow serta meningkatkan Stabilitas dan MQ marshall campuran HRS-WC. Peningkatan nilai Titik lembek aspal akibat penambahan viatop66 ke dalam aspal juga meningkatkan nilai stabilitas marshall sisa di atas 90 % yang mengartikan perubahan nilai Titik Lembek Aspal akibat penambahan viatop66 dapat membuat aspal tahan terhadap perubahan lingkungan sekitar. Kata kunci: Hot Rolled Sheet-Wearing Course (HRS-WC), Titik Lembek, Viatop66 Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 1. Pendahuluan Lapisan perkerasan pada jalan raya pada umumnya menggunakan perkerasan lentur. Campuran yang terdiri dari aspal dan agregat ini terkadang di campuran dalam keadaan panas (hot mix) ataupun dicampur dalam keadaan dingin (cool mix). Sifat aspal yang peka terhadap temperatur memudahkan dalam pengolahan aspal dalam keadaan panas. Namun di sisi lain kepekaan aspal terhadap temperatur menimbulkan masalah lain. Pada saat aspal telah terhampar pada lokasi perencanaan, perubahan peningkatan suhu permukaan aspal akibat cuaca sangat mempengaruhi keadaan aspal. Pada suhu tertentu aspal akan mengalami perubahan bentuk fisik yang semulanya keras, akan mengalami perubahan menjadi lembek. Perubahan aspal menjadi lembek inilah sering disebut dengan titik lembek aspal, dimana aspal mulai menjadi lembek pada titik suhu tertentu. Hal ini memberi tuntutan dimana aspal yang akan dihamparkan harus memiliki titik lembek yang lebih tinggi dari suhu permukaan rata-rata lokasi penghamparan tersebut. Adanya perubahan iklim diberbagai belahan bumi yang mempengaruhi sering terjadinya cuaca ekstrim dan peningkatan suhu bumi secara menyeluruh maka dibutuhkan lapisan Aus yang lebih tahan terhadap kerusakan akibat perubahan suhu yang terjadi. Dimana kerusakan yang terjadi sering menimbulkan retak, aus, bleeding, hingga lubang. Penggunaan Aspal dengan nilai titik lembek pada satu tempat dan tempat lain sering kali berbeda, tergantung pada kebutuhan spesifikasi aspal berdasarkan iklim dan temperatur lokasi penghamparan aspal. Tuntutan nilai titik lembek aspal harus lebih besar dari suhu tertinggi di daerah penghamparan aspal atau suhu permukaan aspal membuat besar nilai titik lembek aspal merupakan bagian penting yang perlu diperhatikan dalam spesifikasi aspal. Bahan aditif sering digunakan sebagai campuran pada aspal guna meningkatkan nilai titik lembek Aspal. Perubahan nilai titik lembek pada aspal dapat berarti lebih baik untuk digunakan pada penghamparan, namun dapat menjadi catatan penting dalam pengolahan atau proses pencampuran aspal. Pencampuran bahan aditif tidak berarti selalu meningkatkan nilai titik lembek aspal, perlu adanya penelitian lebih lanjut terhadap pencampuran bahan aditif dalam aspal. Viatop66 merupakan salah satu bahan aditif berupa serat Seluosa yang dapat dicampurkan dengan aspal dan diharapkan mampu meningkatkan titik lembek aspal. Uraian di atas memberi motivasi khusus pada penulis untuk dapat melakukan penelitian tentang titik lembek aspal yang ditambahkan dengan bahan aditif, dengan metode ring and ball menggunakan sampel aspal penetrasi 60/70 dengan tambahan Viatop66 sebagai bahan aditif dan meninjau perubahan karakteristik aspal pada campuran Aspal Lapis Aus (HRS-WC). Pengaruh Perubahan Titik Lembek …. Pengaruh Perubahan Titik Lembek …. , p , p Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Abstract Increasing the value of the asphalt softening point also affects the characteristics of the HRS-WC mixture. The purpose of this study was to determine the effect of changes in the asphalt softening point due to the addition of viatop66 to Aus layer concrete asphalt pavement characteristics (HRS- WC). Addition of variations in viatop66 levels in bitumen is 0.0%; 1.0%; 2.0%; 3.0%; 4.0%; and 5.0% of asphalt weight. Determination of the Optimum Asphalt Level (KAO) of the HRS-WC mixture using the Marshall Method. Analysis of the relationship between the value of asphalt softening point and HRS-WC mixed characteristic values using the ‘F’ ANOVA test with a confidence level of 95%. The results showed that the increase in asphalt softening point due to the addition of viatop66 to asphalt could increase the density and VFB value of HRS-WC mixture with the highest percentage increase of 0.67% for density and 4.06% for VFB of HRS-WC mixture in the Softening Point value Asphalt 52.48 ° C, decreases the value of VIM and VMA of HRS-WC mixture with a percentage decrease of 11.92% for VIM and 3.24% for VMA of HRS-WC mixture at Asphalt Softening Point value of 52.48 ° C, decreases the value of flow and improving the stability and MQ of the Marshall Hall HRS-WC mix. Increasing the value of asphalt softening point due to the addition of viatop66 to asphalt also increases the value of the remaining marshall stability above 90%, which means that changes in the value of Asphalt Softening Point due to the addition of viatop66 can make asphalt resistant to changes in the surrounding environment. 63 Jurnal Sains dan Teknologi Tadulako Anshory, R., M., Mashuri dan Patunrangi, J. Anshory, R., M., Mashuri dan Patunrangi, J. 1.1. Titik Lembek Aspal Pemeriksaan Titik lembek bertujuan untuk menentukan nilai/suhu titik lembek aspal dan Ter yang berkisar antara 30C – 200C. Titik lembek adalah suhu dimana suatu lapisan aspal dalam cincin yang diletakkan horizontal di atas larutan air atau gliserine yang dipanaskan secara teratur menjadi lembek karena karena beban bola baja dengan diameter 9,53 mm seberat ± 3,5 gram yang diletakkan di atasnya sehingga lapisan aspal tersebut jatuh melalui jarak 25,4 mm (1 inci). Salah satu manfaat pemeriksaan titik lembek dan penetrasi adalah untuk menemukan besarnya nilai PI (Penetration Index) yang merupakan parameter tingkat kepekaan aspal terhadap temperatur. Gambar 1. Pemeriksaan Titik Lembek Gambar 1. Pemeriksaan Titik Lembek Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: 1. Pendahuluan Dan dengan izin Allah Azzawajallah serta berharap terjadinya perubahan trend ke arah yang lebih baik di kalangan ahli transportasi dalam penggunaan bahan tambah pada aspal, penulis memutuskan untuk membuat Tugas Akhir dengan judul “Pengaruh perubahan titik lembek aspal akibat penambahan viatop66 terhadap karakteristik campuran HRS-WC”. Jurnal Sains dan Teknologi Tadulako 64 Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 , , p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: Pengaruh Perubahan Titik Lembek …. pada serat selama proses pencampuran aspal (Ahmad Fatha Abdillah,2018). pada serat selama proses pencampuran aspal (Ahmad Fatha Abdillah,2018). Pada perkerasan jalan, serat selulosa berfungsi untuk meningkatkan kualitas konstruksi perkerasan dengan mengurangi sifat yang merugikan aspal yang diakibatkan oleh perubahan temperatur. Bahan aditif yang digunakan pada penelitian ini adalah serat selulosa berupa jenis Viatop66 yang diproduksi oleh sebuah perusahaan di kota Rosenberg-Jerman, yang juga merupakan produsen aborcel. Viatop66 memiliki spesifikasi seperti yang tertera pada tabel berikut. a. Persentase ARBOCEL®ZZ 8 - 1: 75 – 80% a. Persentase ARBOCEL®ZZ 8 - 1: 75 – 80% b. Panjang rata - rata : 7,5 + 1 : 7,5 + 1 c. Ketebalan rata - rata :5000 mikron :5000 mikron c. Ketebalan rata - rata d. Bulk Density : 1100 mikron d. Bulk Density : 1100 mikron e. Analisa saringan, > 3,55 mm : 45 mikron : 45 mikron 1.4. Kadar Aspal Rencana Penentuan perkiraan awal kadar aspal optimum dapat direncakan setelah dilakukan pemilihan penggabungan pada tiga fraksi agregat. Untuk menentukan perkiraan awal kadar aspal optimum maka digunakan rumus: pKAO = pB = 0,034(%CA) + 0,045(%FA) + 0,18 (%Filler) + K (1 pKAO = pB = 0,034(%CA) + 0,045(%FA) + 0,18 (%Filler) + K pKAO = pB = 0,034(%CA) + 0,045(%FA) + 0,18 (%Filler) + K (1) (1) 1.3. Perancangan Campuran (Mix Design) Perencangan campuran dilakukan untuk memperoleh nilai kadar aspal optimum (KAO) untuk suatu campuran yang ditambahkan Viatop66 dengan kadar yang berbeda pada suhu pengujian yang berbeda sehingga menghasilkan beton aspal yang memiliki nilai tegangan tarik maksimum. , p , p Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 1.2. Bahan Tambah Viatop66 Viatop66 adalah serat selulosa yang dilapisi oleh bitumen dalam suatu proses produksi khusus. Bitumen ini bertindak membantu proses granulasi. Dengan ada nya bitumen ini, memungkinkan untuk mengontrol perilaku proses granulasi yang terjadi pada serat selama proses granulasi. Dalam waktu yang sama bitumen ini mengambil alih fungsi dari pengisi ruang antar serat yang diperlukan untuk kelengkapan proses disperse pada serat selama proses pencampuran aspal. Viatop66 terdiri dari Arbocel dengan bahan tambah lainnya, yang dikemas dalam bentuk butiran silinder berwarna abu- abu kehitaman. Arbocel sendiri adalah serat selulosa yang juga digunakan sebagai bahan aditif campuran aspal panas (Madina. A, 2017). Serat selulosa yang dilapisi oleh bitumen melalui suatu proses produksi yang khusus. Bitumen ini bertindak membantu proses granulasi, yang memungkinkan untuk mengontrol perilaku proses granulasi yang terjadi pada serat selama proses granulasi. Dalam waktu yang sama bitumen ini menganbil alih fungsi pengisi ruang antar serat yang diperlukan untuk kelengkapan proses disperse 65 Vol 6, No.1, April 2020 Pengaruh Perubahan Titik Lembek …. 1.5. Pembuatan Campuran Beraspal Pembuatan campuran aspal terbagi menjadi dua bagian yakni bagian pertama adalah gradasi agregat hasil blending dari dua fraksi batuan atau lebih, yang kedua adalah berapa jumlah kadar aspal yang akan digunakan di dalam campuran. Setiap blending agregat akan memberikan porsi kadar aspal yang berbeda. Campuran hasilan blending agregat dan aspal harus mengikuti spesifikasi yang disyaratkan. (Saodang, H.2005). Pembuatan campuran didasarkan pada nilai PKAO. Untuk menentukan kadar aspal optimum (KAO) suatu campuran, maka dibuat campuran dengan 6 variasi kadar aspal yaitu 2 variasi kadar aspal di bawah PKAO dan 3 variasi kadar aspal di atas PKAO. Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Anshory, R., M., Mashuri dan Patunrangi, J. Anshory, R., M., Mashuri dan Patunrangi, J. 22,2 KN (5000 lbs) dan flowmeter. Proving ring digunakan untuk mengukur nilai stabilitas, dan flowmeter untuk mengukur kelelehan plastis atau flow. Benda uji Marshall berbentuk silinder berdiameter 4 inchi (10,2 cm) dan tinggi 2,5 inchi (6,35 cm). Prosedur pengujian Marshall mengikuti SNI 06- 2489-1991, atau AASHTO T 245-90, atau ASTM D 1559-76. (Algaztmasagala.wordpress.com 2012). Benda uji (campuran agregat dan aspal) dibentuk dengan cara menumbuk campuran di dalam cetakan (mold) berbentuk silinder dengan diameter 10 cm dengan tinggi 7,5 cm (18”). Jumlah pukulan tergantung pada beban rencana lalu lintas. Setelah dibiarkan selama 24 jam dalam suhu ruang, rendam benda uji dalam bak atau dipanaskan dalam oven selama 2 jam dari alat Marshall. Sebelum pembebanan diberikan kepala penekan beserta benda uji dinaikkan hingga menyentuh alas cincin penguji. Berikan pembebanan dengan kecepatan tetap 50 mm/menit, sampai pembebanan maksimum tercapai, atau pembebanan menurun seperti ditunjukkan oleh jarum arloji baca, dan catat pembebanan maksimum yang tercapai. Pada pemeriksaan Marshall akan diperoleh data sebagai berikut: 1.6. Pengujian Marshall dan Volumetric Campuran 66 Jurnal Sains dan Teknologi Tadulako Rancangan campuran berdasarkan metode Marshall ditemukan oleh Bruce Marshall, dan telah distandarisasi oleh ASTM ataupun AASHTO melalui beberapa modifikasi, yaitu ASTM D 1559- 76, atau AASHTO T-245-90. Prinsip dasar metode Marshall adalah pemeriksaan stabilitas dan kelelehan (flow), serta analisis kepadatan dan pori dari campuran padat yang terbentuk. Alat Marshall merupakan alat tekan yang dilengkapi dengan proving ring (cincin penguji) berkapasitas 66 Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 1. Stabilitas Nilai Stabilitas diperoleh berdasarkan nilai yang ditunjukkan oleh jarum dial pada proving ring stabilitas yang dipasang pada alat Marshall Test, kemudian dikonversikan dengan tabel kalibrasi sesuai proving ring yang digunakan. Selanjutnya nilai stabilitas tersebut harus dikoreksi dengan suatu factor koreksi ketebalan benda uji. Flow atau nilai pelelehan diperoleh dari jarum dial flow dalam satuan unit dimana 1 unit = 0,01 mm, sehingga tidak perlu lagi dikonversi. Flow atau nilai pelelehan diperoleh dari jarum dial flow dalam satuan unit dimana 1 unit = 0,01 mm, sehingga tidak perlu lagi dikonversi. 3 M h ll Q i Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: Vol 6, No.1, April 2020 6 , , p p 22 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: 2.1. Pekerjaan Pesiapan Pengecekan alat penelitian di Laboratorium Transportasi dan Jalan Raya Universitas Tadulako, bahan penelitian berupa Aspal Penetrasi 60/70, Viatop66 dan penyuratan tempat lokasi pengambilan agregat. 2.2. Pengambilan Material Aspal yang digunakan adalah Aspal Pen 60/70 yang telah tersedia di dalam Laboratorium Transportasi dan Jalan Raya Universitas Tadulako. Aspal yang digunakan adalah Aspal Pen 60/70 yang telah tersedia di dalam Laboratorium Transportasi dan Jalan Raya Universitas Tadulako. Agregat diambil pada Stone Crusher PT. Palu Batu Madu Loli berupa Agregat Kasar, Halus dan Filler. Viatop66 merupakan zat aditif berupa serat selousa yang bahan tersebut telah tersedia dalam Laboratorium transportasi dan jalan raya Universitas Tadulako yang kemudian akan digunakan dalam penelitian ini. 2.3. Pemeriksaan Material Pemeriksaan agregat meliputi Analisa Saringan menggunakan metode (SNI 03-1968-1990), Keausan Agregat Menggunakan Mesin Abrasi Los Angeles dengan metode (SNI 2417:2008), Kelekatan Agregat Terhadap Aspal berdasarkan (SNI 2439:2011), Berat Jenis dan Penyerapan (SNI 03- 1969-1990). 2. Metode Penelitian Penelitian ilmiah ini berbentuk Eksperimen, dimana penelitian ini dilaksanakan dengan melakukan percobaan-percobaan di Laboratorium, berdasarkan kaidah-kaidah ilmiah dengan prosedur yang sistematis melalui pembuktian yang ilmiah. Penelitian ini dilakukan melalui tahapan-tahapan seperti: Pengaruh Perubahan Titik Lembek …. Pengaruh Perubahan Titik Lembek …. 𝑀𝐴= 100 (1 − 𝐺𝑚𝑏 (1−𝑝𝑏𝑡) 𝐺𝑠𝑏 ( 𝑉𝑀𝐴= 100 (1 − 𝐺𝑚𝑏 (1−𝑝𝑏𝑡) 𝐺𝑠𝑏 (3) 𝐺𝑠𝑏 2. Rongga udara dalam campuran padat (Void in Mix, VIM) 2. Rongga udara dalam campuran padat (Void in Mix, VIM) 2. Rongga udara dalam campuran padat (Void in Mix, VIM) 2. Rongga udara dalam campuran padat (Void in Mix, VIM) VIM dalam campuran beraspal terdiri dari ruang antara partikel agregat yang terselimuti aspal. VIM dapat dihitung menggunakan rumus: VIM dalam campuran beraspal terdiri dari ruang antara partikel agregat yang terselimuti aspal. VIM dapat dihitung menggunakan rumus: VIM dalam campuran beraspal terdiri dari ruang antara partikel agregat yang terselimuti aspal. VIM dapat dihitung menggunakan rumus: 𝑀= 100 𝑥 𝐺𝑚𝑏−𝐺𝑚𝑚 𝐺𝑚𝑚 (4) 𝑉𝐼𝑀= 100 𝑥 𝐺𝑚𝑏−𝐺𝑚𝑚 𝐺𝑚𝑚 (4) 𝑉𝐼𝑀= 100 𝑥 𝐺𝑚𝑏−𝐺𝑚𝑚 𝐺𝑚𝑚 (4) 3. Rongga udara terisi aspal (Void Filled with Asphalt, VFA) VFA adalah persen rongga yang terdapat di antara partikel agregat (VMA) yang terisi loleh aspal yang terserap oleh agregat. VFA dapat dihitung menggunakan rumus: VFA adalah persen rongga yang terdapat di antara partikel agregat (VMA) yang terisi loleh aspal yang terserap oleh agregat. VFA dapat dihitung menggunakan rumus: 𝑉𝐹𝐴= 100 𝑥 𝑉𝑀𝐴−𝑉𝐼𝑀 𝑉𝑀𝐴 (5) 2. Metode Penelitian 𝑉𝐹𝐴= 100 𝑥 𝑉𝑀𝐴−𝑉𝐼𝑀 𝑉𝑀𝐴 (5) (5) , p , p Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 3. Marshall Quotient Untuk mengetahui kekakuan campuran aspal maka dilakukan analisa dengan mencari nilai Marshall Quotient (MQ), yang merupakan hasil bagi antara nilai stabilitas dan nilai perlelehan (flow), Marshall Quotient dapat dihitung dengan rumus: 𝑀𝑄= 𝑀𝑠 𝑀𝑓 (2) (2) 𝑀𝑄= 𝑀𝑠 𝑀𝑓 Pemeriksaan Volumetrik campuran beraspal adalah volume benda uji campuran yang telah dipadatkan.Pada Pemeriksaan ini akan diperoleh data sebagai berikut: Pemeriksaan Volumetrik campuran beraspal adalah volume benda uji campuran yang telah dipadatkan.Pada Pemeriksaan ini akan diperoleh data sebagai berikut: 1. Rongga diantara Mineral Agregat (Void in Mineral Agregat, VMA) gg g g ( g g ) VMA adalah volume rongga udara diantara butir- butir agregat dalam campuran beraspal dalam kondisi padat. VMA meliputi volume rongga udara dalam capuran beraspal dan volume aspal efektif (tidak termasuk volume aspal yang diserap agregat). VMA dihitung berdasarkan jenis bulk (Gsb) agregat yang dinyatakan sebagai persen volume bulk campuran yang dipadatkan. VMA dapat dihitung menggunakan rumus: VMA adalah volume rongga udara diantara butir- butir agregat dalam campuran beraspal dalam kondisi padat. VMA meliputi volume rongga udara dalam capuran beraspal dan volume aspal efektif (tidak termasuk volume aspal yang diserap agregat). VMA dihitung berdasarkan jenis bulk (Gsb) agregat yang dinyatakan sebagai persen volume bulk campuran yang dipadatkan. VMA dapat dihitung menggunakan rumus: 67 Vol 6, No.1, April 2020 2.7. Desain Gradasi Campuran HRS-WC Gradasi dari campuran HRS-WC adalah gradasi senjang dengan fraksi agregat 3/4‟, fraksi ½‟, fraksi 3/8‟ dan filler sebagai agregat penyusun dalam campuran. 2.6. Modifikasi Titik Lembek Aspal dengan Viatop66 Sifat aspal yang dimodifikasi pada penelitian ini adalah titik Lembek Aspal dengan cara menambahkan bahan aditif Viatop66 ke dalam aspal . variasi Viatop66 dalam aspal adalah 0,0%, 1,0%, 2,0%, 3,0%, 4,0% dan 5%. Penambahan Viatop66 pada aspal dilakukan saat pemanasan aspal atau pada saat perataan aspal dalam wadah pemanas aspal, selanjutnya proses eksperimen dilakukan sesuai dengan SNI 2434:2011. 2.9. Pengujian Mashall dan Volumetric Campuran Benda Uji PKAO Pengujian Marshall dan Volumetric menggunakan metode (SNI 06-2489-1991) Hal ini dilakukan untuk mengetahui ketahanan (stabilitas) terhadap kelelehan (flow) dari suatu campuran aspal dan agregat 2.8. Penentuan PKAO dan Pembuatan Benda Uji Setiap Variasi Penentuan perkiraan kadar aspal pada campuran HRS-WC mengacu pada spesifikasi teknis lapis permukaan HRS-WC dengan variasi kadar aspal a- 0,01; a-0,005; PKAO = a; a+0,005; a+0,010 dan a+0,015. perencanaan campuran adalah untuk mendapatkan campuran agregat, aspal dan bahan tambah viatop66 yang optimal sehingga dihasilkan perkerasan dengan kualitas optimal. 2.9. Pengujian Mashall dan Volumetric Campuran Benda Uji PKAO 2.4. Pemeriksaan Aspal 68 Jurnal Sains dan Teknologi Tadulako Jenis pengujian yang dilakukan antara lain Penetrasi (SNI 2456:2011), Berat jenis (SNI 2441:2011), Daktilitas (SNI 2432:2011), Titik lembek (SNI 2434:2011), titik Nyala titik bakar (SNI 2433:2011), Anshory, R., M., Mashuri dan Patunrangi, J. Anshory, R., M., Mashuri dan Patunrangi, J. dan Kehilangan berat minyak dan aspal (SNI 06- 2440-1991). 2.5. Pemeriksaan Viatop66 Pemeriksaan bahan tambah viatop66 meliputi pemeriksaan Kadar air viatop66 menggunakan (SNI 1965:2008), Analisa Saringan menggunakan metode (SNI 03-1968-1990). Pemeriksaan bahan tambah viatop66 meliputi pemeriksaan Kadar air viatop66 menggunakan (SNI 1965:2008), Analisa Saringan menggunakan metode (SNI 03-1968-1990). Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: 2.11. Uji Marshall Kondisi KAO Pengujian mashall dilakukan dalam 2 bagian, bagian pertama merupakan pengujian stabilitas marshall dan pengujian marshall sisa. p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: 2.10. Perhitungan KAO dan Pembuatan Benda Uji KAO Berdasarkan KAO pada setiap variasi Viatop66, dibuat benda uji dilakukan dengan 2 bagian benda uji yakni yang gunakan untuk benda uji rendaman 30 menit dan benda uji rendaman 24 jam. 2.11. Uji Marshall Kondisi KAO Pengaruh Perubahan Titik Lembek …. Tabel 1. Hasil Pemeriksaan Agregat Kasar Sumber: Hasil Pemeriksaan 2018 Tabel 2. Hasil Pemeriksaan Halus Sumber: Hasil Pemeriksaan 2018 Tabel 3. Hasil Pemeriksaan Filler Sumber: Hasil Pemeriksaan 2018 3.2. Pemeriksaan Karakteristik Aspal Tanpa Campuran Viatop66 Pemeriksaan aspal yang pertama dilakukan pada kondisi tanpa campuran viatop66 ,pemeriksaan ini bertujuan untuk melihat karakteristik aspal pada keadaan tanpa campuran viatop66. Hasil dari pemeriksaan karakteristik aspal tersebut disajikan pada Tabel 4. Tabel 4. Hasil Pemeriksaan Aspal Pen 60/70 Tabel 1. Hasil Pemeriksaan Agregat Kasar Sumber: Hasil Pemeriksaan 2018 Tabel 1. Hasil Pemeriksaan Agregat Kasar Tabel 1. Hasil Pemeriksaan Agregat Kasar Sumber: Hasil Pemeriksaan 2018 Tabel 1. Hasil Pemeriksaan Agregat Kasar Sumber: Hasil Pemeriksaan 2018 Sumber: Hasil Pemeriksaan 2018 Tabel 2. Hasil Pemeriksaan Halus Sumber: Hasil Pemeriksaan 2018 Tabel 2. Hasil Pemeriksaan Halus Sumber: Hasil Pemeriksaan 2018 Sumber: Hasil Pemeriksaan 2018 Tabel 3. Hasil Pemeriksaan Filler Sumber: Hasil Pemeriksaan 2018 3.1. Hasil Pemeriksaan Agregat Pemeriksaan Agregat yang dilakukan di Laboratorium Transportasi dan Jalan Raya Universitas Tadulako ini meliputi pemeriksaan agregat kasar, agregat halus, filler yang merupakan agregat dari Stone crusher Palu Batu Madu Loli-Donggala, berikut adalah Tabel - tabel hasil pemeriksaan: 69 Vol 6, No.1, April 2020 Pengaruh Perubahan Titik Lembek …. Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 Jurnal Sains dan Teknologi Tadulako Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 3.3. Pemeriksaan Karakteristik Aspal 60/70 Dengan Campuran Viatop66 Pemeriksaan nilai Titik lembek aspal PEN 60/70 pada beberapa kadar Viatop66 dapat dilihat pada Tabel 5 berikut: Tabel 5. Hasil Pemeriksaan Titik Lembek Aspal Pen 60/70 dengan Variasi Viatop66 Tabel 5. Hasil Pemeriksaan Titik Lembek Aspal Pen 60/70 dengan Variasi Viatop66 Sumber: Hasil Pengujian 2018 Anshory, R., M., Mashuri dan Patunrangi, J. Anshory, R., M., Mashuri dan Patunrangi, J. 3.3. Pemeriksaan Karakteristik Aspal 60/70 Dengan Campuran Viatop66 3.4. Pemeriksaan Karakteristik Serat Seluosa (Viatop66) Pada pengujian serat Seluosa dilakukan pengujian kadar air dan Analisa saringan serat seluosa (Viatop66) yang akan digunakan sebagai bahan tambah dalam campuran HRS-WC. Berikut merupakan hasil pemeriksaan karakteristik Viatop66. Tabel 6. Hasil Pemeriksaan Karakteristik Viatop66 Tabel 6. Hasil Pemeriksaan Karakteristik Viatop66 Tabel 6. Hasil Pemeriksaan Karakteristik Viatop66 Sumber: Hasil Pengujian 2018 Sumber: Hasil Pengujian 2018 Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 , , p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: 3.2. Pemeriksaan Karakteristik Aspal Tanpa Campuran Viatop66 Pemeriksaan aspal yang pertama dilakukan pada kondisi tanpa campuran viatop66 ,pemeriksaan ini bertujuan untuk melihat karakteristik aspal pada keadaan tanpa campuran viatop66. Hasil dari pemeriksaan karakteristik aspal tersebut disajikan pada Tabel 4. Tabel 4. Hasil Pemeriksaan Aspal Pen 60/70 Tabel 4. Hasil Pemeriksaan Aspal Pen 60/70 Tabel 4. Hasil Pemeriksaan Aspal Pen 60/70 Sumber: Hasil Pemeriksaan 2018 Sumber: Hasil Pemeriksaan 2018 Sumber: Hasil Pemeriksaan 2018 Jurnal Sains dan Teknologi Tadulako 70 Anshory, R., M., Mashuri dan Patunrangi, J. p p Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 3. 5. Penentuan Komposisi Agregat dalam Campuran HRS-WC Penentuan komposisi agregat pada campuran HRS-WC dilakukan dengan cara pengukuran, penimbangan berdasarkan ukuran saringan (metode by Sieve),banyaknya fraksi agregat ditentukan melalui penimbangan berdasarkan komposisi untuk masing- masing ukuran saringan. Agregat tidak di kelompokan berdasarkan kelompok agregat (Halus, kasar dan Filler) seperti pada metode by portion. Berikut merupakan tabel perhitungan komposisi agregat dengan metode by sieve gradasi senjang HRS-WC dengan proporsi ideal: Tabel 7. Komposisi Agregat Campuran HRS-WC Senjang Vol 6, No.1, April 2020 71 Tabel 7. Komposisi Agregat Campuran HRS-WC Senjang Vol 6, No.1, April 2020 71 Pengaruh Perubahan Titik Lembek …. Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Pengaruh Perubahan Titik Lembek …. Sumber: Hasil Pemeriksaan 2018 PKAO = 0,035 (% CA) + 0,045 (%FA) + 0,18 (% FF) + C = 0,035 (39 %) + 0,045 (53 %) + 0,18 (8%) + 1,0 = 5,99% ≈ 6,0% Sumber: Hasil Pemeriksaan 2018 PKAO = 0,035 (% CA) + 0,045 (%FA) + 0,18 (% FF) + C = 0,035 (39 %) + 0,045 (53 %) + 0,18 (8%) + 1,0 = 5,99% ≈ 6,0% Hasil pengujian Marshall dan Volumetrik campuran HRS-WC di sajikan dalam Tabel 8 berikut Hasil pengujian Marshall dan Volumetrik campuran HRS-WC di sajikan dalam Tabel 8 berikut: Tabel 8. Hasil Pengujian Marshall dan Volumetrik Campuran HRS-WC Tabel 8. Hasil Pengujian Marshall dan Volumetrik Campuran HRS-WC Sumber: Hasil Pengujian 2018 Sumber: Hasil Pengujian 2018 Berdasarkan Pengujian dan memperhatikan spesifikasi yang berlaku, dibuatlah gambar Barchart untuk menentukan rentang kadar aspal yang memenuhi spesifikasi serta menentukan Kadar Aspal Optimum (KAO). Gambar 2. Barchart Rentang KAO Gambar 2. Barchart Rentang KAO Dari Gambar Barchart rentang KAO didapatkan rentang kadar aspal yang memenuhi spesifikasi campuran HRS-WC yaitu 5,9% - 7,02%. Sehingga nilai KAO (Kadar Aspal Optimum) didapatkan sebagai berikut: 𝐾𝐴𝑂= 𝐴+ 𝐵 2 = 6,235% + 6,772% 2 = 13,007% 2 = 6,504 = 6,5% 3.6. Analisa Perubahan Nilai Titik Lembek Aspal PEN 60/70 Akibat Penambahan Viatop66 Anshory, R., M., Mashuri dan Patunrangi, J. Anshory, R., M., Mashuri dan Patunrangi, J. menunjukkan adanya perubahan nilai Titik lembek pada tiap variasi viatop66, berikut merupakan tabel analisis data pemeriksaan nilai titik lembek: Tabel 9. Hasil Analisis Data Titik Lembek pada Pengujian Sumber : Hasil Analisis 2019 Tabel 9. Hasil Analisis Data Titik Lembek pada Pengujian Pada Gambar 3, merupakan grafik yang memperlihatkan bahwa penambahan Viatop66 cenderung meningkatkan nilai titik lembek aspal dengan persentase perubahan nilai Titik lembek akibat penambahan viatop66 pada setiap variasi kadar viatop66. Gambar 3. Grafik Hubungan antara Titik Lembek dengan Kadar Viatop66 Gambar 3. Grafik Hubungan antara Titik Lembek dengan Kadar Viatop66 3.7. Analisis Hubungan Nilai Titik Lembek Aspal dengan Parameter Volumetrik Campuran HRS- WC 3.7. Analisis Hubungan Nilai Titik Lembek Aspal dengan Parameter Volumetrik Campuran HRS- WC. Komponen volumetrik campuran Aspal yaitu kepadatan campuran, volumetrik rongga dalam campuran (VIM), volume rongga diantara mineral agregat (VMA) dan volume rongga terisi aspal (VFB). Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 , , p p Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: 3.6. Analisa Perubahan Nilai Titik Lembek Aspal PEN 60/70 Akibat Penambahan Viatop 72 Jurnal Sains dan Teknologi Tadulako Hasil pengujian Titik lembek aspal dengan berbagai macam variasi kadar viatop66 Jurnal Sains dan Teknologi Tadulako Pengaruh Perubahan Titik Lembek …. Tabel 10. Hasil Pengujian Kepadatan Sumber: Hasil Pengujian 2018 Gambar 4. Grafik Hubungan Nilai Titik Lembek dengan Nilai Kepadatan Campuran HRS-WC Tabel 10. Hasil Pengujian Kepadatan Tabel 10. Hasil Pengujian Kepadatan Gambar 4. Grafik Hubungan Nilai Titik Lembek dengan Nilai Kepadatan Campuran Gambar 4. Grafik Hubungan Nilai Titik Lembek dengan Nilai Kepadatan Campuran HRS-WC Nilai kepadatan cenderung meningkat seiring dengan peningkatan nilai titik lembek aspal. Penyebabnya yaitu karena Viatop66 dapat mengisi rongga-rongga pada agregat sehingga membuat kerapatan antar agregat dengan aspal menjadi tinggi dan menyebabkan sifat saling mengunci (interlocking) dari partikel-partikel agregat menjadi semakin baik. 3.8. Nilai Titik Lembek Aspal PEN 60 /70 dengan Kepadatan campuran HRS-WC Vol 6, No.1, April 2020 73 Nilai kepadatan campuran dipengaruhi oleh karakteristik dan komposisi agregat, distribusi gradasi agregat, kadar aspal dan jumlah tumbukan. Adapun hasil pengujian kepadatan campuran dan titik lembek aspal dapat dilihat pada Tabel 10, dan penyajian data dalam grafik pada gambar 4. Pengaruh Perubahan Titik Lembek …. Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 C i ht 2020 Dit bitk l h J l S i d T k l i T d l k ISSN 2089 8630 Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 3.10 Nilai Titik Lembek Aspal dengan Void In Mineral Agregat (VMA) Campuran HRS-W Faktor yang mempengaruhi VMA antara lain adalah gradasi agregat (komposisi campuran agregat dan ukuran diameter butir terbesar), energi pemadatan, kadar aspal, pemanasan aspal dan bentuk butir. Anshory, R., M., Mashuri dan Patunrangi, J. Sumber: Hasil Pengujian 2018 Data ini disajikan dalam bentuk grafik berikut Gambar 5. Grafik Hubungan Nilai Titik Lembek terhadap Nilai VIM Campuran HRS-WC Sumber: Hasil Pengujian 2018 Data ini disajikan dalam bentuk grafik berikut Gambar 5. Grafik Hubungan Nilai Titik Lembek terhadap Nilai VIM Campuran HRS-W Nilai Void in Mixture cenderung menurun seiring dengan peningkatan nilai titik lembek aspal yang berarti rongga dalam campuran semakin kecil karena terisi oleh serat Viatop66 yang ada. 3.10 Nilai Titik Lembek Aspal dengan Void In Mineral Agregat (VMA) Campuran HRS-WC Nilai Void in Mixture cenderung menurun seiring dengan peningkatan nilai titik lembek aspal yang berarti rongga dalam campuran semakin kecil karena terisi oleh serat Viatop66 yang ada. 3.9. Nilai Titik Lembek Aspal PEN 60/70 dengan Void In Mixture (VIM) Campuran HRS-WC Nilai VIM yang semakin tinggi menunjukkan semakin besarnya rongga udara dalam campuran. Nilai VIM menjadi indikator durabilitas atau memberi pengaruh terhadap keawetan dari campuran beton aspal. Besar dan kecilnya nilai VIM sangat dipengaruhi oleh distribusi dan gradasi agregat yang akan membuat campuran lebih padat. Adapun hasil pengujian VIM campuran dan titik lembek aspal dapat dilihat pada Tabel 11. 74 Jurnal Sains dan Teknologi Tadulako Diterima 31 Maret 2020 Direvisi 24 Apri 2020 Diterima untuk publikasi 21 Mei 2020 Tabel 11. Hasil Pengujian VIM Tabel 11. Hasil Pengujian VIM 74 Anshory, R., M., Mashuri dan Patunrangi, J. Pengaruh Perubahan Titik Lembek …. Pengaruh Perubahan Titik Lembek …. Diterima 31 Maret 2020, Direvisi 24 Apri 2020, Diterima untuk publikasi 21 Mei 2020 , p , p Copyright 2020 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630 3.11. Marshall Sisa Marshall sisa merupakan pengujian marshall yang dilakukan setelah perendaman benda uji selama 24 jam pada suhu 60±1°C kondisi Kadar Aspal Optimum (KAO). Nilai Marshall sisa diperoleh dari hasil persentase perbandingan Stabilitas uji marshall antara rendaman 24 jam dan rendaman 30 menit pada suhu 60±1°C. Vol 6, No.1, April 2020 75 Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: Tabel 17. Marshall Sisa Campuran HRS-WC Pada Kadar Aspal Optimum 6,5% Sumber: Hasil Analisis 2019 Tabel 17. Marshall Sisa Campuran HRS-WC Pada Kadar Aspal Optimum 6,5% Vol 6, No.1, April 2020 75 Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Gambar 11. Grafik Hubungan Nilai Titik Lembek dan Marshall Sisa Dapat disimpulkan bahwa Stabilitas marshall sisa mengalami peningkatan seiring dengan peningkatan perubahan Nilai Titik Lembek Aspal yang mengartikan penambahan peningkatan Titik Lembek Aspal memberi ketahanan terhadap perubahan lingkungan sekitar. 4.1. Kesimpulan Dari Hasil penelitian “Pengaruh Perubahan Titik Lembek Aspal akibat penambahan Viatop66 terhadap karakteristik Campuran HRS-WC disimpulkan bahwa: Penambahan variasi kadar viatop66 ke dalam aspal mempengaruhi nilai Titik Lembek Aspal, dimana semakin besar penambahan kadar viatop66 berbanding lurus dengan naiknya nilai Titik Lembek aspal. Nilai Titik lembek aspal terbesar ada pada kadar viatop66 tertinggi yaitu 5% dengan nilai titik lembek 53,23 °C dan persentase kenaikan sebesar 10,32%. ● Kepadatan dan VFB Campuran HRS-WC mengalami peningkatan seiring dengan meningkatnya nilai Titik lembek Aspal, nilai tertinggi Kepadatan dan VFB campuran HRS-WC berada pada Nilai Titik Lembek Aspal 52,48 °C yaitu dengan rata-rata nilai kepadatan sebesar 2,275 gr/cm3 dan persentase kenaikan sebesar 0,67%. sedangkan nilai rata- rata VFB 71,402 % dan persentase kenaikan sebesar 4,06%. ● VIM dan VMA Campuran HRS-WC mengalami penurunan seiring dengan meningkatnya nilai Titik lembek Aspal, nilai terendah VIM dan VMA campuran HRS-WC berada pada Nilai Titik Lembek Aspal 52,48 °C dengan rata-rata nilai VIM sebesar 5,108 % dan persentase penurunan sebesar 11,92%, sedangkan nilai rata- rata VMA 17,859 % dan persentase penurunan sebesar 3,24%. ● Flow Marshall campuran HRS-WC akibat perubahan Nilai Titik lembek Aspal mengalami penurunan yakni berkisar antara 2.81 – 9.22 %, atau rata – rata penurunannya 5,472%. Sedangkan stabilitas marshall mengalami peningkatan dari 2,17 – 15,72% dengan rata-rata peningkatan 7,883%. MQ Marshall campuran HRS-WC mengalami kenaikan, seiring dengan perubahan Nilai Titik Lembek Aspal akibat adanya penambahan kadar viatop66 hal ini menunjukkan perubahan campuran yang menjadi lebih kaku. ● Flow Marshall campuran HRS-WC akibat perubahan Nilai Titik lembek Aspal mengalami penurunan yakni berkisar antara 2.81 – 9.22 %, atau rata – rata penurunannya 5,472%. Sedangkan stabilitas marshall mengalami peningkatan dari 2,17 – 15,72% dengan rata-rata peningkatan 7,883%. MQ Marshall campuran HRS-WC mengalami kenaikan, seiring dengan perubahan Nilai Titik Lembek Aspal akibat adanya penambahan kadar viatop66 hal ini menunjukkan perubahan campuran yang menjadi lebih kaku. ● Stabilitas marshall sisa menunjukkan angka kestabilan lebih dari 90% dan semakin meningkat seiring dengan perubahan Nilai Titik Lembek Aspal akibat adanya penambahan variasi kadar viatop66 yang mengartikan bahwa penambahan variasi kadar viatop66 dapat membuat aspal tahan terhadap perubahan lingkungan sekitar. Anshory, R., M., Mashuri dan Patunrangi, J. Anshory, R., M., Mashuri dan Patunrangi, J. yaitu: yaitu: ● Memperhatikan alat uji dengan baik sebelum maupun sesudah melakukan pengujian y ● Memperhatikan alat uji dengan baik sebelum maupun sesudah melakukan pengujian ● Memperhatikan alat uji dengan baik sebelum maupun sesudah melakukan pengujian ● Memperhatikan rentan waktu dan perubahan suhu dengan teliti saat pengujian Titik lembek berlangsung. ● Periksa posisi penempatan bola baja saat melakukan pengujian Titik lembek. ● Memperhatikan suhu pencampuran dan pemadatan pada setiap variasi kadar viatop66 yang digunakan. ● Memperhatikan suhu pencampuran dan pemadatan pada setiap variasi kadar viatop66 yang digunakan. Diterima 1 Februari 2020, Direvisi 4 Maret 2020, Diterima untuk publikasi 1 April 2020 Copyright 2022 Diterbitkan oleh Jurnal Sains dan Teknologi Tadulako, ISSN 2089-8630, DOI: 4.2. Saran Dari penelitian yang sudah dilakukan maka penulis memberikan saran guna penelitian selanjutnya 76 Jurnal Sains dan Teknologi Tadulako Saodang, H. (2005). Perancangan Perkerasan Jalan Raya Nova. Bandung. Daftar Pustaka Fatha Abdillah, A. (2017). Pengaruh Penggunaan Bahan Tambah Viatop66 Pada Campuran Stone matrix Asphalt Terhadap Titik Lembek Aspal Dan Sifat Drain Down Campuran. Tugas Akhir. Universitas Tadulako. Palu. https://algaztmasagala.wordpress.com (diakses maret 2018) Fatha Abdillah, A. (2017). Pengaruh Penggunaan Bahan Tambah Viatop66 Pada Campuran Stone matrix Asphalt Terhadap Titik Lembek Aspal Dan Sifat Drain Down Campuran. Tugas Akhir. Universitas Tadulako. Palu. https://algaztmasagala.wordpress.com (diakses maret 2018) Madina, A. (2017) “Pengaruh Perubahan Penetrasi Aspal Akibat Penggunaan Bahan Tambah Viatop66 Terhadap Sifat Draindown Campuran Stone Matrix Asphalt (SMA)”. Tugas Akhir. Universitas Tadulako, Palu. Madina, A. (2017) “Pengaruh Perubahan Penetrasi Aspal Akibat Penggunaan Bahan Tambah Viatop66 Terhadap Sifat Draindown Campuran Stone Matrix Asphalt (SMA)”. Tugas Akhir. Universitas Tadulako, Palu. Saodang, H. (2005). Perancangan Perkerasan Jalan Raya Nova. Bandung. Saodang, H. (2005). Perancangan Perkerasan Jalan Raya Nova. Bandung. 77 Vol 6, No.1, April 2020 77
https://openalex.org/W2048068953	https://europepmc.org/articles/pmc3497592?pdf=render	English	null	Contextual determinants of health behaviours in an aboriginal community in Canada: pilot project	BMC public health	2,012	cc-by	7,388	© 2012 Joseph 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 Contextual determinants of health behaviours in an aboriginal community in Canada: pilot project Pamela Joseph1,4, A Darlene Davis2, Ruby Miller2, Karen Hill2, Honey McCarthy2, Ananya Banerjee1, Clara Chow3, Andrew Mente1,3,4 and Sonia S Anand1,3,4* * Correspondence: anands@mcmaster.ca 1Department of Medicine and Epidemiology, McMaster University, 1280 Main St, Hamilton, Ontario L8S 4K1, Canada 3Population Health Research Institute, 237 Barton Street East, Hamilton, ON L8L 2X2, Canada Full list of author information is available at the end of the article Abstract Background: Rapid change in food intake, physical activity, and tobacco use in recent decades have contributed to the soaring rates of obesity, type 2 diabetes and cardiovascular disease (CVD) in Aboriginal populations living in Canada. The nature and influence of contextual factors on Aboriginal health behaviours are not well characterized. Methods: To describe the contextual determinants of health behaviours associated with cardiovascular risk factors on the Six Nations reserve, including the built environment, access and affordability of healthy foods, and the use of tobacco. In this cross-sectional study, 63 adults from the Six Nations Reserve completed the modified Neighbourhood Environment Walkability Scale (NEWS), questionnaire assessing food access and availability, tobacco pricing and availability, and the Environmental Profile of Community Health (EPOCH) tool. Results: The structured environment of Six Nations Reserve scored low for walkability, street connectivity, aesthetics, safety, and access to walking and cycling facilities. All participants purchased groceries off-reserve, although fresh fruits and vegetables were reported to be available and affordable both on and off-reserve. On average $151/week is spent on groceries per family. Ninety percent of individuals report tobacco use is a problem in the community. Tobacco is easily accessible for children and youth, and only three percent of community members would accept increased tobacco taxation as a strategy to reduce tobacco access. Conclusions: The built environment, access and affordability of healthy food and tobacco on the Six Nations Reserve are not perceived favourably. Modification of these contextual factors described here may reduce adverse health behaviours in the community. Keywords: Obesity, Aboriginal health, Health behaviours, Environment design Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 Assessment and outcomes Individuals completed questionnaires with a trained interviewer at the SHARE-AP research unit located on the reserve. Standardized tools were specifically tailored for the Aboriginal population living on the Six Nations Reserve to assess built environment, food availability, and tobacco availability, access and beliefs. A modified Neighbourhood Environment Walkability Scale (NEWS) was administered to assess built environment and indi- vidual perceptions of neighbourhood walkability. The scale is used to assess proximity and ease of access to community facilities, walking and cycling facilities, street connectivity, traffic and crime safety, aesthetics, and community satisfaction. Items are rated on a 4-point scale. NEWS has test-retest reliability, and has been vali- dated against Geographic Information Systems, acceler- ometer and anthropometric measures in the United States [20-22]. This instrument has been used in rural communities in Taiwan and in an international prospect- ive cohort study [23,24], and among an Indigenous population of New Zealand the Maori [25]. Food avail- ability and access was assessed with a modified tool which was previously validated in a low socioeconomic sample in the United States [26]. Items include fre- quency and location of grocery shopping, food costs, availability and cost of fruits and vegetables on and off- reserve, and whether participants grow their own vegeta- bles during the summer. An overall score was deter- mined by an unweighted sum of five items. Perceptions of tobacco pricing and availability were measured using a Community Readiness Survey [27]. The perception score is scaled by the unweighted sum of the 8-items, which assess individual perceptions on tobacco use and accessibility among teenagers, problems with tobacco use among adults, willingness to increase tobacco tax, ban tobacco advertisements or enforce more laws on tobacco sales. The scale was found to have good con- struct validity, as scale scores were significantly asso- ciated with community readiness as evaluated by prevention planners [27]. The Environmental Profile of Community Health (EPOCH-2) tool measures commu- nity level determinants of cardiovascular risk factors and The Six Nations Reserve in Brant County, Ontario spans approximately 18,000 hectares of land, and is located 25 km from the major city of Hamilton, between Brantford, Caledonia and Hagersville. The reserve is home to the largest population of First Nations in Canada, with over 12,000 Six Nations people [18,19]. Understanding the association of contextual factors and related CVD health behaviours in high-risk communities is important for the design of future interventions. Background It is well known that control of pricing and advertising is more effective for population control of tobacco use than are individualized smoking cessation programs [17]. However there is little regula- tion of tobacco access on most Aboriginal reserves in Canada. Assessment and outcomes Therefore the objective of this study was to characterize the contextual factors in the Six Nations community in- cluding the built environment, access and affordability of healthy food and tobacco. We hypothesize that con- textual factors exist which constrain the ability of indivi- duals to make and sustain health behaviour changes. Participants community is conceptualized as the extent to which en- vironmental characteristics influence walking for leisure, exercise, recreation, travel and access to services [13]. In a systematic review of environmental determinants of health in adults, both availability of exercise equipment and connectivity of trails were associated with greater physical activity [14]. In Aboriginal communities, forma- tive research has identified environmental factors, such as loose dogs, poor roads, safety and lack of facilities, as potential barriers to physical activity [4,5]. Similar envir- onmental constraints may influence individual dietary behaviors. Aboriginal people living on remote reserves are exposed to high food prices and have limited access to healthy foods [15]. Aboriginal people also face in- creasing promotion and availability of energy-dense, nu- trient poor products in comparison to fruits and vegetables [16]. While tobacco use has a traditional role in many Aboriginal cultures, expansion of tobacco pro- duction and easy access to cigarettes has resulted in a large proportion of adults using this highly addictive substance long-term [1]. It is well known that control of pricing and advertising is more effective for population control of tobacco use than are individualized smoking cessation programs [17]. However there is little regula- tion of tobacco access on most Aboriginal reserves in Canada. Sixty-three individuals from the Six Nations Reserve were included in the present study. Study participants were previously enrolled in the SHARE-AP ACTION household-based randomized trial [4]. Eligibility and ex- clusion criteria are described elsewhere [4]. All 83 adult participants in SHARE-AP ACTION were mailed a letter describing the study, followed by telephone calls inviting them into the research unit to complete the assessment. The main reason cited for non-participation was being “too busy” at work or at home. Background prevalence of obesity and related metabolic complications in adults, the SHARE-AP ACTION household-based intervention was implemented with the goal of changing health behaviours associated with weight gain. However after a six month intervention, only modest changes in dietary and physical activity practices were observed among intervention families [4]. Similarly, school and community-based interventions aimed at intensifying in- dividual health behaviours among American Aboriginal communities have shown little impact on reducing body weight [5-8]. Aboriginal people suffer a higher risk of developing obesity, type 2 diabetes and cardiovascular disease (CVD) compared to the general population [1-3]. A pre- vious study we conducted on the Six Nations Reserve (SHARE-AP) demonstrated that community members have high rates of obesity, diabetes, hypertension, tobacco use, elevated cholesterol and CVD compared to European-origin Canadians [1]. In response to the high * Correspondence: anands@mcmaster.ca 1Department of Medicine and Epidemiology, McMaster University, 1280 Main St, Hamilton, Ontario L8S 4K1, Canada 3Population Health Research Institute, 237 Barton Street East, Hamilton, ON L8L 2X2, Canada Full list of author information is available at the end of the article There is emerging evidence to support the influence of community level factors on health behaviors associated with CVD, such as diet, physical activity and tobacco use [9-12]. The built environment or “walkability” of a Page 2 of 8 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 community is conceptualized as the extent to which en- vironmental characteristics influence walking for leisure, exercise, recreation, travel and access to services [13]. In a systematic review of environmental determinants of health in adults, both availability of exercise equipment and connectivity of trails were associated with greater physical activity [14]. In Aboriginal communities, forma- tive research has identified environmental factors, such as loose dogs, poor roads, safety and lack of facilities, as potential barriers to physical activity [4,5]. Similar envir- onmental constraints may influence individual dietary behaviors. Aboriginal people living on remote reserves are exposed to high food prices and have limited access to healthy foods [15]. Aboriginal people also face in- creasing promotion and availability of energy-dense, nu- trient poor products in comparison to fruits and vegetables [16]. While tobacco use has a traditional role in many Aboriginal cultures, expansion of tobacco pro- duction and easy access to cigarettes has resulted in a large proportion of adults using this highly addictive substance long-term [1]. Methods Using a cross-sectional design, information was collected from individuals residing in the community to charac- terize community level factors of built environment, healthy food access and availability, and tobacco use among individuals from the Six Nations Reserve. The Re- search Ethics Board of McMaster University and Band Council of the Six Nations approved the study protocol and all study participants provided written informed con- sent. This study was funded by a grant from the Heart and Stroke Foundation of Ontario (Grant #PEA6547). Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 Page 3 of 8 Table 2 Mean (SD) subscale scores from the NEWS questionnaire NEWS subscale Six nations (N=63) Possibility range Walkability to Community Facilities 8.9 (4.8) 7-42 Street Connectivity 6.7 (2.3) 4-16 Walking/Cycling Facilities 5.4 (2.7) 4-16 Aesthetics 7.5 (2.5) 5-25 Pedestrian/Traffic Safety 12.2 (2.3) 5-25 Crime Safety 16.4 (3.3) 5-25 Community Satisfaction 31.2 (6.8) 6-36 * Higher scores indicate a more favourable value of the Environmental characteristics. Table 2 Mean (SD) subscale scores from the NEWS Table 2 Mean (SD) subscale scores from the NEWS questionnaire disease. This instrument has been tested in several com- munities in Canada, India, Columbia, China and Iran and was found to have good construct validity [28]. EPOCH-2 includes a survey of community awareness, attitudes and social norms given to all participants. Statistics Statistical analysis was conducted using SPSS v11.0 soft- ware. All study variables were analyzed descriptively by percentage and means. Due to the limited statistical power we did not test the association between percep- tions of the contextual environment and individual health behaviours. Results their household. All individuals reported purchasing gro- ceries off of the Six Nations reserve, which required an average of 5 (SD 2.56) trips per month. The majority of individuals reported that fruits and vegetables were affordable (89%) and available (98%) off of the reserve. Fewer people believed fruits and vegetables were afford- able (76%) and available (67%) on the reserve. Twenty two percent grew their own fruits and vegetables in the summer. Recruitment began in May 2008 and was completed in December 2008. Of the eighty-three participants approached from the previous SHARE-AP ACTION study, 75.9% (n=63) responded and were included in the study. Demographic characteristics of study participants are shown in Table 1. The mean age of participants was 42.3 (SD 10.7) years, with 41% of individuals making an annual income between $30,000 and $60,000. Thirty per- cent of study participants were current smokers. Built environment (NEWS questionnaire) Table 4 summarizes the responses for tobacco availabil- ity and accessibility. The majority of individuals on the Six Nations Reserve reported tobacco use to be a prob- lem in their community (90.5%) and a problem among teenagers (90.5%). Most participants believed that there should be a ban on tobacco advertisement (74.6%) and greater law enforcement on tobacco sales (63.5%). However, only 3.2% of individuals were supportive of increased tobacco taxation. Three quarters of respon- dents indicated that teenagers can easily obtain tobacco from home; a majority believe that adults can easily buy tobacco for teens (87.3%), and teenagers can easily buy tobacco for themselves (66.7%). Table 2 shows the mean and standard deviation of the walkability scores. Participants generally perceived the walkability of the Six Nations environment to be poor. For example, the score reported for walkability to com- munity facilities, street connectivity, walking/cycling fa- cilities, aesthetics, and pedestrian/traffic safety were all lower than the ‘midpoint’ score for each of the NEWS subscale components. Interestingly despite the unfavour- able responses to the built environment, community sat- isfaction was reported to be high. Environmental profile of community health (EPOCH) Environmental profile of community health (EPOCH) Participants were also asked whether they noticed tobacco and anti-tobacco advertisements in their com- munity. (Table 5) Most reported seeing anti-smoking advertisements on television and radio (87.5%). 88.9% of individuals were aware of the laws requiring health warnings on cigarette packages. In terms of nutrition en- vironmental factors, both junk food and healthy food advertisements were observed by study participants. Only 65% were aware of the official dietary guidelines, and 7.9% aware of the laws that lower fruit and vegetable tax (Table 6). to tobacco products was seen as problematic for the community. While most respondents were satisfied living on the Reserve, participants identified the Reserve as being a difficult place in which to walk, having low street con- nectivity, poor aesthetics, and a higher potential for crime and traffic safety concerns. The ratings for neigh- bourhood walkability on the Six Nations reserve are very low in contrast to urban cities in Canada, where street connectivity, aesthetics, access to walking/cycling facilities and safety score above mid-point values on the NEWS questionnaire [29]. The NEWS questionnaire has been used to assess walkability in rural regions also, but not yet in Canada. It may be expected that walkability scores may be lower in rural compared to urban regions, although this is highly context dependent [23,24]. Enhancements to the walkability features of the Reserve may be an important target to increase physical activity among community members. Food availability and affordability Table 3 shows the participant responses for food avail- ability and accessibility. The average individual reported spending $151 (SD $64.4) per week on groceries to feed Table 3 Fruits and vegetables accessibility and availability Fruits & vegetables accessibility and availability N (%) or mean (SD) Average household money spent on Groceries per week $150.66 (64.4) Grocery Shopping done outside reserve 63 (100%) Times per month to go Grocery Shopping 5 (2.56) Grow own Vegetables & Fruits in Summer 14 (22.2%) Fruits & Vegetables available inside the Reserve 42 (66.7%) Fruits & Vegetables affordable inside the Reserve 48 (76.2%) Fruits & Vegetables available outside the Reserve 62 (98.4%) Fruits & Vegetables affordable outside the Reserve 56 (88.9%) Table 3 Fruits and vegetables accessibility and availability Table 1 Six nations participant demographics (N=63) % Female age mean, years (SD) 46 (73.9%) 42.3 (10.7) Individual Income, N (%) <$29,999 16 (27.6%) $30,000 - $59,999 24 (41.4%) >$60,000 18 (31.0%) Smoking Status, N (%) Current 18 (29.5%) Former/Never 43 (70.5%) Table 1 Six nations participant demographics (N=63) % Female age mean, years (SD) 46 (73.9%) 42.3 (10.7) Individual Income, N (%) <$29,999 16 (27.6%) $30,000 - $59,999 24 (41.4%) >$60,000 18 (31.0%) Smoking Status, N (%) Current 18 (29.5%) Former/Never 43 (70.5%) Table 1 Six nations participant demographics (N=63) % Female age mean, years (SD) 46 (73.9%) 42.3 (10.7) Individual Income, N (%) <$29,999 16 (27.6%) $30,000 - $59,999 24 (41.4%) >$60,000 18 (31.0%) Smoking Status, N (%) Current 18 (29.5%) Former/Never 43 (70.5%) Table 1 Six nations participant demographics (N=63) Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 Page 4 of 8 Environmental profile of community health (EPOCH) Participants were also asked whether they noticed tobacco and anti-tobacco advertisements in their com- munity. (Table 5) Most reported seeing anti-smoking advertisements on television and radio (87.5%). 88.9% of individuals were aware of the laws requiring health warnings on cigarette packages. In terms of nutrition en- vironmental factors, both junk food and healthy food advertisements were observed by study participants. Food availability and affordability Table 4 Tobacco pricing and availability factors Tobacco accessibility and availability variable YES % Tobacco Use is a Problem in the Six Nations Community 90.5 There should be a ban on Tobacco Advertising 74.6 There should be more Law Enforcement on Tobacco sales 63.5 Tobacco Use Problem among Teenagers 90.5 Easy to Sneak Tobacco for Teenagers from Home 74.6 Easy for Adults to Buy Tobacco for Teenagers 87.3 Easy for Teenagers to Buy Tobacco 66.7 Willing to Pay More Tax on Tobacco 3.2 Table 6 Nutrition environmental factors predicting daily fruit and vegetable consumption on six nations reserve from EPOCH-II (N=63) Variable YES % Participant Observations Junk Food Advertisements Posters 47.6 TV/Radio 69.8 Newspapers 63.5 Sports/Music 41.3 Healthy Food Advertisements Posters 49.2 TV/Radio 55.6 Newspapers 66.7 Sports/Music 33.3 Participant Awareness Official Dietary Guidelines 65.1 Laws that lower taxes on 7.9 fresh fruits/vegetables Table 4 Tobacco pricing and availability factors Tobacco accessibility and availability variable Table 4 Tobacco pricing and availability factors Tobacco accessibility and availability variable YES % Tobacco Use is a Problem in the Six Nations Community 90.5 There should be a ban on Tobacco Advertising 74.6 There should be more Law Enforcement on Tobacco sales 63.5 Tobacco Use Problem among Teenagers 90.5 Easy to Sneak Tobacco for Teenagers from Home 74.6 Easy for Adults to Buy Tobacco for Teenagers 87.3 Easy for Teenagers to Buy Tobacco 66.7 Willing to Pay More Tax on Tobacco 3.2 Environmental profile of community health (EPOCH) Discussion This study highlights several potential barriers toward adoption of healthy behaviours that exist in the Six Nations community. Specifically the built environment was viewed as being unfavourable to walking, access to healthy foods on the reserve was limited, and easy access y NEWS questionnaire [29]. The NEWS questionnaire has been used to assess walkability in rural regions also, but not yet in Canada. It may be expected that walkability scores may be lower in rural compared to urban regions, although this is highly context dependent [23,24]. Enhancements to the walkability features of the Reserve may be an important target to increase physical activity among community members. All participants purchased their groceries off of the Six Nations reserve, which is likely due to the greater avail- ability and affordability of food. Six Nations respondents travel off of the Reserve more than 5 times per month in order to obtain a variety of food items, and spend ap- proximately $151 per week on groceries alone, in con- trast to the average total food expense of $140 per week in Ontario households [30]. In addition, Aboriginal households in Ontario are reported to have markedly lower household incomes ($46,865) in comparison to the Ontario median ($60,455), suggesting a greater burden of food costs is carried by Aboriginal families [31,32]. Table 5 Tobacco environmental factors Variable YES % Participant Observations Tobacco Advertisements Posters 33.0 TV/Radio 12.0 Newspapers 34.9 Sports/Music 28.6 Anti Smoking Advertisements Posters 58.7 TV/Radio 87.5 Newspapers 63.5 Participant Awareness Support Programs to stop smoking 69.8 Bans of smoking in public 84.1 Bans on smoking advertising 84.1 Laws on health warnings 88.9 Table 5 Tobacco environmental factors Variable YES % Participant Observations Tobacco Advertisements Posters 33.0 TV/Radio 12.0 Newspapers 34.9 Sports/Music 28.6 Anti Smoking Advertisements Posters 58.7 TV/Radio 87.5 Newspapers 63.5 Participant Awareness Support Programs to stop smoking 69.8 Bans of smoking in public 84.1 Bans on smoking advertising 84.1 Laws on health warnings 88.9 Table 5 Tobacco environmental factors Variable YES % Participant Observations Tobacco Advertisements Posters 33.0 TV/Radio 12.0 Newspapers 34.9 Sports/Music 28.6 Anti Smoking Advertisements Posters 58.7 TV/Radio 87.5 Newspapers 63.5 Participant Awareness Support Programs to stop smoking 69.8 Bans of smoking in public 84.1 Bans on smoking advertising 84.1 Laws on health warnings 88.9 Table 5 Tobacco environmental factors Table 5 Tobacco environmental factors The COMMIT randomized control trial of 11 communities in Canada and the United States did not change heavy smoker quit rates, but had a minimally significant influ- ence on light-to-moderate smokers consuming less than 25 cigarettes per day [41]. Prior studies of the association between contextual factors and health behaviours in non-Aboriginal com- munities such as physical activity, dietary intake, BMI, and tobacco use have produced mixed results. A recent systematic review of studies in which community-based interventions were initiated to improve community levels of physical activity reported inconsistent results, and emphasized the need for well-designed intervention studies [33]. Prior studies have reported that food avail- ability and affordability [34] and presence of supermar- kets [35] are associated with healthy food purchases, whereas difficulty accessing produce and high quality groceries promoted the consumption of fast food [36]. A recent systematic review of 28 studies, mostly from the United States showed that greater accessibility to super- markets or less access to takeaway outlets was associated with a lower prevalence of overweight/obesity. However, no strong associations between the food environment and dietary intake were observed, with the exception of area-level deprivation where individuals who lived in low socioeconomic communities had a greater likelihood of having an obesogenic dietary intake [37]. Thus while community perceptions and contextual factors are asso- ciated with weight status, the association between the community environment and health behaviours such as physical activity, dietary intake, and BMI will require lar- ger studies involving more communities [37]. For ex- ample, a fully powered study of the built environment on BMI would require inclusion of 12 Aboriginal com- munities, with at least 50 subjects per community to be able to detect a minimum 0.6 point difference in BMI between high versus low walkable communities (assum- ing an intraclass correlation of 0.09, and alpha of 0.05). Furthermore a recent review of intervention studies con- ducted among reserve dwelling Aboriginal people from the United States identified important components of successful community interventions designed to change health behaviours. Table 5 Tobacco environmental factors All participants purchased their groceries off of the Six Nations reserve, which is likely due to the greater avail- ability and affordability of food. Six Nations respondents travel off of the Reserve more than 5 times per month in order to obtain a variety of food items, and spend ap- proximately $151 per week on groceries alone, in con- trast to the average total food expense of $140 per week in Ontario households [30]. In addition, Aboriginal households in Ontario are reported to have markedly lower household incomes ($46,865) in comparison to the Ontario median ($60,455), suggesting a greater burden of food costs is carried by Aboriginal families [31,32]. Page 5 of 8 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 longevity of the health promotion program. They also highlight the importance of future research studies to study the effectiveness of community- based programs to change health behaviours and outcomes [38]. Overwhelmingly 90% of the community members reported tobacco use as being a problem in the Six Nations community especially among teenagers. Chil- dren and teenagers were perceived to have easy access to tobacco on the Reserve. Most participants are aware of smoking support programs, bans in public, advertise- ment laws, and health warnings on tobacco packaging. While most individuals indicated that bans on tobacco advertisements or increased law enforcement would be acceptable for reducing rates, only 3% were willing to tolerate increased taxation. Current literature regarding effective tobacco-related community interventions in Aboriginal communities is sparse. Our observation that advertisements promoting smoking and easy access to smoking for adults and chil- dren on the Six Nations reserve, together with the previ- ous observation we reported of the high prevalence of cigarette smoking, makes it highly likely that the two characteristics are linked. However data from individual studies vary. For example, a community and school- based intervention among Native American children from 27 elementary schools and 5 states showed no in- fluence of a community-based intervention including classroom lessons, media, and information sessions on rates of cigarette use [39]. In a Cochrane review of com- munity interventions targeting adult smoking by mass media, smoking policy, and smoke-free environments, had no effect on the prevalence of smoking [40]. Table 5 Tobacco environmental factors These successful components include interventions targeted toward multiple levels including households, schools, community food suppliers as well as individuals, engaging and partnering with multiple community stakeholders and community members, and having an institutional base of support to ensure g p y There is strong evidence that increased taxation from a population perspective is associated with lower smok- ing rates, and it seems reasonable to assume that with increased taxation and decreased access to cigarettes, the rates of smoking on the Six Nations reserve would decline. The use of tobacco on the Six Nations reserve is complex. Registered Indians or bands on reserve are ex- empt from sales taxes for goods and services as outlined by section 87 of the Indian Act [42]. Although First Nations can implement taxes on tobacco sales under the First Nations Sales Tax (FNST), only nine communities have chosen to do so [43]. Many First Nations govern- ments are dependent on federal fiscal transfers to ac- count for more than 90 percent of their revenue. While local taxation of cigarettes could serve as a means for al- ternative and stable revenue of the elected Six Nations council, without a clear mechanism to enforce this policy, this strategy for tobacco control is not likely to be successful [44]. Increased price of tobacco is asso- ciated with lower tobacco use (price elasticity) in non- Aboriginal communities where smoking rates have decreased 2-4% for every 10% increase in price [45,46]. Opposition to tobacco taxation among First Nations is partly attributed to the strong historical, cultural, and economic ties to the tobacco industry. Successful tobacco control policies such as those advocated by the World Health Organization’s Framework Convention on Tobacco Control include common features such as Page 6 of 8 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 banning all forms of promotion of tobacco products, price increases through taxation, strong prominent health warnings on packaging, banning sales to children, and enforcement of tobacco policy by law including im- plementation of quotas [47]. Presently on the Six Nations reserve these control policies do not exist – which does not bode well for the future health of this community given that the use of tobacco use among adults, pregnant mothers and children is substantially higher than in most other communities in Canada. factors, may overpower the independent influence of physical environment on behaviours. Table 5 Tobacco environmental factors Inter-household sharing is a common practice in First Nations communi- ties, suggesting the importance of studying the social en- vironment on individual health behaviours [15]. banning all forms of promotion of tobacco products, price increases through taxation, strong prominent health warnings on packaging, banning sales to children, and enforcement of tobacco policy by law including im- plementation of quotas [47]. Presently on the Six Nations reserve these control policies do not exist – which does not bode well for the future health of this community given that the use of tobacco use among adults, pregnant mothers and children is substantially higher than in most other communities in Canada. This study highlights potential areas of interest for fu- ture study of contextual factors and community interven- tions. Our results suggest that interventions to improve reserve walkability, increase healthy food advertisements and nutrition education, and to reduce access and afford- ability of tobacco products may reduce the burden of chronic diseases faced by this community. Efforts should be made to find culturally appropriate community inter- ventions that target health behaviours. Modifications to contextual factors have been attempted by other Aboriginal communities with successful intervention leading to increased access to fitness centers, use of walking trails, development of gardening programs, and improved food stores [53,54]. The World Health Organization and other health ad- vocacy groups cite the aggressive global marketing of risky products and behaviors, particularly those targeting children and youth, as key factors in bringing tobacco, alcohol, and unhealthy processed foods into households worldwide [48]. In our survey, Six Nations community members recognized the excessive advertising of tobacco products and easy access for teenagers. However they did not embrace the idea that taxation may decrease use of tobacco. The community leadership in health must galvanize the community sentiment toward the adverse effects of tobacco on health, and persuade the local gov- ernment to take a tough stand against the easy availabil- ity and access tobacco, especially among children and youth. The future health of Six Nations depends on the community’s involvement and ownership of new initia- tives to target maladaptive health behaviors [48]. Authors’ contribution PJ AB d AM i d PJ, AB and AM assisted with the analysis, interpretation and drafting of the manuscript. HM, participated in data collection and provided comments of the manuscript drafts, CC assisted in the analysis of the EPOCH questionnaire data and provided comments on the manuscript drafts, RM helped to coordinate the study, and provided critical revisions on the manuscript drafts, SA conceived the study, and participated in analysis, interpretation and writing of the manuscript. All authors read and approved the final manuscript. Acknowledgements This study was funded by a pilot grant from the Heart and Stroke Foundation of Ontario (Grant #PEA6547). *Dr. Anand holds the Michael G DeGroote and Heart and Stroke Foundation of Ontario Chair in Population Health, the May Cohen Eli Lilly Endowed Chair in Women’s Health Research, and a Canada Research Chair in Ethnicity and Cardiovascular Disease at McMaster University. Conclusion The present study characterizes the contextual determi- nants of key health behaviours associated with cardio- vascular risk on the Six Nations reserve. This study provides preliminary information to assist in the design, implementation and evaluation of contextual studies and interventions to determine if they are effective in im- proving health behaviours. It is likely that multiple pre- vention strategies including individualized programs as well as community interventions are needed to improve the future health of this community. Limitations The small sample size of this study limited examination of direct associations between community level factors and individual behaviours. The evaluation tools we used may be too narrow to encompass all potential commu- nity level factors which impact on individuals health behaviours, as we relied on participants perception of their environment and not objective measures of the community. Other studies have shown that collection of both perception and objective measures of contextual factors enhances the ability to study influences of envir- onment on health, and future studies should consider in- cluding both types of measures [49-51]. Furthermore our participants likely represented a more health con- scious segment of the community (based on comparison of their higher household income and lower smoking rates to the general population) which may have influ- enced their responses to our questions. The effects of the off-reserve environment on Aboriginal health beha- viours were not considered. In addition, the on-reserve environment is also extensive. The influence of context- ual factors in schools and at the workplace would be relevant for analysis, as well as micro environmental components such as food quality, grocery store set-up, and restaurant menus [52]. 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Ho LS, Gittelsohn J, Rimal R, Treuth MS, Sharma S, Rosecrans A, Harris SB: An integrated multi-institutional diabetes prevention program improves knowledge and healthy food acquisition in Northwestern Ontario First Nations. Health Educ Behav 2008, 35:561–573. 26. Author details 1 1Department of Medicine and Epidemiology, McMaster University, 1280 Main St, Hamilton, Ontario L8S 4K1, Canada. 2Six Nations Health Services, 1745 Chiefswood Road, Ohsweken, ON N0A 1M0, Canada. 3Population Health Research Institute, 237 Barton Street East, Hamilton, ON L8L 2X2, Canada. 4Chanchlani Research Centre, McMaster University, 1280 Main St. W., MDCL Rm. 3204, Hamilton, Ontario L8S 4K1, Canada. Page 7 of 8 Page 7 of 8 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 Joseph et al. BMC Public Health 2012, 12:952 http://www.biomedcentral.com/1471-2458/12/952 References 2006 census. Statistics Canada. Catalogue no. 92-596-XWE.Released December 4, 2007. 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Received: 29 June 2012 Accepted: 31 October 2012 Published: 7 November 2012 Received: 29 June 2012 Accepted: 31 October 2012 Published: 7 November 2012 census-recensement/2006/dp-pd/prof/92-591/index.cfm?Lang=E (accessed January 19, 2012). census-recensement/2006/dp-pd/prof/92-591/index.cfm?Lang=E (accessed January 19, 2012). 20. Saelens BE, Sallis JF, Black JB, Chen D: Neighbourhood-based differences in physical activity: an environmental scale evaluation. Am J Public Health 2003, 93:1552–1558. 20. Saelens BE, Sallis JF, Black JB, Chen D: Neighbourhood-based differences in physical activity: an environmental scale evaluation. Am J Public Health 2003, 93:1552–1558. References Am J Clin Nutr 2011, 93(5):1179S–1183S. 19. Statistics Canada: Six nations (part) 40, Ontario (Code3528037) (table)2006 Community Profiles. 2006 Census. Statistics Canada. Catalogue no. 92-591- XWE. Ottawa. Released March 13, 2007 2007. http://www12.statcan.ca/ Page 8 of 8 Page 8 of 8 Joseph et al. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • 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/W2153187598	https://eprint.ncl.ac.uk/fulltext.aspx?url=208734/62FB4E80-AD59-4F83-9F41-8178D9D79AE8.pdf&pub_id=208734	English	null	Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials	BMC public health	2,013	cc-by	8,292	Bhattarai N, Prevost AT, Wright AJ, Charlton J, Rudisill C, Gulliford MC. Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials. BMC Public Health 2013, 13: 1203. Copyright: © 2013 Bhattarai 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. http://dx.doi.org/10.1186/1471-2458-13-1203 Abstract Background: A diet rich in fruit, vegetables and dietary fibre and low in fat is associated with reduced risk of chronic disease. This review aimed to estimate the effectiveness of interventions to promote healthy diet for primary prevention among participants attending primary care. Methods: A systematic review of trials using individual or cluster randomisation of interventions delivered in primary care to promote dietary change over 12 months in healthy participants free from chronic disease or defined high risk states. Outcomes were change in fruit and vegetable intake, consumption of total fat and fibre and changes in serum cholesterol concentration. Results: Ten studies were included with 12,414 participants. The design and delivery of interventions were diverse with respect to grounding in behavioural theory and intervention intensity. A meta-analysis of three studies showed an increase in fruit consumption of 0.25 (0.01 to 0.49) servings per day, with an increase in vegetable consumption of 0.25 (0.06 to 0.44) serving per day. A further three studies that reported on fruit and vegetable consumption together showed a pooled increment of 0.50 (0.13 to 0.87) servings per day. The pooled effect on consumption of dietary fibre, from four studies, was estimated to be 1.97 (0.43 to 3.52) gm fibre per day. Data from five studies showed a mean decrease in total fat intake of 5.2% of total energy (1.5 to 8.8%). Data from three studies showed a mean decrease in serum cholesterol of 0.10 (−0.19 to 0.00) mmol/L. Conclusion: Presently-reported interventions to promote healthy diet for primary prevention in primary care, which illustrate a diverse range of intervention methods, may yield small beneficial changes in consumption of fruit, vegetables, fibre and fat over 12 months. The present results do not exclude the possibility that more effective intervention strategies might be developed. Keywords: Diet, Health promotion, Primary care, Systematic review, Meta-analysis intake of fruit, and 59% of the recommended intake of vegetables [2]. A higher intake of dietary fibre is associated with lower risk of all-cause mortality [3], as well as lower incidence of colorectal cancer [4] and stroke [5]. The esti- mated mean fibre intake for American adults is 15.9 gram per day, lower than the recommended intake of at least 25–38 gram per day [6]. RESEARCH ARTICLE Open Access * Correspondence: nawaraj.bhattarai@glasgow.ac.uk 1Department of Primary Care and Public Health Sciences, King’s College London, London, UK 3Health Economics and Health Technology Assessment Unit, Institute of Health and Wellbeing, University of Glasgow, 1 Lilybank Gardens, Glasgow G12 8RZ, UK Full list of author information is available at the end of the article Abstract Cardiovascular diseases and dia- betes are associated with obesity and high dietary intakes of fat and sugars [7] but a typical American diet includes 280% of the recommended intake of calories from solid fats and sugars [2]. Obesity imposes a significant burden of morbidity and mortality on populations. The health care costs associated with obesity are substantial and the © 2013 Bhattarai 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. Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials Nawaraj Bhattarai1,3, A Toby Prevost1, Alison J Wright1, Judith Charlton1, Caroline Rudisill2 and Martin C Gulliford1 © 2013 Bhattarai 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. Date deposited: Newcastle University ePrints - eprint.ncl.ac.uk Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Background Comparators included usual care or no intervention. We excluded those trials comparing one type of diet promotion intervention with another only because our aim was to estimate the effect size differ- ence between a diet promotion intervention and the existing usual care or no intervention; we did not aim to compare any two methods of diet promotion interven- tions. A minimum follow- up period of 12 months after randomisation was required. Only English language publi- cations were included. vast majority of the costs are attributable to treating health consequences of obesity including type 2 diabetes, cancer and cardiovascular diseases [8]. There is evidence for the effectiveness of primary care-based interventions to promote physical activity [9], alcohol reduction [10] and smoking cessation [11]. The regularity of patient consultations in primary care [12], and the value that patients place on medical advice [13], offer opportunities for general practitioners to play important roles in promoting health and preventing disease. This may include the provision of advice on healthy eating. Several randomised trials have evaluated the potential to modify patients dietary habits through primary care based interventions. However, earlier system- atic reviews of the effectiveness of dietary interventions for primary prevention are limited in their applicability to primary care by the inclusion of studies set in work places, shopping centres and churches. Some reviews have included non-randomised studies [14] and trials with short follow-up, as well as participants with estab- lished medical conditions, or patients with defined high- risk status [14-17], with the possibility of diet restrictions and which would limit the participation in diet promotion intervention. Recent work on understanding the effectiveness of interventions to increase healthy diet has focused on the importance of using behaviour science theory to understand determinants of behaviour. Use of specific intervention techniques including setting goals, moni- toring behaviour, and reviewing progress towards goals in the light of feedback may be key to dietary behaviour change [18,19]. The effectiveness of behavioural interven- tions may also depend on factors such as the frequency of contacts, the type of professional involved, and whether delivered individually or in a group setting. We report a systematic review and meta-analysis of randomised controlled trials of primary care-based diet promotion interventions for primary prevention in adults with minimum 12 months follow up. Background An increase in intake of fruit and vegetables of one por- tion per day (80 g/day) may be associated with a 10% rela- tive reduction in risk of ischaemic heart disease and 6% reduction in stroke, with between 1% and 6% reduction in risk of certain cancers [1]. However, a typical American diet includes only includes 42% of the recommended daily Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 2 of 14 in primary care, including dietary counselling, motiv- ational interviews, advice for behaviour change, computer- delivered dietary information, reminder telephone calls and postal newsletters. Primary care in this context refers to interventions delivered through the first point of contact in a health care system, where the service provider acts as the principal source of advice to patients, rather than through specialist referral. Dietary promotion intervention in this context means any methods which are used to promote healthy diet, including healthy eating advice and counselling, telephone calls, group lectures or use of any other dietary education materials in- cluding posters, booklets and guidelines. We excluded multifaceted interventions including those with physical activity promotion along with diet promotion and we did not set any threshold amount of physical activity for exclusion. Target populations included the general popula- tion of adults aged 16 years or over, including both men and women. We excluded studies in pregnant women, patients at high risk of, or diagnosed with, cardiovascu- lar diseases, type 2 diabetes, cancer or other chronic conditions, as well as studies in first or second degree relatives of affected individuals. We also excluded studies that included participants at high risk of colorectal cancer (because of adenomatous polyps) [20] or breast cancer (with mammographic abnormalities) [21,22]. In order to focus on a population approach to primary prevention, we excluded trials which included participants who were pregnant, or with existing chronic conditions, or at high risk of diseases such as colorectal or breast cancer, or with participants who were relatives of family members with chronic health problems linked to diet. Such high risk participants or those with established chronic conditions linked with diet, may be more motivated to make dietary behaviour changes which may apparently show higher effectiveness of interventions promoting healthy diet in primary care. Also, there may be possibilities of diet restrictions which may limit the participation in diet promotion intervention and may apparently show lower effectiveness. Background The aim was to quantify whether diet promotion for primary prevention in primary care is effective in sustained dietary modifi- cations over at least one year. We also aimed to charac- terise existing interventions in terms of their theoretical basis and intervention techniques employed, and explore whether these were related to intervention effectiveness. Eligibility criteria The review included reports of randomised or cluster controlled trial study designs. Outcome measures included fruit and vegetable intake (servings/day), fat (% of total energy intake), fibre consumption (gram per day) and change in serum cholesterol level (mg/dl or mmol/l). Interventions included any diet promotion intervention Methodological quality assessment Methodological quality assessment NB appraised each study for methodological quality using a standard guidance and checklist [25]. We assessed the methodological quality and risk of bias in terms of ran- domisation, allocation concealment, blinding, loss to fol- low up and outcome assessment tool validity. Search methods, study selection and data extraction search terms “dietary intervention AND primary care”, “diet promotion intervention AND primary care”, “diet advice AND primary Care”, “counselling AND diet AND primary care”, “diet promotion AND primary care”, “diet advice AND behaviour change”, “advice in primary care AND behaviour AND diet”, “nutritional counselling AND primary care”, “lifestyle counselling AND cardiovascular risk AND primary care”, “fruit AND vegetable AND primary care”, “nutritional counselling AND general practice”, “dietary intervention AND general practice”, “dietary intervention AND primary care practice”, “nutritional advice AND general practice”, “primary care and diet modification”, “fruit and vegetables consumption AND general practice” “primary care AND fiber consump- tion” and “fruit and vegetable consumption AND diet intervention”. We also reviewed reference lists of relevant articles and previous systematic reviews. The search was carried out initially in September 2012 and again in March 2013. NB carried out initial screening of title and abstracts against inclusion criteria and retrieved those potentially eligible. NB and MCG independently assessed the re- trieved full text articles and any differences were reviewed and agreed. NB extracted data concerning participants, interventions, and outcomes in a tabular form designed for this review. AJW and NB extracted data on the nature of each intervention, including total number of contacts with participants, mode(s) of administration and intervention techniques used [23] and coded the extent to which intervention was based on psychological theories of the determinants of behaviour change, using a published coding scheme [24]. NB and MCG cross checked the extracted data. We used random effects meta-analysis to pool the estimates from individual studies. We used the I2 stat- istic to describe the variation in effect size attributable to heterogeneity among studies; higher values suggest- ing greater heterogeneity. We also constructed funnel plots to assess for the publication bias for the studies included in the review. We used the metan command in STATA version 12 for the analysis. Search methods, study selection and data extraction We searched Medline, PsycINFO, EMBASE, Centre for Reviews and Dissemination, and the Cochrane Library, with no restrictions in the date and year, using the combined Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 3 of 14 in mg/dl, the conversion factor of 38.6598 was used to express them as mmol/L. search terms “dietary intervention AND primary care”, “diet promotion intervention AND primary care”, “diet advice AND primary Care”, “counselling AND diet AND primary care”, “diet promotion AND primary care”, “diet advice AND behaviour change”, “advice in primary care AND behaviour AND diet”, “nutritional counselling AND primary care”, “lifestyle counselling AND cardiovascular risk AND primary care”, “fruit AND vegetable AND primary care”, “nutritional counselling AND general practice”, “dietary intervention AND general practice”, “dietary intervention AND primary care practice”, “nutritional advice AND general practice”, “primary care and diet modification”, “fruit and vegetables consumption AND general practice” “primary care AND fiber consump- tion” and “fruit and vegetable consumption AND diet intervention”. We also reviewed reference lists of relevant articles and previous systematic reviews. The search was carried out initially in September 2012 and again in March 2013. NB carried out initial screening of title and abstracts against inclusion criteria and retrieved those potentially eligible. NB and MCG independently assessed the re- trieved full text articles and any differences were reviewed and agreed. NB extracted data concerning participants, interventions, and outcomes in a tabular form designed for this review. AJW and NB extracted data on the nature of each intervention, including total number of contacts with participants, mode(s) of administration and intervention techniques used [23] and coded the extent to which intervention was based on psychological theories of the determinants of behaviour change, using a published coding scheme [24]. NB and MCG cross checked the extracted data. Identification of trials We screened titles and abstracts of 2,932 papers and identified 49 full text articles. We further identified 3 full text articles from cross-checking references of 49 full text articles. We then identified 10 trials [27-36] for inclusion in this study after excluding trials not meeting the eligibility criteria. The details are shown in the flow diagram (Figure 1). Study and participants characteristics We present the study and participant characteristics in Table 1. Ten studies included in the systematic review were published between 1988 and 2006. These studies were conducted in samples representing the primary care general population in Japan [27] (1 study), USA [28,31,32,34-36] (6 studies), Italy [30] (1 study) and UK [29,33] (2 studies).The randomised study sample size varied among studies and ranged from 213 to 3,179 participants with 12,414 participants randomised in total. Participants were men and women, but three studies [28,31,36] included women only, with the age ranging from 18 to 79 years. Participants were generally healthy without established chronic diseases. Participants in one study [28] were postmenopausal women consuming at least 36% energy from fat; participants in another study [31] had serum cholesterol values of 200 mg/dl (5.17 mmol/l) or more but as this is close to the population mean value, this study was not excluded as being directed at high risk individuals. We present the study and participant characteristics in Table 1. Ten studies included in the systematic review were published between 1988 and 2006. These studies were conducted in samples representing the primary care general population in Japan [27] (1 study), USA [28,31,32,34-36] (6 studies), Italy [30] (1 study) and UK [29,33] (2 studies).The randomised study sample size varied among studies and ranged from 213 to 3,179 participants with 12,414 participants randomised in total. Participants were men and women, but three studies [28,31,36] included women only, with the age ranging from 18 to 79 years. Participants were generally healthy without established chronic diseases. Participants in one study [28] were postmenopausal women consuming at least 36% energy from fat; participants in another study [31] had serum cholesterol values of 200 mg/dl (5.17 mmol/l) or more but as this is close to the population mean value, this study was not excluded as being directed at high risk individuals. Intervention and control characteristics For each trial, we extracted the intervention effects at 12 months. We estimated the intervention effect as the difference in the change in mean outcome values (follow up value minus baseline value) between the intervention group and control group. If the baseline data were not reported in the trials, we used the difference in the mean outcomes between groups at follow up. If not supplied, we followed standard procedure [26] to derive standard error (SE) for each measure. Where fruit and vegetable consumption was expressed in grams, the conversion factor of one serving = 80 gm was used to express them as servings and where the serum cholesterol was expressed The diet promotion interventions in the trials varied in number of contacts with participants, mode of delivery and behaviour change techniques employed (Table 2). The number of scheduled contacts for intervention with the participants in the intervention groups ranged between one and twenty. Most involved at least one face to face contact, but two [32,34] involved only a combination of telephone calls and mailed intervention materials. Of the interventions using face-to-face sessions, only one [28] was solely delivered in a group format, while the others used a combination of group and individual contacts. Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 4 of 14 Figure 1 Study selection flow diagram. Figure 1 Study selection flow diagram. Many of the interventions also involve printed mate- rials. The interventions involved between two and eight intervention techniques. Interventions that involved a greater number of contacts with participants did not necessarily employ a greater number of techniques. In each study, the control group was not enrolled in any intervention, but four had minimal interventions: dietary guidelines [28], standard health education from leaflets [29], a non-personalised conversation without diet counselling [30] and breast self-examination counselling [31] (Table 2). Four [31,32,34,35] of the ten interventions were expli- citly described as being based on at least one psycho- logical theory of behaviour change. While none of the four reports explicitly linked all components of the inter- vention to all the relevant constructs of the theoretical model(s) upon which they claimed to be based, they all explicitly linked at least one intervention technique to at least one determinant of behaviour specified by relevant psychological theory. Intervention and control characteristics In three [31,32,34] of the interventions, the intervention was tailored for partici- pants according to how they varied on a psychological construct specified by a theory. In each trial, previously validated self-administered food frequency questionnaires, or modified simpler versions, were used to measure the study outcomes. Diet intakes were estimated using the average of the diet consump- tion in the previous 24 hours to 1 month, collected using food frequency questionnaires. No on 8 2 oval our) on of theory techniques CT tech used 0 2 theory Methodological quality of included studies The funnel plots and Egger’s test for potential publication bias were not informative as insufficient studies were identified for each outcome. Table 3 presents a summary Number of practices Participants randomised (% Female) Eligible age range (mean) years Ethnicity and socioeconomic status Diet assessment tool One group general practice 437 randomised 368 participated (49) 25-60 (41.7) Social class 1 or 2: controls, 30% men, 24% women; intervention 39% men, 43% women. Self-administered food frequency questionnaire 28 physician practices within 6 clinics 2121 (68) 26% > 65 years White: 91%; Some college education: 73%. Family income below $25000 per year: 28%. Telephone interview administered food frequency questionnaire University clinical centres in three states 2208 (100) 50-79 (60) White (55%), Black (28%), Hispanic (16%); <High School (11%), High School (20%), Post high school with no college degree (35%), graduate /post graduate (33%) Self-administered food frequency questionnaire Three rural Virginia physician practices 754 (64) 18-72 (46.34) White: 61%, African American: 37%; 8th grade: College degree: 24%; Income < $10,000: 14.69%, ≥$41,000:19%. Telephone interview administered fat and fibre behaviour questionnaire One clinic 213 (100) 20-40 (33.4) 76% White, 13.5% Black, 4% Hispanic, 5.5% Asian, 1.5% other; 85% completed college Telephone interview administered food frequency questionnaire, based on 24 hr diet recall on each of three days Health maintenance organisation 1459 (50) 18-69 (45.8) White (85.9%), Black (4.5%), Asian (5.8%), Hispanic (3.0%), Other (0.8%); Household income < $25,000 12.2%, ≥$70,000 21.7%. Telephone interview administered Food Frequency Questionnaire (FFQ) and Diet Habits Questionnaire 8 family practices 956 (50) 35-59 (47.3) Non-manual occupation, intervention 60%, control 49%; rented accommodation intervention 11%, control 25%. Self-administered food frequency questionnaire. 33 general practitioners 3179 (50) 18-65 (44.5) Not reported Family physician administered food frequency questionnaire Health maintenance organization (HMO) 616 (100) 40-70 (53.8) Minority groups: 7%; College graduates: 40% Self-administered fat and fibre behaviour questionnaire (FFBQ) Not reported 550 (68) 40-69 (56) Not reported Self-administered diet history questionnaire (DHQ) aire. aire and t omote dietary proving health, ging social norm her fibre foods, ng positive for n of booklet taking 2 weeks later, a ed by physician ants who had ntion. theory consequences of behaviour usual care 3. provide information regarding others’ approval 5. goal setting (behaviour) 8. Barrier identification and problem solving? 9. Set graded tasks 19. Provide feedback on performance 21. provide information on how to do the behaviour 27. sed g, Yes – stage change from the TTM nt on effort/ owards r and on l behaviour pt self monitoring de information o perform the 8 s – stage ange from e TTM Methodological quality of included studies Use of follow up prompts sessions in groups 6 weeks, bi-weekly y for 9 months and 18 months. Group periences. None N/A 5. goal setting – behaviour 8 2 Not counselled, but given Dietary Guidelines for Americans 8. problem solving 12. prompt rewards contingent on effort/ success towards behaviour and on successful behaviour 16. prompt self monitoring 21. provide information on how to perform the behaviour 22. model/demonstrate the behaviour 26. prompt practice 29. plan social support usual care p behaviour 22. model/demonstrate the behaviour 26. prompt practice 29 plan social support 21. provide information on how to perform the behaviour 2 22. model/demonstrate the behaviour No intervention until after end of study ention) tting – behaviour 8 m solving pt rewards nt on effort/ owards r and on l behaviour pt self monitoring de information o perform the without the use of a brochure. No dietary advice, however advised on Breast Self Examination(BSE) e y ed iew . on, nd he Non rint ng ed d e Soc the Non onsistent w 4 techn p rmation sequences ur 7 2 Usual care (No intervention) – behaviour asks edback on on how to ehaviour monstrate (?) w-up - behaviour 3 or 4* 2 or 3 No intervention until after end of study behaviour 22. model/demonstrate the behaviour 26. prompt practice 29 plan social support 21. provide information on how to perform the behaviour 2 22. model/demonstrate the behaviour N/A new onstr ons in ce of ventio elp m h be Standard health education leaflet, Guide to healthy eating 10 prompt review of behavioural goals 16. or 17.(For some) self- monitoring – not quite clear if this was of the behaviour or of weight. 21. instruction on how to perform the behaviour 1. provide information about the consequences of behaviour 2 0 A simpler and non personalized conversation without the use of a brochure. 21. provide instruction on how to perform the behaviour ersonal self and 5. goal setting – behaviour 7 3 No dietary advice, however advised on Breast Self Examination(BSE) 8. barrier identification and problem solving 9. set graded tasks 10. prompt review of behavioural goals 19. provide feedback on performance 21. provide instruction on how to perform the behaviour 37. motivational interviewing 5. goal setting – behaviour 3 2 No intervention 19. provide feedback on performance 21. provide instruction on how to do the behaviour e following four intervention techniques: prompt specific goal setting, prompt review of behavioural s overweight, otherwise three techniques. http://www.biomedcentral.com/1471 2458/13/1203 p p p Standard health education leaflet, Guide to healthy eating review of l goals or some) self- – not quite was of the or of weight. ion on how to e behaviour nformation consequences ur 2 0 A simpler and non personalized conversation without the use of a brochure. e instruction on rform the ng – behaviour 7 3 No dietary advice however about the consequences of behaviour non personalized conversation without the use of a brochure. 21. provide instruction on how to perform the behaviour cognitive TTM Yes – personal barriers, self efficacy and stage of change 5. goal setting – behaviour 7 3 No dietary advice, however advised on Breast Self Examination(BSE) 8. barrier identification and problem solving 9. set graded tasks 10. prompt review of behavioural goals 19. provide feedback on performance 21. provide instruction on how to perform the behaviour 37. motivational interviewing N/A 5. goal setting – behaviour 3 2 No intervention 19. provide feedback on performance 21. provide instruction on how to do the behaviour control theory (out of the following four intervention techniques: prompt specific goal setting, prompt review of behavioural es used if participant was overweight, otherwise three techniques. without the use of a brochure. No dietary advice, however advised on Breast Self Examination(BSE) e y ed iew . n, nd he Non rint ng ed d e Soc theo Non onsistent w *4 techn men d ding Participation at 12 months Outcome assessm validity reported l. Nurse assessment was blinded 448/550 (81%) Yes stated 1,141/2,208 (52%) Yes stated Intervention 86%; control 74% Yes ome assessors and participants d to be blinded 2,977/3,179 (93%) Yes al. Clinic staff conducting data ction were blinded Intervention 89%; control 85% Yes stated 1,205/1,459 (83%) Partial stated 329/368 (89%) Not stated stated 516/754 (68%) Yes 1,818/2,121 (86%) Yes stated 177/213 (83%) Yes Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 10 of 14 Vegetable consumption was increased by 0.25 (0.06 to 0.44, p = 0.01) servings per day, with less evidence of hetero- geneity among studies was I2 = 58.4%, p = 0.091 (Figure 2). Three studies [30-32] reporting intervention effects on consumption of fruit and vegetable combined together showed a pooled effect of 0.50 (0.13 to 0.87, 0.008) servings per day. p There was evidence of substantial heterogeneity (I2 = 90.5%; p < 0.001) (Figure 2). The sum- mary measure had a conservatively wide 95% confidence interval (0.13 to 0.87) for the pooled effect by use of the random effects model. of the methodological quality of included studies. In general, description of randomisation method, blinding of the outcome assessment and allocation concealment were poor in the trials. None of the studies reported what steps were taken to ensure intervention fidelity [37]. Only one study assessed the psychological con- structs targeted by the intervention, and briefly reported a mediation analysis to see if the intervention worked by changing the targeted beliefs. Therefore, the ability of the studies to test the psychological mechanisms of the interventions’ effects on dietary intakes was seriously compromised [24]. Intervention effect on fibre intake Six studies [27,29,33-36] reported the intervention effect on fibre intake, but two [34,35] of these studies were not included in the meta-analysis. The estimated inter- vention effects in the study [35] reporting the fibre intake in grams per 1000 kcal was 0.32 (SE 0.19) grams per 1000 kcal, whereas in the study [34] reporting the fibre intake score, the estimated intervention effect was −0.04 (SE 0.04), with a negative score indicating increased Fruit and vegetable intake Pooled analysis of three studies [27-29], which reported intervention effects on consumption of fruits and vege- tables separately, showed a mean difference of 0.25 (95% confidence interval (CI) 0.01 to 0.49, p = 0.04) serving per day for fruit consumption. There was evidence of ap- preciable heterogeneity (I2 = 88.0%, p < 0.001) (Figure 2). Figure 2 Individual study and pooled effects of diet promotion on intakes of fruit, vegetables, and combined fruit and vegetable at 12 months. ES = effect size, 95% CI = 95% confidence intervals. Figure 2 Individual study and pooled effects of diet promotion on intakes of fruit, vegetables, and combined fruit and vegetable at 12 months. ES = effect size, 95% CI = 95% confidence intervals. Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 11 of 14 0.19 mmol/L, p = 0.049) more mean decrease in serum cholesterol level in people receiving the diet promotion intervention compared to the comparators (heterogeneity among studies I2 = 3%, p = 0.36) (Figure 3C). fibre intake. Pooled analysis of the remaining four studies showed evidence of increase of 1.97 (0.43 to 3.52, p = 0.012) grams of fibre consumption per day. There was some evi- dence of heterogeneity (I2 = 70.4%, p = 0.017) (Figure 3A). The impact of intervention characteristics on observed effects A descriptive analysis was implemented because the small number of included studies meant that it was not possible to conduct meta-regression analyses to formally test the impact of intervention characteristics on observed effect sizes. For fruit and vegetable intake, assessed either singly or in combination, and for serum cholesterol, effect sizes did not clearly increase with increasing participant contact. In contrast, for fibre and fat intake, the interventions with the most contacts clearly had the largest effects. None of the interventions that contributed data to pooled effect size estimates for fruit, vegetable or fibre intake were based on theory. However, for fruit and vegetable consumption con- sidered together, interventions based on theory appeared to have larger effects than those not based on theory. In contrast, the most effective interventions for reducing fat intake were not based on theory, nor was there any clear benefit of using theory for reducing serum choles- terol. Fruit and vegetable intake There was no clear relationship between the total number of intervention techniques used in an interven- tion and the effect sizes observed. Intervention effects on fat intake Seven studies reported the effects of intervention on fat intake, but two [32,34] of these studies were not included in the meta-analysis. The estimated intervention effect in one [32] of these studies was −0.1(SE0.02) and in the other [34] study was −0.06 (SE 0.041), both expressed in scores scales with negative score indicating decreased fat intake. Pooled analysis from five studies [28,29,31,35,36] showed a mean decrease of 5.16% (95% CI −8.81 to −1.52, p = 0.005) in fat intake, expressed as percentage of total energy intake per day. There was heterogeneity between studies (I2 = 98.8%, p < 0.001) with one study [28] show- ing a very large change (Figure 3B). Intervention effects on cholesterol Dietary cholesterol intake was evaluated in one study [28] with a 79.2 mg (95% CI 61.9 to 96.5) more decrease in dietary cholesterol per day when compared to those not receiving the diet promotion intervention. Three studies [29,31,33] analysed the intervention effects in the serum cholesterol level. There was a 0.10 mmol/L (0 to Figure 3 Individual study and pooled effects of diet promotion on fiber and fat intake and serum cholesterol level at 12 months. ES = effect size, 95%CI = 95% confidence intervals. Figure 3 Individual study and pooled effects of diet promotion on fiber and fat intake and serum cholesterol level at 12 months. ES = effect size, 95%CI = 95% confidence intervals. Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 12 of 14 Page 12 of 14 assessors and trial personnel may have introduced bias in trials thereby overestimating the effectiveness of interven- tion. We restricted the inclusion of trials investigating the effects of multi-factorial interventions to avoid the potential confounding effect of other health promoting interventions. However, assessing effectiveness of only diet promotion interventions may have overestimated the effects in practice where diet promotion may run simultaneously with other health promotion activities. Where reported, most of the trial participants were white and had some college education, while few reported socio- economic status. We cannot confirm whether our results can be applied to populations with different ethnic, edu- cational and socio-economic characteristics. We do not know whether it is cost-effective to implement diet pro- motion interventions with similar intensity as reported in the trials in the primary care general population. Par- ticipants with unknown previous exposures to health campaigns and media may increase the selection bias. We may have missed some unpublished or published trials. Discussion The results of this systematic review of randomised con- trolled trials suggest that moderately sustained but small effects on diet can be achieved through diet promotion interventions in primary care. The heterogeneity in observed effect sizes suggests that these interventions, despite all being delivered or deliverable in primary care, varied a great deal in their impact on behaviour. The studies employed a range of behaviour change techniques in- cluding one-to-one counselling along with variety of media with variable theoretical under-pinning. Incomplete report- ing makes it difficult to establish how interventions were intended to achieve their effects. No study reported monitoring treatment fidelity, so it is unclear whether interventions were delivered as their designers planned. If interventions failed to change behaviour, was this because the components of the intervention were not effective? or because these were not successfully delivered? Comparison with other studies A Cochrane systematic review [17] reported that diet promotion intervention in health care setting increased fruit and vegetable intake by 1.88 (95% CI1.07 to 2.70) servings per day; and irrespective of setting fruit intake alone increased by 0.67 (95% CI 0.007 to 1.28) servings per day and vegetable intake alone by 0.92 (95% CI0.34 to 1.49) servings per day, fibre intake increased by 6.51 (95% CI2.20 to 10.82) grams per day, in health care setting total dietary fat intake expressed as percentage of total calories fell by 5.38% (95% CI −7.84 to −2.92) and total blood cholesterol level reduced by 0.11 mmol/l (95% CI – 0.19 to −0.03). Effect sizes were generally con- siderably smaller in our study compared to the Cochrane review. The Cochrane review included studies with partic- ipants with chronic conditions, as well as participants at high risk of colorectal cancer and breast cancer. Studies were carried out in faith and work settings as well as in primary care. These differences in participant characteris- tics, resulting from differences in inclusion/exclusion cri- teria between our study and Cochrane review may explain these differences in results. Another review [16] published in 1998, reported 5.5% reduction in total blood cholesterol level; expressed in a different measurement unit than our study and we could not compare the changes in blood cholesterol with our results. We acknowledge several limitations. Most of the included trials used self-reported measures of dietary change, and there are possibilities that intervention effects may have suffered responder bias. Furthermore, we cannot rule out the possibility of contamination in open trials which could have resulted in the exchange of information on diet mod- ifications and may have modified outcomes in controls. Dietary interventions cannot be completely blinded; how- ever, outcome assessors can be blinded to avoid the chance of selection bias in trials. Not blinding outcome Strengths and limitations Our review has several strengths compared with earlier reviews [14-17]. We estimated the long-term effective- ness of dietary interventions in primary care using only randomised controlled trials or cluster randomised trials with at least 12 months follow-up, where interventions were delivered mostly by health care professionals. In order to focus on a population approach to primary pre- vention, we did not include trials which included partic- ipants with existing chronic conditions or at high risk of diseases such as colorectal or breast cancer and partici- pants with relatives or family members with chronic health problems linked to diet. We did not include trials implemented in the workplace or faith settings where characteristics of participants may be different from those attending the primary health care. We did not restrict the inclusion of trials on the basis of percentage loss to follow up which limited the evidence to trials with more adherent participants and we included two trials [29,34] in our review which were not included in the Cochrane review. The restriction of studies with more than 20% loss to follow up may introduce bias in the findings limited to trials with more adherent participants. We also explored the existing interventions in terms of behaviour change techniques used. Author details 1 1Department of Primary Care and Public Health Sciences, King’s College London, London, UK. 2Department of Social Policy, London School of Economics and Political Science, London, UK. 3Health Economics and Health Technology Assessment Unit, Institute of Health and Wellbeing, University of Glasgow, 1 Lilybank Gardens, Glasgow G12 8RZ, UK. 21. Boyd NF, Martin LJ, Beaton M, Cousins M, Kriukov V: Long-term effects of participation in a randomized trial of a low-fat, high-carbohydrate diet. Cancer Epidemiol Biomarkers Prev 1996, 5(3):217–222. 22. Lee-Han H, Cousins M, Beaton M, McGuire V, Kriukov V, Chipman M, Boyd N: Compliance in a randomized clinical trial of dietary fat reduction in patients with breast dysplasia. Am J Clin Nutr 1988, 48(3):575–586. Authors’ contributions MCG conceived the study. All authors contributed to the design of the study. NB carried out the search, extracted the data, performed the analysis and prepared the first draft. AJW and NB extracted the data in terms of the behavioural theories and coded the interventions. MCG and NB cross checked the extracted data and analysis. All authors contributed to the revisions, read and approved the final manuscript. 14. Ammerman AS, Lindquist CH, Lohr KN, Hersey J: The efficacy of behavioral interventions to modify dietary fat and fruit and vegetable intake: a review of the evidence. Prev Med 2002, 35(1):25–41. 14. Ammerman AS, Lindquist CH, Lohr KN, Hersey J: The efficacy of behavioral interventions to modify dietary fat and fruit and vegetable intake: a review of the evidence. Prev Med 2002, 35(1):25–41. 15. Pomerleau JL K, Knai C, McKee M: Interventions designed to increase adult fruit and vegetable intake can be effective- a systematic review of literature. J Nutr 2005, 135:2486–2495. 16. Tang JL, Smith GD, Armitage JM, Lancaster T, Silagy CA, Fowler GH, Neil HAW, Ebrahim S: Systematic review of dietary intervention trials to lower blood total cholesterol in free-living subjectsCommentary: Dietary change, cholesterol reduction, and the public health—what does meta-analysis add? Bmj 1998, 316(7139):1213–1220. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. 12. Royal College of General Practitioners: Healthy people: promoting health and preventing disease. London: Royal College of General Practitioners; 2009. 13. Boyce T, Peckham S, Hann A, Trenholm S: A proactive approach. Health promotion and ill health prevention. London: The King’s Fund; 2010. Acknowledgements This study was supported by the UK National Prevention Research Initiative whose funding partners include the Alzheimer’s Research Trust; Alzheimer’s Society; Biotechnology and Biological Sciences Research Council; British Heart Foundation; Cancer Research UK; Chief Scientist Office, Scottish Government Health Directorate; Department of Health; Diabetes UK; Economic and Social Research Council; Engineering and Physical Sciences Research Council; Health & Social Care Research & Development Office for Northern Ireland; Medical Research Council; The Stroke Association; Welsh Assembly Government; and World Cancer Research Fund. 17. Rees K, Dyakova M, Ward K, Thorogood M, Brunner E: Dietary advice for reducing cardiovascular risk. Cochrane Database Syst Rev 2013. Mar 28(3): CD002128. doi:10.1002/14651858.CD002128.pub4. 18. Michie S, Abraham C, Whittington C, McAteer J, Gupta S: Effective techniques in healthy eating and physical activity interventions: a meta-regression. Health Psychol 2009, 28(6):690–701. This research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. 19. Dombrowski SU, Sniehotta FF, Avenell A, Johnston M, MacLennan G, Araújo-Soares V: Identifying active ingredients in complex behavioural interventions for obese adults with obesity-related co-morbidities or additional risk factors for co-morbidities: a systematic review. Health Psychol Rev 2010, 6(1):7–32. 20. Smith-Warner SA, Elmer PJ, Tharp TM, Fosdick L, Randall B, Gross M, Wood J, Potter JD: Increasing vegetable and fruit intake: randomized intervention and monitoring in an at-risk population. Cancer Epidemiol Biomarkers Prev 2000, 9(3):307–317. Conclusions 9. Orrow G, Kinmonth A-L, Sanderson S, Sutton S: Effectiveness of physical activity promotion based in primary care: systematic review and meta-analysis of randomised controlled trials. BMJ 2012, 344:e1389. 10. Kaner EF, Beyer F, Dickinson HO, Pienaar E, Campbell F, Schlesinger C, Heather N, Saunders J, Burnand B: Effectiveness of brief alcohol interventions in primary care populations. Cochrane Database Syst Rev. 2007 Apr 18(2): CD004148. doi: 10.1002/14651858.CD004148.pub3. 10. Kaner EF, Beyer F, Dickinson HO, Pienaar E, Campbell F, Schlesinger C, Heather N, Saunders J, Burnand B: Effectiveness of brief alcohol interventions in primary care populations. Cochrane Database Syst Rev. 2007 Apr 18(2): CD004148. doi: 10.1002/14651858.CD004148.pub3. 11. Stead LF, Bergson G, Lancaster T: Physician advice for smoking cessation. Cochrane Database Syst Rev 2008, 16(2):CD000165. 11. Stead LF, Bergson G, Lancaster T: Physician advice for smoking cessation. Cochrane Database Syst Rev 2008, 16(2):CD000165. 11. Stead LF, Bergson G, Lancaster T: Physician advice for smoking cessation. Cochrane Database Syst Rev 2008, 16(2):CD000165. 12. Royal College of General Practitioners: Healthy people: promoting health and preventing disease. London: Royal College of General Practitioners; 2009. 13. Boyce T, Peckham S, Hann A, Trenholm S: A proactive approach. Health promotion and ill health prevention. London: The King’s Fund; 2010. 14. Ammerman AS, Lindquist CH, Lohr KN, Hersey J: The efficacy of behavioral inter entions to modif dietar fat and fr it and egetable intake a re ie Conclusions Our review suggests that diet promotion interventions in the primary care population yield modest positive effects on diet intake over one year. Opportunities to promote dietary change may be taken by general practice based health care staff during patient general practice Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 13 of 14 visits. Our findings should be interpreted with caution because of some of the limitations of the review and the included studies. The present studies may offer limited potential to inform the science of dietary behaviour change in primary care. For example, it is unclear whether inter- ventions are ineffective or whether these are difficult to deliver in routine practice, possibly requiring greater training to deliver consistently. Our findings raise ques- tions concerning whether a brief single diet counselling can be effective as more intensive diet promotion inter- ventions; whether diet promotion interventions will have the same effect in minority ethnic groups and in a deprived general population; and whether the limited effects sizes are of long-term benefit in relation to resources used. 5. 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https://openalex.org/W2559304157	https://respiratory-research.biomedcentral.com/track/pdf/10.1186/s12931-016-0475-8	English	null	Pleural inhibition of the caspase-1/IL-1β pathway diminishes profibrotic lung toxicity of bleomycin	Respiratory research	2,016	cc-by	8,395	© The Author(s). 2016 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. Pleural inhibition of the caspase-1/IL-1β pathway diminishes profibrotic lung toxicity of bleomycin Olivier Burgy1, Pierre-Simon Bellaye1, Sebastien Causse1, Guillaume Beltramo1,2, Guillaume Wettstein1, Pierre-Marie Boutanquoi1, Françoise Goirand1, Carmen Garrido1,3 and Philippe Bonniaud1,2* Abstract Background: Idiopathic and toxic pulmonary fibrosis are severe diseases starting classically in the subpleural area of the lung. It has recently been suggested that pleural mesothelial cells acquire a myofibroblast phenotype under fibrotic conditions induced by TGF-β1 or bleomycin. The importance and role of inflammation in fibrogenesis are still controversial. In this work, we explored the role of IL-1β/caspase-1 signaling in bleomycin lung toxicity and in pleural mesothelial cell transformation. Methods: C57BL/6 mice were intravenously injected with either bleomycin or nigericin or NaCl as control. In vitro, the Met5A cell line was used as a model of human pleural mesothelial cells. Results: Intravenous injections of bleomycin induced lung fibrosis with histologically-proven peripheral distribution, collagen accumulation in the pleural and subpleural area, and overexpression of markers of myofibroblast transformation of pleural cells which migrated into the lung. These events were associated with an inflammatory process with an increase in neutrophil recruitment in pleural lavage fluid and increased caspase-1 activity. TGF-β1 was also overexpressed in pleural lavage fluid and was produced by pleural cells following intravenous bleomycin. In this model, local pleural inhibition of IL-1β with the IL-1β inhibitor anakinra diminished TGF-β1 and collagen accumulation. In vitro, caspase-1 inhibition interfered with Met5A cell transformation into the myofibroblast-like phenotype induced by bleomycin or TGF-β1. Moreover, nigericin, a caspase-1 activator, triggered transformation of Met5A cells and its intra-pleural delivery induced fibrogenesis in mice. Conclusions: We demonstrated, after intravenous bleomycin injection in mice, the role of the pleura and highlighted the key role of IL-1β/caspase-1 axis in this fibrogenesis process. Keywords: Idiopathic Pulmonary Fibrosis, Bleomycin, Pleural cells, Caspase-1, TGF-β1 Keywords: Idiopathic Pulmonary Fibrosis, Bleomycin, Pleural cells, Caspase-1, TGF-β1 only a slight impact on disease progression [1–3]. IPF typically starts from subpleural areas of the lung and then progresses deeper towards the lung parenchyma, where it disturbs alveolar architecture and gas exchange. Bleomycin (BLM) is an effective chemotherapeutic agent widely used intravenously in humans mainly in patients with Hodgkin’s lymphoma. However, the pulmonary toxicity of BLM has restricted its use in clinical practice (www.pneumotox.com). Burgy et al. Respiratory Research (2016) 17:162 DOI 10.1186/s12931-016-0475-8 Burgy et al. Respiratory Research (2016) 17:162 DOI 10.1186/s12931-016-0475-8 * Correspondence: philippe.bonniaud@chu-dijon.fr 1INSERM, LNC UMR866, LipSTIC LabEx team, Université Bourgogne Franche-Comté, 21000 Dijon, France 2Service de Pneumologie et Soins Intensifs Respiratoires, Centre Hospitalo-Universitaire de Bourgogne, 21000 Dijon, France Full list of author information is available at the end of the article Background Idiopathic Pulmonary Fibrosis (IPF) is a devastating disease characterized by matrix accumulation in the lung leading to death with a mean survival of three to 5 years after diagnosis [1]. To date, the causes of IPF remain elusive and the therapeutic options available to patients are limited, with only two drugs recently approved, pirfe- nidone and nintedanib, both of which have demonstrated * Correspondence: philippe.bonniaud@chu-dijon.fr 1INSERM, LNC UMR866, LipSTIC LabEx team, Université Bourgogne Franche-Comté, 21000 Dijon, France 2Service de Pneumologie et Soins Intensifs Respiratoires, Centre Hospitalo-Universitaire de Bourgogne, 21000 Dijon, France Full list of author information is available at the end of the article The role of mesothelial cells in animal models of pul- monary fibrosis and in human IPF has recently been reported [4–11]. In these diseases, they differentiate into myofibroblast-like cells via a cellular mechanism Burgy et al. Respiratory Research (2016) 17:162 Page 2 of 10 Page 2 of 10 lavage fluid (BALF) and pleural lavage fluid (PLF) were then collected as previously described [5]. For histological analysis, lungs were harvested, inflated and fixed in for- malin. Intrapleural injection of β-Galactosidase coding adenoviruses (AdLacZ) were administered as previously described [4, 5]. After being harvested, the lungs were placed in a solution containing β-Galactosidase sub- strate. Hydrolysis of this substrate released a blue-colored product highlighting pleural adenovirus-infected cells. called Mesothelio-Mesenchymal Transition (MMT) [5]. However, the exact mechanisms that lead to the in- volvement of the pleura and pleural mesothelial cells in the onset and progression of fibrosis are not yet fully understood. y The role of chronic inflammation in human IPF is still controversial. Inflammatory cytokines and the infiltra- tion of immune cells are found in IPF [12, 13]. IL-1β overexpression in rodent lung induced the upregulation of TGF-β1, a major pro-fibrotic growth factor, and lung fibrosis [14, 15]. IL-1β is synthesized as a latent form and is activated following cleavage by caspase-1. Procaspase-1 is activated in multiprotein complexes, called the inflammasome, which contains a NOD-like receptor and a scaffold protein such as the apoptosis associated speck-like protein containing a CARD (ASC). The importance of IL-1β/caspase-1 signaling has been shown in the BLM model of lung fibrosis [16]. Mice deficient for inflammasome components such as the NOD-like receptor NLRP3 and thereby unable to activate caspase-1 and thus produce active IL-1β develop less severe fibrosis after BLM challenge [17]. Histology and fibrosis assessment gy ibrosis in pleural and subpleural areas was assessed on lung sections after H&E staining using a scoring method based on the modified Ashcroft score previously described [20]. Lung sections were analyzed in a double- blinded test to assess the pleural area using ×100 mag- nification and were graded from 0 (normal lung) to 8 (completely fibrotic lung). When varying degrees of fibrosis were present, the highest score was retained. Collagen was quantified using a histomorphometric assay on picrosirius red stained sections [5]. Ten areas targeting the pleura were randomly acquired under polarized light (×100. Nikon Eclipse E600) with a high- resolution microscope camera (Nikon DS-Ri1). Signal intensity was quantified using a homemade ImageJ macro and plotted as signal intensity according to distance to the pleura (up to 500 μm into the lung parenchyma) (Additional file 2: Figure S2). Briefly, the user inputs a pleura limit, a parenchymal side (above, below, left, right), a background area and areas to ex- clude from the analysis (staining artifacts, vessels and large airways). The program adjusts the pleural limits, the beginning of the pleura being set as the first of two consecutive pixels (in an order from the background to the parenchyma) with an intensity higher than the mean background signal plus two standard deviations from the background. From there on, the program maps the distance from the parenchymal area to the pleura (Additional file 2: Figure S2A). It then measures the intensity of each parenchymal pixel in the picrosirius red channel. The program returns a text file with the in- tensity and distance to pleura of each measured pixel. The data are plotted to form a curve with the mean intensity of all pixels with a given distance to the pleura (Additional file 2: Figure S2B). Statistical analyses were performed by comparing areas under the curve over the whole range in the pleura (0–60 μm) or subpleural (60–500 μm) area. Picrosirius red was also monitored in the parenchymal area as previously described [21]. For collagen quantifica- tion, vessels and large airways were excluded. Total TGF- β1 levels in BALF or PLF were assessed with a specific ELISA for mouse TGF-β1 (Quantikine® ELISA. R&D Systems) according to the manufacturer’s guidelines. In the present study, using repetitive intravenous in- jections of BLM in mice, we highlighted the pleural acti- vation of IL-1β/caspase-1 signaling as a seminal event in BLM-induced pleural and subpleural fibrotic toxicity. Histology and fibrosis assessment Our work suggests that caspase-1 could be a therapeutic target for IPF as well as BLM lung fibrotic toxicity. Background Even though most studies on IL-1β/caspase-1 signaling focused on immune cells, some studies highlighted the involvement of this signaling pathway in structural cells [18, 19]. The role of caspase-1 on lung structural cells and its involvement during fi- brotic processes is still poorly understood. Cell culture Human pleural mesothelial cells Met5A were cultured in Medium-199 (Lonza) supplemented with 10% serum (Hyclone, Fisher Scientific). Proliferating cells were treated with BLM (Calbiochem). For Il-1β as- sessment in supernatant, cells were treated with BLM in serum-free medium. When indicated, caspase-1 was blocked using YVAD (Bachem AG) as previously described [19]. RT-qPCR analysis y Total RNA from mouse lung was extracted using TRIzol (Invitrogen). Reverse transcription was performed on the total RNA using the M-MLV kit (Promega) and quanti- tative RT-PCR was performed on the cDNA using SYBR green master mix (Promega). The forward and reverse primer sequences were the following: for mouse samples: TGF-β1, 5′-CGTGGCTTCTAGTGCTGACGC-3′ and 5′-CCATGTCGATGGTCTTGCAGGT-3′; PAI-1, 5′-G GCCGTGGAACAAGAATGAGAT-3′ and 5′-GCTTGA AGAAGTGGGGCATGAAG-3′; E-cadherin, 5′-GGAG AGGCACCTGGAGAG-3′ and 5′-TCCGAAAAGAAG GCTGTC-3′; L32, 5′-GAAACTGGCGGAAACCCA-3′ and 5′-GGATCTGGCCCTTGAACCTT-3′, for human samples: E-cadherin, 5′-ACA GCC CCG CCT TAT GAT T-3′ and 5′-CTTCGGAACCGCTTCCTTCA-3′; α-SMA, 5′-TGGTCGGTATGGGTCAGAAAG-3′ and 5′-TCAGGGTCAGGATACCTCTCTTG-3′; procolla- gen, 5′-GCTACCCAACTTGCCTTCATG-3′ and 5′-GC AGTGGTAGGTGATGTTCTAAGA-3′; L32, 5′-TGTC CTGAATGTGGTCACCTGA-3′ and 5′-CTGCAGTCT CCTTGCACACCT-3′. Animal procedures Eight-week-old C57BL/6J mice (Charles River, Saint Germain-sur-l’Arbresle, France) were housed accordingly to the guidelines of the “Ministère de la Recherche et de la Technologie”. All experiments were approved by the “Comité d’Ethique de l’Expérimentation Animale (C2EA) du grand campus Dijon, n°105” (ref number : 4612). Mice were intravenously injected three times per week with bleomycin (BLM. Calbiochem) at a dose of 20 mg/kg for a total of 6 injections (Additional file 1: Figure S1A). Il-1β signaling was blocked with IL-1ra (anakinra) that was injected (0.1, 1 or 5 mg) intra- pleura every other day from day 0 to day 14 (Addi- tional file 1: Figure S1B). Caspase-1 was activated by intrapleural injection of nigericin (Sigma Aldrich). Three, 14 or 21 days after the beginning of the injections and after a sample of blood was collected, mice were euthanized by abdominal aortic bleeding. Bronchoalveolar Burgy et al. Respiratory Research (2016) 17:162 Page 3 of 10 Page 3 of 10 methanol-chloroform method before denaturation. To evaluate active MMP expression, a non-denaturant load- ing buffer was used to deposit BALF or PLF for the SDS- PAGE. 30 μg of proteins for lysates or constant volume for supernatant were deposed for migration on 12–15% acryl- amide gels according to the molecular weight of the targeted proteins. After electrophoresis, proteins were transferred on to PVDF membranes (GE Healthcare Europe GmbH) in a boric acid containing buffer. For IL- 1β transfer, an ethanol-supplemented glycin buffer was used. Membranes were saturated with BSA before incuba- tion with primary antibodies. MMP-9 (BML-SA620) from Enzolife Science, MMP-2 (sc-13594) and HSC70 (clone B- 6) from Santa Cruz Biotechnology, HSP47 (LF-PA41903) from Abfrontier, Caspase-1 (AHZ0082) from Invitrogen, Il-1β (clone 166926) from R&D, α-SMA (ab5694) from Abcam, E-Cadherin (24E10) from Cell Signaling and β- actin (AC-74) from Sigma-Aldrich. After washing in TBS- Tween 0.1%, membranes were incubated with HRP- coupled secondary antibody (Jackson Immunoresearch Laboratories). The signal was revealed using a chemilu- minescent reagent (Western Blotting Luminol Reagent. Santa Cruz Biotechnology) and was followed by a Chemi- Doc XRS System (Bio-Rad). Immunohistology gy Formalin fixed, paraffin embedded lung sections were dewaxed with xylene and endogenous peroxidases were inhibited (H2O2 3%). Frozen sections were fixed with ice-cold acetone and cells fixed with PFA and perme- abilized with triton. Before saturation (BSA 5%), sam- ples were incubated overnight at 4 °C with specific antibody. HSP47 (LF-PA41903) was from Ab Frontier, WT-1 (ab89901) from Abcam, Ki-67 (PA1-21520) from Thermo, HSP27 (SPA-803) and αB-crystallin (SPA-223) from Enzo Life Sciences, α-SMA (ab5694) from Abcam and NLRP3 (Cryo-2) from Adipogen. After washing, sections were incubated with HRP-coupled anti-rabbit antibody (Jackson Immunoresearch Laboratories) or Alexa568- or Alexa488-coupled antibody (Invitrogen). NovaRED (Vector Labs) was used to detect HRP followed by counterstaining with hematoxylin for im- munohistochemistry. DAPI-containing ProLong® Gold (Invitrogen) was used for immunofluorescence experi- ments. For caspase-1 fluorescent staining, frozen lung sections were labeled according to the manufacturer’s instructions (FAM-FLICA YVAD. ImmunoChemistry Technologies). TGF-β1 expression was followed by a FISH analysis on dewaxed sections. Briefly, sections were incubated in hybridization solution (Formamide 20%, SSPE 2X) and then incubated with a mix of Quasar 570-coupled probes (“Stellaris”, Biosearch Tech- nologies) specifically targeting murine TGF-β1 mRNA. After incubation, excess was eliminated from the probes and sections were mounted. Images were acquired with a Nikon Eclipse E600 with a high-resolution micro- scope camera (Nikon DS-Ri1). Fluorescent images were visualized using an Axio Imager.M2 microscope (Zeiss). Western blot Proteins from cells or lung tissues were extracted using a Triton X-100 and anti-protease (Roche) containing buffer for 30 to 40 min on ice. Proteins were quantified using a colorimetric method (Bio-Rad Protein Assay. Bio-Rad) before thermic denaturation in loading buffer containing SDS and β-mercaptoethanol. For Il-1β as- sessment, supernatants were collected and centrifuged to pellet debris. Samples were concentrated using a Statistical analysis All experiments were repeated at least three times inde- pendently. The different groups were compared using a two-tailed nonparametric unpaired Mann Whitney test. P values below 0.05 were considered statistically significant. Fig. 1 Intravenous bleomycin induces subpleural fibrosis associated with pleural cell differentiation and migration. a Histological analysis of lung sections from mice receiving either NaCl or BLM intravenously at D14 and D21. Representative images (insert: NaCl at the corresponding time point. Scale bars : 500 μm). b Ashcroft scoring of lung sections. c Profiles of Picrosirius Red signal according to distance to the pleura at D3, D14 and D21. d Area Under Curve comparison of areas targeting the pleura (left) or subpleural (right) areas at D3, D14 and D21. For collagen quantification, data are represented as mean of Picrosirius Red signal normalized to the NaCl condition at the corresponding time point. Data expressed as mean ± SEM. n = 4 for NaCl groups, n = 6 for BLM groups at D3 and D21, and n = 6 for NaCl and n = 8 for BLM at D14. e Representative images of immunostaining for HSP27, αB-crystallin and KI-67 of lung sections from mice receiving NaCl or BLM at D14 (arrows indicate positive cells. Counterstaining: Harris hematoxylin. Scale bars : 100 μm). f Levels of proMMP-9 and active MMP-2 in PLF was determined by western blot. Representative results from NaCl (n = 3) or BLM (n = 3) treated mice at D14 are shown. g AdLacZ-labeled pleural cells were stained for β-galactosidase activity (blue staining) in lung section from mice receiving intravenously NaCl or BLM at D5 or D10. Representative images are shown (n = 4 NaCl, n = 6 BLM. Scale bars: 200 μm), Insert: magnification; scale bars: 100 μm Arrows indicate pleural cells with modified morphology. Counterstaining: Nuclear fast red). p ≤0.05, p ≤0.01 Fig. 1 Intravenous bleomycin induces subpleural fibrosis associated with pleural cell differentiation and migration. a Histological analysis of lung sections from mice receiving either NaCl or BLM intravenously at D14 and D21. Representative images (insert: NaCl at the corresponding time point. Scale bars : 500 μm). b Ashcroft scoring of lung sections. c Profiles of Picrosirius Red signal according to distance to the pleura at D3, D14 and D21. d Area Under Curve comparison of areas targeting the pleura (left) or subpleural (right) areas at D3, D14 and D21. Results Subpleural fibrosis induced by intravenous bleomycin administration is associated with pleural cell migration Histological analysis of lung sections, quantified by modified Ashcroft scoring and picorsirius red quantifica- tion, showed that repeated intravenous injections of bleomycin (BLM) induced progressive subpleural mor- phological changes in mouse lungs, compared with NaCl Proteome profiler Relative levels of several cytokines were assessed with the “Mouse Cytokine Array Panel A Array kit” (R&D Systems). Mouse plasma was tested as recommended by the manufacturer. Signal intensity was quantified by Page 4 of 10 Page 4 of 10 Page 4 of 10 Burgy et al. Respiratory Research (2016) 17:162 Burgy et al. Respiratory Research (2016) 17:162 Burgy et al. Respiratory Research (2016) 17:162 ImageJ software and data expressed as a percentage of intensity normalized to positive controls. Statistical analysis We set up a novel method to quantify picro- sirius red staining by taking into consideration the distance to the pleura, and performed in-depth ana- lyses (Additional file 2: Figure S2). We thus showed that, following intravenous administration of BLM, collagen started to accumulate in the pleura at D3 and progressed towards the lung parenchyma in a time-dependent manner (Fig. 1c, d). In line with this, in the lungs of BLM-treated mice but not in those of control mice, HSP47 was overexpressed in pleural cells as early as D3 and in fibrotic subpleural areas at D14 (Additional file 2: Figure S2F, G). Intravenous BLM promoted the upregulation of HSP27 and αB- crystallin in the fibrotic areas (Fig. 1e). These proteins Fig. 2 Pleural cells are involved in bleomycin-induced inflammation. a Cell recruitment in PLF was assessed by total cell count at D3, D14 and D21. Results are expressed as mean ± SEM. b Inflammation was estimated by differential count in PLF after May-Grünwald Giemsa staining. Results are expressed as percentages of total cells. n = 4 for NaCl groups, n = 6 for BLM groups at D3 and D21, n = 6 for NaCl and n = 8 for BLM at D14. c, d Caspase-1 activation was assessed at D14 by c western blot in whole lung extracts (n = 3 mice/group) or by d specific caspase-1 FLICA analysis on lung section from NaCl or BLM injected mice (Representative images are shown. Active caspase-1: green, DAPI: blue, dotted lines represent the pleura, scale bars: 50 μm). e, f Met5A cells were treated with BLM (100 μM) and e activation of caspase-1 was followed (western blot; left, densitometric analysis; right). f active IL-1β was assessed in the supernatant of Met5A cells after 24 h of culture. Representative results from 3 independent experiments. HSC70 serves as a loading control. p ≤0.05, p ≤0.01, p ≤0.001 Fig. 2 Pleural cells are involved in bleomycin-induced inflammation. a Cell recruitment in PLF was assessed by total cell count at D3, D14 and D21. Results are expressed as mean ± SEM. b Inflammation was estimated by differential count in PLF after May-Grünwald Giemsa staining. Results are expressed as percentages of total cells. n = 4 for NaCl groups, n = 6 for BLM groups at D3 and D21, n = 6 for NaCl and n = 8 for BLM at D14. Statistical analysis For collagen quantification, data are represented as mean of Picrosirius Red signal normalized to the NaCl condition at the corresponding time point. Data expressed as mean ± SEM. n = 4 for NaCl groups, n = 6 for BLM groups at D3 and D21, and n = 6 for NaCl and n = 8 for BLM at D14. e Representative images of immunostaining for HSP27, αB-crystallin and KI-67 of lung sections from mice receiving NaCl or BLM at D14 (arrows indicate positive cells. Counterstaining: Harris hematoxylin. Scale bars : 100 μm). f Levels of proMMP-9 and active MMP-2 in PLF was determined by western blot. Representative results from NaCl (n = 3) or BLM (n = 3) treated mice at D14 are shown. g AdLacZ-labeled pleural cells were stained for β-galactosidase activity (blue staining) in lung section from mice receiving intravenously NaCl or BLM at D5 or D10. Representative images are shown (n = 4 NaCl, n = 6 BLM. Scale bars: 200 μm), Insert: magnification; scale bars: 100 μm Arrows indicate pleural cells with modified morphology. Counterstaining: Nuclear fast red). p ≤0.05, *p ≤0.01 Burgy et al. Respiratory Research (2016) 17:162 Page 5 of 10 Page 5 of 10 have been reported to promote pleural mesothelial cell transformation [4, 9]. We also observed an in- crease in the expression of the marker of Ki-67 pro- liferation in pleural cells next to the fibrotic areas (Fig. 1e). Moreover, we detected an increase in proMMP-9 expression and MMP-2 activation in the pleural lavage fluid (PLF) from BLM-treated mouse lung, compared with the control group (Fig. 1f). We thus investigated the migration of pleural cells after intravenous BLM injection. Following intrapleural in- jection of LacZ-coding adenovirus (AdLacZ), which specifically labels pleural cells [5], we noticed a change in pleural cell morphology (arrows) at D5 in BLM-treated mice, compared with control mice (Fig. 1g). Furthermore, at D10 after the BLM injec- tion, pleural cells were found within the subpleural lung parenchyma whereas in control animals the pleural cells remained in the pleural layer. These controls (Fig. 1a, Additional file 3: Figure S3A, B). By D14, morphological changes were mainly observed in the subpleural areas, and progressed towards the inner parenchyma by D21 (Fig. 1a, Additional file 3: Figure S3C–E). Pleural cells are involved in bleomycin-induced inflammation Compared with controls, intravenous BLM induced an increase in the total number of cells in PLF starting at D14 (Fig. 2a). At D14 and D21 after the BLM systemic administration, we also found an increase in the percent- age of neutrophils and leucocytes in the PLF (Fig. 2b). This pro-inflammatory profile at the pleural level was also found in the broncho-alveolar lavage fluid (BALF) following BLM (Additional file 4: Figure S4A, B). This suggests that the inflammatory response to the intraven- ous administration of BLM may involve the lung as well as the pleural area. Moreover, no differences were found in total cell count in the blood (monocytes, granulocytes, lymphocytes) or in serum levels of the major pro-inflam- matory cytokines. (Additional file 4: Figure S4C, D). We then focused on the caspase-1/IL-1β pathway. Systemic administration of BLM in mice triggered the activation of caspase-1 in the lung in particular in the pleural and sub- pleural area (Fig. 2c, d). In the human pleural mesothelial Met5A cell line, BLM induced caspase-1 activation as well as active IL-1β secretion into the supernatant (Fig. 2e, f), We observed at D14 that cells localizing in the pleura exhibited transformation features such as Ki-67 or HSP27/αB-crystallin overexpression. To investigate if pleural cells were also involved in the establishment of a profibrotic milieu, we investigated TGF-β1 production at this same time-point (e.g. D14). We observed an upregu- lation of TGF-β1 in the PLF from BLM-injected mice compared with controls (Fig. 3a). A Fluorescent In Situ Hybridization (FISH) analysis of lung sections showed that BLM treatment increased the expression of TGF-β1 mRNA in pleural cells whereas barely any expression was observed in control animals (Fig. 3b). A similar up- regulation of TGF-β1 production was observed at D21 (Additional file 6: Figure S6). As inflammation is linked to TGF-β1 upregulation, we next investigated the rela- tionship between BLM-induced IL-1β production by pleural cells and fibrosis markers such as TGF-β1 upreg- ulation. Repeated intra-pleural administration of a spe- cific inhibitor of IL-1β (IL-1ra) during the inflammation step (from day 0 to day 14) decreased TGF-β1 induction Fig. 3 Pleural delivery of IL-1ra hampers bleomycin-induced TGF-β1 upregulation. a TGF-β1 accumulation in the PLF of NaCl- or BLM-injected mice at D14 was measured by ELISA. Results are expressed as mean ± SEM. n = 8 NaCl, n = 10 BLM. Statistical analysis c, d Caspase-1 activation was assessed at D14 by c western blot in whole lung extracts (n = 3 mice/group) or by d specific caspase-1 FLICA analysis on lung section from NaCl or BLM injected mice (Representative images are shown. Active caspase-1: green, DAPI: blue, dotted lines represent the pleura, scale bars: 50 μm). e, f Met5A cells were treated with BLM (100 μM) and e activation of caspase-1 was followed (western blot; left, densitometric analysis; right). f active IL-1β was assessed in the supernatant of Met5A cells after 24 h of culture. Representative results from 3 independent experiments. HSC70 serves as a loading control. p ≤0.05, p ≤0.01, p ≤0.001 Burgy et al. Respiratory Research (2016) 17:162 Page 6 of 10 and caspase-1/IL-1β activation correlated with the pres- ence of NLPR3 aggregates, suggesting inflammasome formation (Additional file 5: Figure S5). results demonstrate that systemic BLM injections trig- ger the transformation of pleural cells, which then migrate into the lung parenchyma. Pleural cells are involved in bleomycin-induced inflammation in the lungs (both mRNA and protein) induced by the intravenous administration of BLM at D21 (Fig. 3c, d). FISH analysis showed that the injection of IL-1ra was able to inhibit TGF-β1 expression in both pleural cells and cells localized in subpleural areas (Fig. 3e). Pleural cells are involved in bleomycin-induced inflammation b Representative pictures from TGF-β1 mRNA expression analysis at D14 by FISH in lung sections from mice receiving NaCl (n = 4) or BLM (n = 6). TGF-β1 mRNA: red, DAPI: blue, scale bars: 50 μm. Pleura (dotted line) and TGF-β1 mRNA expression spots (arrows) are indicated. c TGF-β1 protein level assessed by western blot on whole lung from mice co-injected intravenously with NaCl or BLM together with intrapleural IL-1ra (from day 0 to 14) at the indicated doses or NaCl as a control at D21. HSC70 serves as a loading control, n = 3 mice/group. d qPCR analysis for the expression of TGF-β1 mRNA at D21. Expression relative to L32 and normalized with the NaCl group is shown, n = 5 mice/group. e FISH staining for TGF-β1 mRNA expression on lung sections from the above described mice co-injected with BLM and IL-1ra (n = 5 mice/group, scale bars: 50 μm). p ≤0.05, *p ≤0.01 Fig. 3 Pleural delivery of IL-1ra hampers bleomycin-induced TGF-β1 upregulation. a TGF-β1 accumulation in the PLF of NaCl- or BLM-injected mice at D14 was measured by ELISA. Results are expressed as mean ± SEM. n = 8 NaCl, n = 10 BLM. b Representative pictures from TGF-β1 mRNA expression analysis at D14 by FISH in lung sections from mice receiving NaCl (n = 4) or BLM (n = 6). TGF-β1 mRNA: red, DAPI: blue, scale bars: 50 μm. Pleura (dotted line) and TGF-β1 mRNA expression spots (arrows) are indicated. c TGF-β1 protein level assessed by western blot on whole lung from mice co-injected intravenously with NaCl or BLM together with intrapleural IL-1ra (from day 0 to 14) at the indicated doses or NaCl as a control at D21. HSC70 serves as a loading control, n = 3 mice/group. d qPCR analysis for the expression of TGF-β1 mRNA at D21. Expression relative to L32 and normalized with the NaCl group is shown, n = 5 mice/group. e FISH staining for TGF-β1 mRNA expression on lung sections from the above described mice co-injected with BLM and IL-1ra (n = 5 mice/group, scale bars: 50 μm). p ≤0.05, *p ≤0.01 Burgy et al. Respiratory Research (2016) 17:162 Page 7 of 10 IL-1β signaling is involved in the transformation process occurring in the subpleural area, which represents a trig- gering event in fibrogenesis. Pleural IL-1ra prevents bleomycin-induced lung fibrosis We then investigated the role of caspase-1 in pleural cell differentiation in vitro. In the presence of BLM, Met5A cells exhibited a mesenchymal phenotype with the over- expression of α-SMA (Fig. 5a). The specific caspase-1 in- hibitor YVAD was able to counteract these changes. Furthermore, YVAD also abolished the E-Cadherin downregulation induced by TGF-β1 (Fig. 5b). To further confirm the involvement of caspase-1 in the process of pleural cell transformation, Met5A cells were cultured in the presence of the caspase-1 activator nigericin. As shown in Fig. 5c, E-cadherin expression gradually de- creased as caspase-1 was activated. Compared with the control, nigericin induced other MMT-like changes such as α-SMA overexpression (Fig. 5d). In the same way, intra-pleural delivery of nigericin in mice induced a fi- brotic response in the pleura (Fig. 5e). Moreover, when injected into the pleura, nigericin induced an increase in cell recruitment in the pleura of these animals (Fig. 5f). Collectively, these data indicate that caspase-1 is in- volved in the activation and subsequent transformation of pleural cells triggered by BLM or TGF-β1. Pleural IL-1ra prevents bleomycin-induced lung fibrosis We studied whether the intra-pleural delivery of IL-1ra interfered with BLM pro-fibrotic toxicity in the lung. Pleural delivery of IL-1ra increased expression levels of E-Cadherin and decreased those of PAI-1 (Fig. 4a). In line with this finding, IL-1ra reduced collagen accumula- tion in mouse lung following BLM injection in a dose- dependent manner as observed by histomorphometric analysis (Fig. 4b). Further, as shown in Fig. 4c, in BLM- injected mice compared with the NaCl control, α-SMA was overexpressed in lung areas where caspase-1 was activated. Altogether, these data suggest that caspase-1/ Fig. 4 Pleural IL-1β/caspase-1 inhibition interferes with bleomycin- induced lung fibrosis. Mice were injected intravenously with NaCl or BLM together with intrapleural delivery of IL-1ra at the indicated doses or NaCl as a control (n = 5 mice/group). a qPCR analysis for the expression of E-Cadherin and PAI-1. Expression relative to L32 and normalized with the NaCl group is shown. b Histomorphometric quantification of collagen in lung parenchyma on lung sections from the above described mice. c Double staining for FLICA caspase-1 (green) and α-SMA (red) on lung sections of mice intravenously injected with BLM or NaCl. Representative images, n = 5/group. Nuclear staining: DAPI (blue), dotted lines represent the pleura, scale bars: 50 μm. p ≤0.05, *p ≤0.01 Discussion In recent decades, chronic inflammation has been considered a non-essential factor in the development of IPF. However, there is evidence that inflammation plays a role not only at the onset of the fibrotic process but also during fibrosis progression [22]. IPF progression is interspersed with stable periods inter- rupted by acute exacerbation, in which TGF-β1 plays a key role [23, 24]. The cause of these exacerbations remains unknown, though inflammation is thought to be a triggering event [25]. A retrospective study highlighted the beneficial effect of soluble thrombo- modulin on mortality in patients with an acute ex- acerbation of IPF [26]. Interestingly, thrombomodulin, besides its role in the modulation of intravascular co- agulation, also inhibits HMGB1, a trigger of the NLRP3 inflammasome and subsequent caspase-1 acti- vation [27]. The involvement of caspase-1/IL-1β in BLM-induced lung toxicity has already been reported in animal models [16, 17]. Recently, structural cells have been reported to be involved in capsase-1 activa- tion and IL-1β secretion in the lung following differ- ent profibrotic stimuli [18, 19]. Mesothelial cells have already been shown to be involved in the initiation of the inflammatory response in a model of talc-induced pleurodesis [28]. Furthermore, asbestos can induce Fig. 4 Pleural IL-1β/caspase-1 inhibition interferes with bleomycin- induced lung fibrosis. Mice were injected intravenously with NaCl or BLM together with intrapleural delivery of IL-1ra at the indicated doses or NaCl as a control (n = 5 mice/group). a qPCR analysis for the expression of E-Cadherin and PAI-1. Expression relative to L32 and normalized with the NaCl group is shown. b Histomorphometric quantification of collagen in lung parenchyma on lung sections from the above described mice. c Double staining for FLICA caspase-1 (green) and α-SMA (red) on lung sections of mice intravenously injected with BLM or NaCl. Representative images, n = 5/group. Nuclear staining: DAPI (blue), dotted lines represent the pleura, scale bars: 50 μm. p ≤0.05, *p ≤0.01 Burgy et al. Respiratory Research (2016) 17:162 Page 8 of 10 Fig. 5 Caspase-1 inhibition hampers pleural structural cell transformation. a, b Met5A cells were cultured with a BLM or b TGF alone or in combination with caspase-1 inhibitor YVAD (western blot; left, densitometric analysis; right). Expression of α-SMA and E-Cadherin were assessed by western blot. HSC70 and β-actin: loading control. Representative results from three independent experiments. Discussion c, d qPCR analysis of E-Cadherin and α-SMA mRNA on Met5A cells cultured in the presence of nigericin (0.1 μM) or control c at the indicated times or d for 24 h. C57Bl/6 mice were given intrapleural injections of nigericin (1.25 mg/kg) or control. e Histological analysis at D21 post-intrapleural injection. Representative observations after H&E staining, scale bars: 100 μm. f Total cell count in the PLF at D14 after intrapleural injection of nigericin or control. p ≤0.05, *p ≤0.01 Fig. 5 Caspase-1 inhibition hampers pleural structural cell transformation. a, b Met5A cells were cultured with a BLM or b TGF alone or in combination with caspase-1 inhibitor YVAD (western blot; left, densitometric analysis; right). Expression of α-SMA and E-Cadherin were assessed by western blot. HSC70 and β-actin: loading control. Representative results from three independent experiments. c, d qPCR analysis of E-Cadherin and α-SMA mRNA on Met5A cells cultured in the presence of nigericin (0.1 μM) or control c at the indicated times or d for 24 h. C57Bl/6 mice were given intrapleural injections of nigericin (1.25 mg/kg) or control. e Histological analysis at D21 post-intrapleural injection. Representative observations after H&E staining, scale bars: 100 μm. f Total cell count in the PLF at D14 after intrapleural injection of nigericin or control. p ≤0.05, **p ≤0.01 from angiotensin II-induced cardiac fibrosis [31]. NLRP3 hampers TGF-β–induced EMT in vitro in renal tubular epithelial cells [32]. In line with these findings, we showed here that activation of caspase-1 was able to trigger transformation/differentiation of pleural cells in vitro. Moreover, intra-pleural delivery of nigericin in mice elicits fibrogenesis with a restrictive pleural localization at D21, suggesting that pleural activation of caspase-1 could induce pleural cells transdifferentia- tion. Supporting this hypothesis, caspase-1 inhibition inhibited transdifferentiation of Met5A cells induced by either BLM or TGF-β1. Thus, targeting caspase-1 in pulmonary fibrotic disorders might represent an interesting therapeutic option by interfering with two pro-fibrotic events: inflammation and the transformation of pleural structural cells that acquire a myofibroblast-like phenotype. caspase-1 activity through the NLRP3 inflammasome and thus triggers an inflammatory signal in mesothe- lial cells [29]. Our work highlighted the fact that local intrapleural treatment with an IL-1β inhibitor such as IL-1ra (to counteract BLM-induced pleural inflammation) limited fibrosis progression and could thus be a thera- peutic option notably in the management of acute exacer- bation of IPF. Discussion It worth to note that IL-1ra effect on established fibrosis still needs to be investigated. g We and others have demonstrated the involvement of pleural mesothelial cells in the process of pleuro- pulmonary fibrosis in animal models as well as in human IPF [5, 6, 8]. We previously described in several animal models that pleural mesothelial cells can differentiate into myofibroblast-like cells under TGF-β1 through a transformation process called MMT [4–6, 9]. Interestingly, recent studies revealed the presence of calretinin and mesothelin expressing cells, two markers of pleural mesothelial cells, in IPF lung parenchyma sug- gesting the migration of pleural mesothelial cells [7, 8, 30]. Our present work further endorses these results as we described the differentiation and migration of pleural cells together with increased collagen production after systemic administration of BLM. Moreover, we provide evidence regarding the direct role of caspase-1 in BLM- and TGF-β1-induced pleural cell transform- ation. NLRP3 has been reported to interfere with TGF-β signaling [31, 32]. NLRP3 can regulate cardiac fibroblast differentiation, and its deficiency seems to protect mice Conclusion Using a murine model of pulmonary fibrosis induced by repetitive intravenous injections of BLM, which corre- sponded to a robust experimental model of pulmonary fibrosis mimicking human IPF and human BLM-induced lung toxicity, we confirmed the direct role of pleural cells in the observed fibrotic process. Further, our results suggest that caspase-1 should be considered as a thera- peutic target in the management of pulmonary fibrotic disorders. Page 9 of 10 Burgy et al. Respiratory Research (2016) 17:162 Page 9 of 10 Page 9 of 10 Funding Tucker TA, Jeffers A, Alvarez A, Owens S, Koenig K, Quaid B, Komissarov AA, Florova G, Kothari H, Pendurthi U, et al. Plasminogen activator inhibitor-1 deficiency augments visceral mesothelial organization, intrapleural coagulation, and lung restriction in mice with carbon black/bleomycin-induced pleural injury. Am J Respir Cell Mol Biol. 2014;50:316–27. 11. Tucker TA, Jeffers A, Alvarez A, Owens S, Koenig K, Quaid B, Komissarov AA, Florova G, Kothari H, Pendurthi U, et al. Plasminogen activator inhibitor-1 deficiency augments visceral mesothelial organization, intrapleural coagulation, and lung restriction in mice with carbon black/bleomycin-induced pleural injury. Am J Respir Cell Mol Biol. 2014;50:316–27. Funding 7. Karki S, Surolia R, Hock TD, Guroji P, Zolak JS, Duggal R, Ye T, Thannickal VJ, Antony VB. Wilms’ tumor 1 (Wt1) regulates pleural mesothelial cell plasticity and transition into myofibroblasts in idiopathic pulmonary fibrosis. FASEB J. 2014;28:1122–31. This work has received funding from the European Union, 7th Framework Programme, HEALTH-F2-2007-202224 European IPF Network, the Fonds Européen de Développement Économique et Régional, Agence Nationale de la Recherche (11-BSV-011-01 meso-IPF and Investissement d’Avenir ANR-11-LABX-0021-01-LipSTIC LabEx), La Ligue Régionale Grand Est Contre le Cancer, Le conseil Régional de Bourgogne, l’Institut National du Cancer (INCa), and the Fonds de Dotation “Recherche en Santé Respiratoire“, the Société de Pneumologie de Langue Française and the Fondation du Souffle. O. B. and P.B. are lauréat 2014 de la Fondation du Souffle et du Fonds de dotation Recherche en Santé Respiratoire. O.B., PS.B. and G.W. are supported by Fonds de Dotation « Recherche en Santé Respiratoire » et la Société de Pneumologie de Langue Française. O.B. is supported by La Ligue National Contre le Cancer. The team of C.G. has been awarded the « label d’excellence » by La Ligue National Contre le Cancer and L’Association pour la Recherche sur le Cancer and belongs to the LabEx LipSTIC and GR-Ex. 8. Mubarak KK, Montes-Worboys A, Regev D, Nasreen N, Mohammed KA, Faruqi I, Hensel E, Baz MA, Akindipe OA, Fernandez-Bussy S, et al. Parenchymal trafficking of pleural mesothelial cells in idiopathic pulmonary fibrosis. Eur Respir J. 2012;39:133–40. 9. Wettstein G, Bellaye PS, Kolb M, Hammann A, Crestani B, Soler P, Marchal- Somme J, Hazoume A, Gauldie J, Gunther A, et al. Inhibition of HSP27 blocks fibrosis development and EMT features by promoting Snail degradation. FASEB J. 2013;27:1549–60. 9. Wettstein G, Bellaye PS, Kolb M, Hammann A, Crestani B, Soler P, Marchal- Somme J, Hazoume A, Gauldie J, Gunther A, et al. Inhibition of HSP27 blocks fibrosis development and EMT features by promoting Snail degradation. FASEB J. 2013;27:1549–60. 10. Chen LJ, Ye H, Zhang Q, Li FZ, Song LJ, Yang J, Mu Q, Rao SS, Cai PC, Xiang F, et al. Bleomycin induced epithelial-mesenchymal transition (EMT) in pleural mesothelial cells. Toxicol Appl Pharmacol. 2015;283:75–82. 10. Chen LJ, Ye H, Zhang Q, Li FZ, Song LJ, Yang J, Mu Q, Rao SS, Cai PC, Xiang F, et al. Bleomycin induced epithelial-mesenchymal transition (EMT) in pleural mesothelial cells. Toxicol Appl Pharmacol. 2015;283:75–82. 11. Received: 2 August 2016 Accepted: 22 November 2016 Additional file 3: Figure S3. Intravenous BLM injections trigger collagen accumulation mainly in the subpleural areas by D14 with overexpression of HSP47. (PNG 2604 kb) Additional file 4: Figure S4. BLM promotes an inflammation profile of the BALF but not in the blood. (PNG 539 kb) Availability of data and materials Essential datasets supporting the conclusions are included in this published article. Authors’ contributions 12. Boomars KA, Schweizer RC, Zanen P, van den Bosch JM, Lammers JW, Koenderman L. Eosinophil chemotactic activity in bronchoalveolar lavage from idiopathic pulmonary fibrosis is dependent on cytokine priming of eosinophils. Eur Respir J. 1998;11:1009–14. 12. Boomars KA, Schweizer RC, Zanen P, van den Bosch JM, Lammers JW, Koenderman L. Eosinophil chemotactic activity in bronchoalveolar lavage from idiopathic pulmonary fibrosis is dependent on cytokine priming of eosinophils. Eur Respir J. 1998;11:1009–14. OB and PSB designed and performed all experiments and analyzed the data. SC wrote the ImageJ program for picrosirius red quantification and set up FISH staining. GB, GW, PMB and FG helped with the in vivo experiments. CG supervised the overall project and revised the manuscript. PB and OB designed the study, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. 13. Daniil Z, Kitsanta P, Kapotsis G, Mathioudaki M, Kollintza A, Karatza M, Milic-Emili J, Roussos C, Papiris SA. CD8+ T lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis. Respir Res. 2005;6:81. 13. Daniil Z, Kitsanta P, Kapotsis G, Mathioudaki M, Kollintza A, Karatza M, Milic-Emili J, Roussos C, Papiris SA. CD8+ T lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis. Respir Res. 2005;6:81. 14. Kolb M, Margetts PJ, Anthony DC, Pitossi F, Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 2001;107:1529–36. 14. Kolb M, Margetts PJ, Anthony DC, Pitossi F, Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 2001;107:1529–36. Abbreviations h BALF: Broncho-alveolar lavage fluid; BLM: Bleomycin; EMT: Epithelial to mesenchymal transition; FISH: Fluorescence in situ hybridization; HMGB1: High mobility group box 1; IL-1β: Interleukin-1β; IPF: Idiopathic pulmonary fibrosis; MMT: Mesothelio-mesenchymal transition; NLRP3: BALF: Broncho-alveolar lavage fluid; BLM: Bleomycin; EMT: Epithelial to mesenchymal transition; FISH: Fluorescence in situ hybridization; HMGB1: High mobility group box 1; IL-1β: Interleukin-1β; IPF: Idiopathic pulmonary fibrosis; MMT: Mesothelio-mesenchymal transition; NLRP3: NOD-like receptor family, pyrin domain containing 3; PAI-1: Plasminogen activator inhibitor-1; PLF: Pleural lavage fluid; TGF-β: Transforming growth factor-β1; α-SMA: α-smooth muscle actin 4. Bellaye PS, Burgy O, Colas J, Fabre A, Marchal-Somme J, Crestani B, Kolb M, Camus P, Garrido C, Bonniaud P. Antifibrotic role of alphaB-crystallin inhibition in pleural and subpleural fibrosis. Am J Respir Cell Mol Biol. 2015; 52:244–52. NOD-like receptor family, pyrin domain containing 3; PAI-1: Plasminogen activator inhibitor-1; PLF: Pleural lavage fluid; TGF-β: Transforming growth factor-β1; α-SMA: α-smooth muscle actin 5. Decologne N, Kolb M, Margetts PJ, Menetrier F, Artur Y, Garrido C, Gauldie J, Camus P, Bonniaud P. TGF-beta1 induces progressive pleural scarring and subpleural fibrosis. J Immunol. 2007;179:6043–51. References 1 R h G 1. Raghu G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, Brozek JL, Collard HR, Cunningham W, Homma S, et al. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline: Treatment of Idiopathic Pulmonary Fibrosis. An Update of the 2011 Clinical Practice Guideline. Am J Respir Crit Care Med. 2015;192:e3–19. Additional file 5: Figure S5. BLM triggers the accumulation of NLRP3 protein in Met5A cells. (PNG 464 kb) Additional file 6: Figure S6. Intravenous BLM induces TGF-β1 overproduction in mouse lung at D21. (PNG 425 kb) 2. Mazzei ME, Richeldi L, Collard HR. Nintedanib in the treatment of idiopathic pulmonary fibrosis. Ther Adv Respir Dis. 2015;9(3):121–9. 3. Noble PW, Albera C, Bradford WZ, Costabel U, Glassberg MK, Kardatzke D, King Jr TE, Lancaster L, Sahn SA, Szwarcberg J, et al. Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. Lancet. 2011;377:1760–9. Acknowledgements W h k V S G We thank V. Saint Giorgio, A. Bouchot for their invaluable help and P. Bastable for correcting the manuscript. We thank Dr M. Kolb for providing the AdLacZ. We thank V. Saint Giorgio, A. Bouchot for their invaluable help and P. Bastable for correcting the manuscript. We thank Dr M. Kolb for providing the AdLacZ. 6. Decologne N, Wettstein G, Kolb M, Margetts P, Garrido C, Camus P, Bonniaud P. Bleomycin induces pleural and subpleural fibrosis in the presence of carbon particles. Eur Respir J. 2010;35:176–85. Author details 1 1INSERM, LNC UMR866, LipSTIC LabEx team, Université Bourgogne Franche-Comté, 21000 Dijon, France. 2Service de Pneumologie et Soins Intensifs Respiratoires, Centre Hospitalo-Universitaire de Bourgogne, 21000 Dijon, France. 3Anticancer Centre Georges François Leclerc, CGFL, 21000 Dijon, France. Additional file 1: Figure S1. Scheme of the different models used in this work. (PNG 145 kb) Additional file 2: Figure S2. In depth subpleural collagen quantification. (PNG 1209 kb) Additional file 3: Figure S3. Intravenous BLM injections trigger collagen accumulation mainly in the subpleural areas by D14 with overexpression of HSP47. (PNG 2604 kb) Additional file 4: Figure S4. BLM promotes an inflammation profile of the BALF but not in the blood. (PNG 539 kb) Additional file 5: Figure S5. BLM triggers the accumulation of NLRP3 protein in Met5A cells. (PNG 464 kb) Additional file 6: Figure S6. Intravenous BLM induces TGF-β1 overproduction in mouse lung at D21. (PNG 425 kb) Additional file 1: Figure S1. Scheme of the different models used in this work. (PNG 145 kb) Additional file 1: Figure S1. Scheme of the different models used in this work. (PNG 145 kb) Additional file 2: Figure S2. In depth subpleural collagen quantification. (PNG 1209 kb) Received: 2 August 2016 Accepted: 22 November 2016 Consent for publication 16. Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S, Schnyder B, Akira S, Quesniaux VF, Lagente V, et al. IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice. J Clin Invest. 2007;117:3786–99. 16. Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S, Schnyder B, Akira S, Quesniaux VF, Lagente V, et al. IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice. J Clin Invest. 2007;117:3786–99. Competing interests The authors declare that they have no competing interests. 15. 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Respiratory Research (2016) 17:162 Ethics approval and consent to participate A l f d d l • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W1965952128	https://europepmc.org/articles/pmc3206009?pdf=render	English	null	Challenges in Estimating Insecticide Selection Pressures from Mosquito Field Data	PLoS neglected tropical diseases	2,011	cc-by	9,885	Introduction compromise these efforts. The widespread use of a small portfolio of compounds against large mosquitoes populations with many generations per year (estimated at 12 per year for Anopheles gambiae [3] and 20 for Aedes aegypti [4]) have raised fears that high levels of resistance may arise very quickly. Mosquito-borne diseases are prevalent in the tropics and subtropics and constitute a large proportion of the health problems in developing countries. The major mosquito vectors occur in the genera Culex, Aedes, and Anopheles, which transmit Filaria spp, Japanese encephalitis, dengue and yellow fever viruses and malaria. The control of vector populations is often based on insecticides, such as larviciding, indoor residual spraying (IRS) and personal protection through insecticide treated materials (ITM) and their use has been shown to have a powerful impact on mosquito abundance and disease transmission [1]. The foraging and resting behaviors of mosquitoes ensure a number of potentially lethal interactions with insecticide-treated surfaces during parts of the mosquito lifecycle [2], but prolonged exposure to an insecticide over many generations runs the risk that mosquitoes will develop resistance. In general only four different chemical classes of synthetic insecticides are used in the field: organochlorines, organophosphates, carbamates and pyrethroids. Pyrethroids are particularly important because they are the only class of insecticides recommended by The World Health Organization to use on ITM. Since ITM are being widely distributed across malaria and dengue affected countries and pyrethroids are employed in some areas for agricultural pest control, there is concern that the emergence of resistance will We consider the problem of measuring the strength of selection for insecticide resistance in mosquito field populations and show how changes in the frequencies of the alleles at a single locus can be used to estimate the selection acting on each genotype. This type of data is collected for the identification of genetic mechanisms of resistance and/or during monitoring programs of vector control campaigns. The method we developed extends that described earlier by DuMouchel and Anderson in 1968 [5] for laboratory populations. Laboratory based conditions differ significantly from the field. In the laboratory insecticide assays are conducted over standardized range of doses and concentra- tions that do not account for field situations such as decay rates and exposure characteristics. Susana Barbosa1, William C. Black IV2, Ian Hastings1 rasitology Group, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 2 Department of Microbiology, Colorado Stat , United States of America 1 Molecular and Biochemical Parasitology Group, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 2 Departm University, Fort Collins, Colorado, United States of America 1 Molecular and Biochemical Parasitology Group, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 2 Department of Microbiology, Colorado State University, Fort Collins, Colorado, United States of America Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: sbarbosa@liv.ac.uk Competing Interests: The authors have declared that no competing interests exist. * E-mail: sbarbosa@liv.ac.uk * E-mail: sbarbosa@liv.ac.uk Abstract Insecticide resistance has the potential to compromise the enormous effort put into the control of dengue and malaria vector populations. It is therefore important to quantify the amount of selection acting on resistance alleles, their contributions to fitness in heterozygotes (dominance) and their initial frequencies, as a means to predict the rate of spread of resistance in natural populations. We investigate practical problems of obtaining such estimates, with particular emphasis on Mexican populations of the dengue vector Aedes aegypti. Selection and dominance coefficients can be estimated by fitting genetic models to field data using maximum likelihood (ML) methodology. This methodology, although widely used, makes many assumptions so we investigated how well such models perform when data are sparse or when spatial and temporal heterogeneity occur. As expected, ML methodologies reliably estimated selection and dominance coefficients under idealised conditions but it was difficult to recover the true values when datasets were sparse during the time that resistance alleles increased in frequency, or when spatial and temporal heterogeneity occurred. We analysed published data on pyrethroid resistance in Mexico that consists of the frequency of a Ile1,016 mutation. The estimates for selection coefficient and initial allele frequency on the field dataset were in the expected range, dominance coefficient points to incomplete dominance as observed in the laboratory, although these estimates are accompanied by strong caveats about possible impact of spatial and temporal heterogeneity in selection. k WC IV, Hastings I (2011) Challenges in Estimating Insecticide Selection Pressures from Mosquito Field Data. PLoS Negl Trop Dis 5(11) al.pntd.0001387 ation: Barbosa S, Black WC IV, Hastings I (2011) Challenges in Estimating Insecticide Selection Pressures from Mosquito Field Data. PL 387. doi:10.1371/journal.pntd.0001387 Editor: Rhoel Ramos Dinglasan, Johns Hopkins Bloomberg School of Public Health, United States of America Received June 15, 2011; Accepted September 19, 2011; Published November 1, 2011 Received June 15, 2011; Accepted September 19, 2011; Published November 1, 2011 Copyright: 2011 Barbosa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: S.B. was supported by research grant SFRH/BD/33534/2008 from Fundac¸a˜o para a Cieˆncia e Tecnologia, Portugal and Siemens Portugal under a Ph.D. Program in Computational Biology of the Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal. www.plosntds.org www.plosntds.org Author Summary Author Summary The emergence and spread of insecticide resistance compromise the control of mosquito borne diseases such as dengue or malaria, which are responsible for millions of deaths every year in tropical and subtropical areas. There are currently no easily implemented methodologies to quantify the strength of selection for resistance occurring in nature. Using field data from Mexico on the frequency of an allele mutation conferring resistance in the mosquito Aedes aegypti we use maximum likelihood (ML) to estimate the selection and dominance coefficients driving the evolution of resistance. We explored the impact of poor data collection, data that combine information from different locations and the consequences of selection and dominance coefficients varying over the sampling time period. The ML method can accurately estimate these parameters with simulated data in ideal sampling situa- tions but it is difficult to recover true values when spatial and temporal heterogeneity occurs. The analysis high- lighted factors relevant to field work such as the need for frequent surveillance in discrete sentinel sites. Introduction Following insecticide deployment in the field, concentration decreases and there is a selective window of time at lower concentrations (see Figure 1), where resistant heterozygotes do not die but susceptible homozygotes are still killed, therefore acting as dominant when under more standard- ized conditions it may appear to be recessive. This is relevant because dominance relationships between susceptible and resis- tance alleles affect the rate of spread of resistance. November 2011 \| Volume 5 \| Issue 11 \| e1387 1 November 2011 \| Volume 5 \| Issue 11 \| e1387 Estimating Selection from Mosquito Field Data period of dynamics (typically early and late stage of spread) or may be pooled from different locations. In this paper we discuss the challenges associated with this approach. We used published data on pyrethroid resistance from Aedes aegypti, throughout Mexico [6] on the frequency of the Ile1,016 mutation, one of the mutations in the voltage-gated sodium channel gene known to confer resistance to pyrethroids (this is known as knockdown resistance, a term applied to insects that fail to lose coordinated activity immediately after exposure). pt: frequency of the resistant allele at time/generation t qt = 12pt: frequency of the susceptible allele at time/ generation t pt: frequency of the resistant allele at time/generation t qt = 12pt: frequency of the susceptible allele at time/ generation t This recursive equation is the basic formula of selection of a favourable gene [7,8]. We defined the allele initial frequency p0, as the frequency in the first sampling time point, set as generation 0. Each subsequent generation can be converted onto a real timescale of years by assuming a constant number of generations per calendar year. The ML approach to estimate the unknown parameters h and s and p0, based on this genetic model, involved selecting initial values of s, h, and p0, and then testing how well the predicted allele frequencies matched those observed in the dataset. Field datasets usually consist of the number of resistant alleles xt and the total number of sampled alleles n at different time points t. The probability of observing x resistant alleles among n alleles follows a binomial distribution, with a probability of success (being a resistance allele) p for each sampled time point t. Figure 1. The typical change in insecticide concentration in the field over time. As concentration decays with time after deployment there is a differential survival of genotypes. In period A the RR genotype will survive while the RS and SS dies: this makes the R allele recessive in this period. In period B both RR and RS survive making the R allele dominant in this period. In period C all genotypes can survive so no selection occurs. These are windows of selection, adapted from [37]. doi:10.1371/journal.pntd.0001387.g001 Figure 1. The typical change in insecticide concentration in the field over time. As concentration decays with time after deployment there is a differential survival of genotypes. In period A the RR genotype will survive while the RS and SS dies: this makes the R allele recessive in this period. In period B both RR and RS survive making the R allele dominant in this period. In period C all genotypes can survive so no selection occurs. These are windows of selection, adapted from [37]. doi:10.1371/journal.pntd.0001387.g001 Figure 1. The typical change in insecticide concentration in the field over time. As concentration decays with time after deployment there is a differential survival of genotypes. In period A the RR genotype will survive while the RS and SS dies: this makes the R allele recessive in this period. Model and Methods The genetic model we employ assumes a single autosomal locus conferring insecticide resistance in a diploid sexually reproducing population, with non-overlapping generations and assuming random mating; these are standard assumptions in population genetics models. There are two possible alleles, resistant (R) or susceptible (S), and three possible genotypes (SR, RR, SS). The fitness coefficient, which is a measure of survival and reproduction of the different genotypes, was defined as 1 for the susceptible homozygotes SS, 1+s (s is the selection coefficient) for resistant homozygotes RR and 1+hs (h is the dominance coefficient) for heterozygotes SR. The level of dominance is a measure of the relative position of the phenotype of the heterozygote relative to the phenotype of the two corresponding homozygotes. Complete dominance for susceptible allele is represented by h = 0 and complete dominance for resistance allele by h = 1, alleles are codominant or additive when h = 0.5. The fitness coefficients are composite measures of fitness in both the exposed and unexposed mosquitoes groups and are assumed to be the same for males and females. Using a maximum likelihood (ML) procedure and a recursive genetic model that tracks the changes in the resistance allele frequencies at a single locus it is possible to estimate a selection coefficient (s), a coefficient quantifying dominance (h) and the initial frequency of the resistance allele (p0,), key parameters that determine the dynamics of resistance. The model provides a straightforward way to obtain these values with the least complex dataset possible. However, field data on the spread of resistance is often suboptimal: datasets may be small, may only track one We also assume a large population, so that genetic drift can be ignored, which enabled us to predict the frequency of the resistant allele at any time t according to the recursion expression: Figure 1. The typical change in insecticide concentration in the field over time. As concentration decays with time after deployment there is a differential survival of genotypes. In period A the RR genotype will survive while the RS and SS dies: this makes the R allele recessive in this period. In period B both RR and RS survive making the R allele dominant in this period. In period C all genotypes can survive so no selection occurs. These are windows of selection, adapted from [37]. doi:10.1371/journal.pntd.0001387.g001 www.plosntds.org ptz1~ p2 t (1zs)zptqt(1zhs) 1zs(p2 t z2hptqt) ð1Þ ð1Þ www.plosntds.org Estimating Selection from Mosquito Field Data pt: probability of sampling an R allele, i.e, probability of a success was no temporal or spatial variation in parameter values and populations sizes were sufficiently large that stochastic changes in alleles frequencies could be ignored. Data were available for each generation, 100 alleles (50 mosquitoes) were sampled each generation (Equation 2) and the simulations were run until the resistance allele frequency exceeded 0.99. Accuracy of analysis was gauged by the correlation coefficient between true and estimated parameter values, and by checking how frequently the true values fell with the estimated 95% confidence intervals. nt xt : combinatorial term to account for the number of ways of sampling x resistant alleles among n total alleles The corresponding binomial likelihood function is: L(ptjxt,nt)~f (xtjnt,pt) ð3Þ ð3Þ Next we examined the impact of suboptimal datasets. Equation 1 was used to predict allele frequency for 120 generations and we assumed that 100 alleles were sampled in each generation. Two optimal datasets with different dominance values were produced to check if the ML method accurately recovered the parameters when data from all generations was available (as above) and to investigate the effect of different degrees of dominance on estimations. Subsets of the data were used to examine the influence of incomplete sampling when only a few generations of data are available, or when only the initial stages of spread are available for analysis. The likelihood function returns the likelihood of the value pt given the observed data of xt resistant alleles among the sample of nt at each generation. Essentially, it tells us how consistent the data are with predicted values of pt (Equation 1). The likelihood value for the dataset is the product of the likelihoods across the entire sample: The likelihood function returns the likelihood of the value pt given the observed data of xt resistant alleles among the sample of nt at each generation. Essentially, it tells us how consistent the data are with predicted values of pt (Equation 1). The likelihood value for the dataset is the product of the likelihoods across the entire sample: L pjx,n ð Þ~ P t i~1 ni xi p xi i 1{pi ð Þni{xi ð4Þ ð4Þ y Field data, collected and analyzed by Garcia et al. [6], was available for analysis. Estimating Selection from Mosquito Field Data There were a total of 78 field collections containing 3,808 Aedes aegypti (some as much as 2000 km apart). Each mosquito was genotyped at the Ile1,016 locus. We pooled data from the different locations and analysed it assuming different number of generations of mosquitoes per year (6,9,12,16 and 20), to check the consistency of the estimations. Intuitively, we would expect spatial and temporal variation in the selection parameter in the Garcia et al. dataset and in many other datasets obtained under field conditions. It was therefore vital to ascertain how heteroge- neity would affect the algorithm’s ability to recover the underlying parameters from pooled data. Spatial heterogeneity was investi- gated by simulating allele frequencies for 80 different locations over 50 generations using Equation 1; 100 alleles were sampled from each generation (Equation 2) and data from each generation in each location were used in the analyses. Parameters p0 and h were randomly selected from a uniform probability distribution (p0,<(0,1), h,<(0,1)) while s was randomly drawn from a normal distribution (s,N(0.15,0.025)), the constraints on s coefficient are within a reasonable range for a field setting. Once selected for a location, the values of h and s did not change, i.e., there was no temporal heterogeneity. Two simulation strategies were used: (i) p0 and h were allowed to vary while s was held constant at 0.1, 0.3, 0.6, 0.8 or 1, (ii) p0, and s was allowed to vary while h was held constant at 0, 0.25, 0.5. 0.75 or 1. The data across the simulated locations were pooled for analyses. Each simulation strategy was run 300 times giving a total of 30065 = 1500 per strategy. The mean values of each parameter over all simulated locations was assumed to be the true value and the accuracy of the program was, as before, gauged by the correlation coefficient between the estimated and true values, and by the proportion of the true values falling within the 95% CI. We implemented this ML methodology in R [9] using constrOptim() function (from stats package) for which it is not necessary to provide analytic derivatives and that can minimize/maximize a function subject to linear inequality constraints. Three constraints on the parameter values were enforced: 0,p,1, 0,s,1, 0,h,1, except when analysing the field data when a constraint on h (0–1.5) was imposed. Estimating Selection from Mosquito Field Data The Nelder-Mead optimization method algorithm was used, that generates a new test position by extrapolating the behavior of the objective function measured at each test point arranged as a simplex. The algorithm then chooses to replace one of these test points with the new test point and the algorithm progresses. The simplest step is to replace the worst point with a point reflected through the centroid of the remaining points. If this point is better than the best current point, then it will expand exponentially along this line. On the other hand, if this new point is not much better than the previous value the simplex returns the previous point. The standard error (s.e) of the estimates was determined by inverting the Hessian matrix evaluated at the ML estimate and the 95% confidence interval endpoints were calculated as Parameter estimate61.96s.e. Maximum likelihood estimation is an optimization technique and there is no guarantee that the set of parameters that uniquely maximizes the likelihood will always be found because the algorithm may converge onto local optima whose likelihood is below the global maximum. To overcome this problem 1000 runs of the ML iteration procedure were performed in every estimation, with random starting values of the parameter estimates used to initialise the optimization routine [10]. In the analyses described here, the runs that converged to other estimates had ML values sufficiently less than the global maximum that a likelihood ratio test considered them different, so that the set of parameters could be safely discarded. However a small percentage of the runs converged to a set of different parameters with a similar likelihood value that could not be considered different using a likelihood ratio test. The criteria used to exclude these results as potential best estimates was that the estimated value of h lay on the boundary of the constrained parameter range and is expected to reflect erratic behavior of the algorithm when using a small sample. The effect of temporal heterogeneity in estimations was also investigated by varying s and h over 50 generations in a single location, i.e., different s and h values in different generations. The distribution of values were the same as those used for spatial heterogeneity. Three scenarios were considered: (i) s and h both varied over generations, (ii) h could vary while s was held constant, (iii) h could vary while s was held constant. pt: frequency of the resistant allele at time/generation t qt = 12pt: frequency of the susceptible allele at time/ generation t In period B both RR and RS survive making the R allele dominant in this period. In period C all genotypes can survive so no selection occurs. These are windows of selection, adapted from [37]. doi:10.1371/journal.pntd.0001387.g001 f (xtjnt,pt)~ nt xt pxt t (1{pt)nt xt ð2Þ ð2Þ Where: Where: November 2011 \| Volume 5 \| Issue 11 \| e1387 November 2011 \| Volume 5 \| Issue 11 \| e1387 2 www.plosntds.org Estimating Selection from Mosquito Field Data www.plosntds.org www.plosntds.org Estimating Selection from Mosquito Field Data Estimating Selection from Mosquito Field Data scenario but because the fixed values of h and s could be drawn from a distribution, the total number of runs was 30063 = 900. As before, the performance of the algorithm under conditions of temporal heterogeneity was assessed by defining the true value as mean over the generations, and calculating the correlation coefficient between the estimated and true values and how frequently the true value was included in the 95% confidence interval. with the inclusion of the last generation, the only sampling point in the subset that captures the incipient frequency rise. The ML parameter estimates in Table 2 were achieved in 34 to 83% of the 1000 ML runs indicating that a significant proportion of the estimation routines converged onto local maxima. Analysis of the Aedes aegypti dataset resulted in the parameter estimates in Table 3. These ML estimations were obtained assuming 6, 9, 12, 16 and 20 generations per year. The estimation converged on the same ML value 76 to 96% of the runs. With a small percentage of the runs (0.005 to 0.16%) converged to a set of different parameters with a similar likelihood value but which were excluded because the estimated value of h was on the boundary of the constrained parameter range. The estimates of p0 and h were highly consistent irrespective of assumed number of generations per year and ranged from 0.0032 to 0.0035 and from 0.77 to 0.78, respectively. As expected the s was strongly dependent on the assumed number of generations per year and ranged from 0.042 to 0.15. Finally, it is important to note two features of our analyses that may not be obvious to non-specialists. Firstly, that the genetic parameters h and s describe the overall, net rate of spread of resistance alleles through natural populations and cannot formally distinguish where selection is acting. For example, they cannot determine whether selection was acting differentially on the adult or larval stages, whether fitness costs were associated with resistance, whether there was differential selection on the sexes, nor whether killing was likely to be in early or later adult stages, the latter being a topic of contemporary interest given recent suggestion by Koella and colleagues [11] that killing older adults will reduce the selective pressures for insecticide resistance. Results The analysis of idealized datasets (Table 1) suggest ML can accurately recover the underlying parameter values from optimal simulated data. Additionally, if most of the values were in the confidence interval, the mean range of the interval was as wide as the parameter range. For example in Figure 4 note that when dominance is constant at 0.75, 100% of the true values are in the confidence interval, but the average mean range is 0.86 (the parameter range is 0–1). The plotted simulated data and estimates do not traverse the entire range of the parameters values because they are the mean over the 80 locations, the central limit theorem predicts that these estimates will converge to the center of the distribution. Figure 2 shows six example simulations of the increase in resistance allele frequencies over 120 generations, under two dominance conditions (semi-recessive, h = 0.2 bottom panel, and semi-dominant, h = 0.8 top panel). Values of p0, s, and h appear in Table 2. The program appears less accurate when analysing subsets of the original data, particularly if the resistance allele is semi-recessive. When the resistance allele was semi-dominant, resistance increased rapidly and the true estimates were recovered if the subset included points that captured the pattern of increase, such as the subset 1. When the resistance allele was semi-recessive, the frequency was maintained at low levels for a long period, the true parameters values were either recovered (Subset 1) but within confidence intervals that were so large as to be uninformative, or were not even contained in the confidence intervals (Subset 2) even Simulations of a location with temporal heterogeneous selection pressure (dominance and/or selection changing in every genera- tion) are shown on Figure 5. Again, the model does not accurately recover the true parameters under conditions of temporal heterogeneity. The exception was the dominance parameter when it was held constant in a particular location with selection varying in each generation, the correlation coefficient between the estimate and the mean dominance value over the 80 locations was 0.86. Table 1. Details of 100 idealized simulated datasets. p0 h s Parameter range 0.01–0.04 0.2–0.8 0.1–0.3 r 0.94 0.99 0.99 TV (%)* 91 92 97 [ ]* 0.021 0.014 0.002 The simulated datasets were used to check the precision and accuracy of the ML procedure. Results *r correlation coefficient between original value and estimate, TV percentage of true values in the estimates 95% confidence interval and [ ] mean range value of the confidence interval. doi:10.1371/journal.pntd.0001387.t001 Table 1. Details of 100 idealized simulated datasets. Estimating Selection from Mosquito Field Data Secondly, the analyses were designed to recover the genetic parameters that resulted from past control program and, as such, they cannot explore the issue of how differing patterns of insecticide deployment drive resistance. This require a separate, formal modelling approach explicitly designed to investigate the differing impact of deployment strategies on driving resistance. These analyses have been described elsewhere, particularly for the agriculture pesticides [12–15]. Results from spatially heterogeneous datasets pooling data from 80 different locations are shown in Figures 3 and 4. The algorithm appears unable to consistently obtain accurate estimations of the parameters s and h under such heterogeneous settings, manifested by low values of correlation coefficients and many true values outside the 95% confidence interval of the estimate. For example, with the dominance estimations in Figure 3 when selection was constant at 0.6, only 12% of the true values fell within the confidence interval. Initial frequency values were accurately recovered in all simulated scenarios, possibly due to the the recursion dependency on the initial frequency. However, the estimation of selection and dominance coefficients was achieved with very low values of correlation coefficients between the estimates and the mean of the parameter over the 80 simulated locations (not very precise), in all different hypothetical scenarios. Estimating Selection from Mosquito Field Data In the simulations of spatial heterogeneity the values of h and s had to be fixed across locations (e.g. h = 0, 0.25, 0.5. 0.75 or 1) but in the simulation of temporal heterogeneity only one location was examined in each simulation so the values could be drawn from the underlying distributions. As before, 300 datasets were produced for each We tested the algorithm and program by analyzing 100 datasets simulated under idealized conditions using Equations 1 and 2. Initial frequency, dominance and selection coefficient were in the ranges 0.01–0.04, 0.3–0.8 and 0.1 to 0.3 respectively, all distributions were uniform. Three parameters values were selected for each dataset and held constant during the simulation, i.e., there November 2011 \| Volume 5 \| Issue 11 \| e1387 November 2011 \| Volume 5 \| Issue 11 \| e1387 3 Discussion Insecticide resistance research is largely focused on the identification of the mechanisms responsible for resistance, and whether the genetic mechanism is monogenic or polygenic, general or population specific and if there are associated fitness costs and developmental patterns [16]. The emergence and spread of resistance is well documented, but there is still a worrying lack of quantification of the evolution dynamics in populations under control [17] and its persistence in populations following cessation of control. The quantification of the strength of selection acting in November 2011 \| Volume 5 \| Issue 11 \| e1387 November 2011 \| Volume 5 \| Issue 11 \| e1387 4 www.plosntds.org Estimating Selection from Mosquito Field Data Figure 2. Simulated evolution of resistance allele frequency over 120 generations under two different scenarios of dominance relationship and analysing the full dataset or subsets of data. Specifications in Table 2. doi:10.1371/journal.pntd.0001387.g002 Figure 2. Simulated evolution of resistance allele frequency over 120 generations under two different scenarios of dominance relationship and analysing the full dataset or subsets of data. Specifications in Table 2. doi:10.1371/journal.pntd.0001387.g002 the wild has previously been attempted using direct laboratory and field trials, and indirect approaches using a variety of data, including patterns of DNA variability and spatial and temporal changes in allele frequencies [17,18]. Selection acting on insecticide resistance genes in the field was first estimated using genetic models for species in the genera Anopheles by Curtis et al. [19] and Wood and Cook [20], both were based on the observed changes in gene frequency over regular intervals and the latter also discussed estimation by deviations from the expected Hardy- Weinberg equilibrium frequencies. Both methods assumed a fixed level of effective dominance under field conditions. A recent example is the estimation of relative fitness by Livingston and Fackler [21] for pyrethroid resistance in insects that infest crops. In this case the magnitude of the estimates were similar to those obtained using traditional laboratorial direct approaches using non linear least squares estimation. The most refined work that we are the wild has previously been attempted using direct laboratory and field trials, and indirect approaches using a variety of data, including patterns of DNA variability and spatial and temporal changes in allele frequencies [17,18]. Selection acting on insecticide resistance genes in the field was first estimated using genetic models for species in the genera Anopheles by Curtis et al. Sampled generations and true parameter values and ML parameter estimates with respective 95% confidence intervals. doi:10.1371/journal.pntd.0001387.t002 mpled generations and true parameter values and ML parameter estimates with respective 95% confidence intervals. 10 1371/journal pntd 0001387 t002 generations and true parameter values and ML parameter estimates with respective 95% confidence intervals. 371/journal.pntd.0001387.t002 November 2011 \| Volume 5 \| Issue 11 \| e1387 Estimating Selection from Mosquito Field Data doi:10.1371/journal.pntd.0001387.t003 Pooling data from different locations can be seen as a reasonable option to minimize the lack of sampled generations and small sample size. The Ile1,016 mutation frequency dataset of Garcia et al. [6] provided the opportunity to apply the model to real field data. This data contains allele frequencies of mosquitoes collected in 78 different locations around Mexico since 1999. Insecticide use was not uniform across cities and towns in Mexico and will probably differ between years and in addition migration will probably lead to different initial resistance allele frequency. The estimates obtained from simulated pooled data demonstrated that this kind of data-pooling, which is probably inevitable in most surveys, is not very robust. The coefficients reported in Table 3 should simply be recognized as a rough estimate between the years 1999 to 2008 and that they may vary, albeit by an unknown amount, over time and space. aware of, quantifies selection coefficients and costs associated with resistance for Culex pipiens in Southern France, using spatial information from clines to estimate selective advantages and costs, and temporal information from a long-term survey to estimate the selection coefficients of alleles in each environment using a standard ML estimation approach [22,23]. We have described a ML method for simultaneously estimating the selection and dominance coefficients and an initial resistance allele frequency similar to that of [5], but we also tackled the effects of spatial and temporal differences in selection intensity that can arise as a result of different strategies of deployment of the insecticide, migration patterns and/or infrequent and sparse field sampling of mosquitoes. The approach described in this paper was accurate with simulated data but proved less robust when analysing few intermediate allele frequencies, especially when the resistance allele is recessive. The reason is that all resistance dynamics start from the same point (very low frequency) and end at the same point (very high frequencies) but in the absence of intermediate time points it is impossible to reconstruct the dynamics in between. If the sampling period covers only the onset of resistance or the final stages, when resistance is close to fixation, the accurate estimation of selection and dominance coefficients can be difficult. Estimating Selection from Mosquito Field Data The estimation is problematic because in the early stages heterozygotes prevail in the population, with a fitness Wrs = 1+hs for which there are a range of values of h and s that yield the same product hs. This is illustrated using subsets in Figure 2. The true values of s and h were 0.2 and 0.2 but the estimates were 0.45 and 0.07 (Table 2). The situation was even worse for subset 2 of the data (Figure 2) where the analysis inferred a completely different trajectory of resistance spread and the true values of h = s = 0.2 were estimated as h = 0.02 and s = 1.0 (Table 2). Once again, note that the fitness of the heterozygote was estimated as 1+hs = 1.02 which was relatively close to the true value of 1.04 and that it is the predicted value of the homozygotes, which were largely absent from this subset of the data, that were badly estimated (as Wrr = 2.0 rather than the true value of 1.2). p Equation 1 describes a highly idealized population, i.e., one that is large, randomly mating, and homogenous in time and space. It is therefore important to consider the extent to which our population differs from this paradigm and what consequence this may have for the results. A large population is required so that we can ignore genetic drift, i.e., random fluctuations in allele frequency around our predicted values. Drift is important in laboratory studies (see [24] for discussion) but in natural populations there is a consensus that genetic drift can be ignored provided 4Ne~s.10 where Ne is effective population size [25] and ~s is the weighted mean fitness of the resistance heterozygotes and homozygotes. Estimates of Ne provided by [26] for Aedes Aegypti ranged from 10–22 in different regions of Mexico. These estimates seem intuitively to be very small. The most likely explanation is that they are measure of historical population size, so may have been caused by founder effects and population bottlenecks in the distant past. Estimates of contemporary population sizes are more appropriate in the current context and most estimates of contemporary effective population sizes of vectors are much higher, for example, in the region of 1000+ for Anopheles gambiae [27–29]. It would be possible to introduce the effects of drift by simulating small populations sizes and sampling (with replacement) the parents of the next generation. Estimating Selection from Mosquito Field Data Nevertheless, the calculated fitness (1+hs) is very similar (1.04 and 1.03). On the other extreme, when resistance is almost fixed, there will be mainly homozygotes in the dataset (with fitness Wrr = 1+s), so estimating a dominance value will also be problematic because of the lack of heterozygotes (with fitness Wrs = 1+hs). Unfortunate- ly, this is a very common type of data where genetic surveys initially indicate resistance was absent then, once its presence was detected, a second survey was run and higher levels were detected. Our analyses indicate that it is highly unlikely that any robust genetic parameters can be obtained from these kind of fragmented datasets. Future surveillance surveys should consequently be optimized by choice of a proper sampling strategy and timeframe. It is therefore of extreme importance to sample as many generations as possible, even if it means collecting fewer individuals. There is an important difference between standard statistics and ML estimation. In standard statistics, the 95% CI should capture the likely variation in magnitude of parameter estimates. In ML it only captures the likely variation provided the model has identified the correct trajectory of allele frequency changes. This is problematic in incomplete datasets where many trajectories may provide similar fits to the observed data. It is absolutely essential to run numerous analyses from randomly selected starting parameter values to check for the presence of numerous trajectories of similar ML but with widely different parameter estimates. Table 3. Estimated p0, h and s parameters from field data. Table 3. Estimated p0, h and s parameters from field data. Parameter Generations/ year Best value 95% Confidence interval p0 6 0.0032 0.0032 0.0032 9 0.0033 0.0033 0.0033 12 0.0034 0.0034 0.0034 16 0.0034 0.0034 0.0034 20 0.0035 0.0035 0.0035 h 6 0.77 0.76 0.78 9 0.77 0.76 0.78 12 0.77 0.76 0.78 16 0.78 0.77 0.78 20 0.78 0.77 0.78 s 6 0.15 0.14 0.16 9 0.096 0.090 0.101 12 0.071 0.060 0.081 16 0.053 0.048 0.057 20 0.042 0.038 0.046 The dataset corresponds to field collected data on Ile1,016 resistance allele frequencies in Ae. Aegypti from Mexico. Assuming 6, 9, 12, 16 and 20 generations per year. doi:10.1371/journal.pntd.0001387.t003 The dataset corresponds to field collected data on Ile1,016 resistance allele frequencies in Ae. Aegypti from Mexico. Assuming 6, 9, 12, 16 and 20 generations per year. Discussion [19] and Wood and Cook [20], both were based on the observed changes in gene frequency over regular intervals and the latter also Table 2. Specifications of datasets of Figure 2. True Estimates [95% CI] Dataset Generations p0 h s p0 h s Complete 1:120 0.001 0.2 0.2 0.0010 [0, 0.0018] 0.17 [0.13, 0.38] 0.24 [0, 0.48] 0.001 0.8 0.2 0.0009 [0.0005, 0.0013] 0.81 [0.77, 0.84] 0.20 [0.19, 0.21] Subset 1 1:5,15:18,116:120 0.001 0.2 0.2 0.0025 [0, 0.0062] 0.45 [0, 1.5] 0.07 [0, 1] 0.001 0.8 0.2 0.0016 [20.0017, 0.0049] 0.77 [0.36, 1.18] 0.19 [0.08, 0.29] Subset 2 1,13,25,37,73,85,97,120 0.001 0.2 0.2 0.0014 [0, 0.0030] 0.02 [0, 0.18] 1.00 [0.96, 1] 0.001 0.8 0.2 0.0009 [20.0007, 0.0025] 0.80 [0.64, 0.96] 0.20 [0.16, 0.25] Sampled generations and true parameter values and ML parameter estimates with respective 95% confidence intervals. doi:10.1371/journal.pntd.0001387.t002 Table 2. Specifications of datasets of Figure 2. Table 2. Specifications of datasets of Figure 2. November 2011 \| Volume 5 \| Issue 11 \| e1387 November 2011 \| Volume 5 \| Issue 11 \| e1387 5 www.plosntds.org www.plosntds.org www.plosntds.org Estimating Selection from Mosquito Field Data However one of the key conclusions of this study is the difficulty of www.plosntds.org November 2011 \| Volume 5 \| Issue 11 \| e1387 November 2011 \| Volume 5 \| Issue 11 \| e1387 6 Estimating Selection from Mosquito Field Data Figure 3. Effect of spatial heterogeneity (pooled data from 80 simulated locations) on estimates of initial allele frequency, dominance and selection parameters. The value of the selection coefficient was held constant at 0.1, 0.3, 0.6, 0.8 or 1 in all locations and in every generation, hence there are five rows of results corresponding to each of the 5 values of the selection coefficient. Dominance (h,<(0,1)) varied between simulated locations, but was constant over time within each location. The true value is the mean parameter value over all locations. The Pearson correlation coefficient (r) is between estimated and true values. TV is the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is the mean width of the 95% confidence interval in all runs. doi:10.1371/journal.pntd.0001387.g003 Figure 3. Effect of spatial heterogeneity (pooled data from 80 simulated locations) on estimates of initial allele frequency, dominance and selection parameters. The value of the selection coefficient was held constant at 0.1, 0.3, 0.6, 0.8 or 1 in all locations and in every generation, hence there are five rows of results corresponding to each of the 5 values of the selection coefficient. Dominance (h,<(0,1)) varied between simulated locations, but was constant over time within each location. The true value is the mean parameter value over all locations. The Pearson correlation coefficient (r) is between estimated and true values. TV is the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is the mean width of the 95% confidence interval in all runs. doi:10.1371/journal.pntd.0001387.g003 obtaining good quality estimates of genetic parameters from field data, so we prefer to ignore the effects of drift, and simply point out that the stochastic variation introduced by drift will likely further decrease our ability to recover accurate genetic parameter values from field data. The second requirement, that mating occurs at random is unlikely to be true given the geographical scale of our surveys. It would be relatively straightforward to incorporate this effect by including Wrights F statistics in Equation 1. www.plosntds.org www.plosntds.org Estimating Selection from Mosquito Field Data However, there was no evidence of significant departure from Hardy-Weinberg in our November 2011 \| Volume 5 \| Issue 11 \| e1387 November 2011 \| Volume 5 \| Issue 11 \| e1387 7 Estimating Selection from Mosquito Field Data Figure 4. Effect of spatial heterogeneity on estimates of initial allele frequency, dominance and selection parameters. The value of the dominance coefficient was held constant at 0, 0.25, 0.5, 0.75 or 1 in all locations and in every generation, hence there are five rows of results corresponding to each of the 5 values of the dominance coefficient. The value of the selection coefficient (s,N(0.15, 0.025)) varied between locations, but was held constant over time in each location. The true value is the mean parameter value over all locations. The Pearson correlation coefficient (r) is between estimated and true values. TV is the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is the mean width of the 95% confidence interval in all runs. doi:10.1371/journal.pntd.0001387.g004 Figure 4. Effect of spatial heterogeneity on estimates of initial allele frequency, dominance and selection parameters. The value of the dominance coefficient was held constant at 0, 0.25, 0.5, 0.75 or 1 in all locations and in every generation, hence there are five rows of results corresponding to each of the 5 values of the dominance coefficient. The value of the selection coefficient (s,N(0.15, 0.025)) varied between locations, but was held constant over time in each location. The true value is the mean parameter value over all locations. The Pearson correlation coefficient (r) is between estimated and true values. TV is the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is the mean width of the 95% confidence interval in all runs. doi:10.1371/journal.pntd.0001387.g004 vectors in Africa show unpredictably high levels of heterogeneities in resistance even across relatively small distances [30]. As mentioned by [5] simple models cannot account for the alteration of selection pressure by long term changes in the environment. More complex models that consider geographic clines and the antagonist effect of selection-migration, should be more accurate, dataset (results not shown) so this strategy was not required. The assumption that the population is homogenous in space is clearly untrue. www.plosntds.org www.plosntds.org Estimating Selection from Mosquito Field Data Pooling of data from different regions was required to increase sample size and frequency because mosquito collections were not uniform at the same location. The simulation results demonstrate the dangers of this approach and work on malaria November 2011 \| Volume 5 \| Issue 11 \| e1387 8 Estimating Selection from Mosquito Field Data Figure 5. Effect of temporal heterogeneity on estimates of initial allele frequency, dominance and selection coefficients parameters. Three different scenarios were simulated: (A) dominance and selection are different in every generation, (B) selection coefficient was held constant in all generations but dominance was allowed to vary, (C) dominance was held constant in all generations while the selection coefficient was allowed to vary. The Pearson correlation coefficient (r) between estimate and true value is shown. TV refers to the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is the mean range of the 95% confidence interval in all runs. doi:10.1371/journal.pntd.0001387.g005 Figure 5. Effect of temporal heterogeneity on estimates of initial allele frequency, dominance and selection coefficients parameters. Three different scenarios were simulated: (A) dominance and selection are different in every generation, (B) selection coefficient was held constant in all generations but dominance was allowed to vary, (C) dominance was held constant in all generations while the selection coefficient was allowed to vary. The Pearson correlation coefficient (r) between estimate and true value is shown. TV refers to the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is the mean range of the 95% confidence interval in all runs. doi:10.1371/journal.pntd.0001387.g005 Figure 5. Effect of temporal heterogeneity on estimates of initial allele frequency, dominance and selection coefficients parameters. Three different scenarios were simulated: (A) dominance and selection are different in every generation, (B) selection coefficient was held constant in all generations but dominance was allowed to vary, (C) dominance was held constant in all generations while the selection coefficient was allowed to vary. The Pearson correlation coefficient (r) between estimate and true value is shown. TV refers to the percentage of the true values that are included in the 95% confidence interval of the estimate. [ ] is the mean range of the 95% confidence interval in all runs. www.plosntds.org www.plosntds.org Estimating Selection from Mosquito Field Data This resulted in estimates of p0 = 0.0006 (95% CI: 0.0001–0.0010 ), h = 0.89 (95% CI: 0.84– 0.95), s = 0.20 (95% CI: 0.18–0.21) which are similar to those obtained using data from all generations (Table 2, the dominance coefficient is higher but the confidence intervals overlap). The second point is that dominance levels acting in the field may be much higher than those observed in the laboratory. The most plausible explanation is that mosquitoes in the wild are encountering low levels of insecticide that are insufficient to kill heterozygotes. Increasing dominance greatly increases the rate at which resistance develops. This suggests that insecticide applica- tions should be enforced in such a way that ensure high coverage with high doses. Our results suggest that the doses being applied may be inadequate and that pursuing the current deployment settings will lead to the rapid increase of resistant mosquitoes and eventually to the complete inefficiency of permethrin in the combat of dengue in Mexico. The estimated values of h, 0.77 to 0.78, point to partial dominance of the resistance allele under field settings. Alleles conferring knockdown resistance were found to be to be recessive or semi-recessive in their influence in Anopheles gambiae s.s. [34], but there is strong evidence for partial dominance or additive effects of Ile1,016 from two laboratory studies of knockdown and survival in strains or families of Aedes aegypti segregating for the Ile1,016 allele. Saavedra-Rodriguez et al. [35] found that 127 of 221 heterozygotes recovered from permethrin knockdown and showed later [36] that when considering overall survival the differences among the three phenotypes appear additive. Dominance in the field is dependent on the concentration and decay of the insecticide (see Figure 1), under this situation the resistant allele will be effectively dominant and we think that our results of intermediate dominance of Ile1,016 reflect this effect [37]. This interpretation is supported by Roush and Tabashnik [31], who reported the same situation of partial dominance for cyclodienes and lindane, diazinon, malathion and also for pyrethroids, where 20–60% of the heterozygotes survived exposure in a field setting. There is ongoing debate about differences between laboratory and field settings that extended to the evolution of insecticide resistance itself, some suggesting that resistance in the fields tends to be based on an allele of major effect at a single locus whereas resistance obtained in the laboratory is usually polygenically based [38]. Estimating Selection from Mosquito Field Data doi:10.1371/journal.pntd.0001387.g005 Nevertheless the estimated value for p0 (0.0032–0.0035), was in the higher range of 1022 to 10213 expected when a pesticide is first introduced, based on mutation-selection equilibrium [31]. This initial p0 value reflects the frequency prior to the sampling period. Since 1950, vector control programs in Mexico have used a series of insecticides. DDT was used extensively for indoor but the amount of data necessary make the implementation unlikely in most settings. This model in its simplicity presents a straightforward way to obtain estimates of fitness parameters. The fact that only information about resistant allele frequencies is necessary should make it easier to apply, and yet even such a simple data design is difficult to implement. November 2011 \| Volume 5 \| Issue 11 \| e1387 November 2011 \| Volume 5 \| Issue 11 \| e1387 9 Estimating Selection from Mosquito Field Data house spraying from 1950–1960 and was still used in some locations up until 1998. Malathion was later used for ultra-low volume space spraying of wide areas from 1981 to 1999. In 2000, programs switched to permethrin based insecticides [32]. The spread of resistance genes in a treated region will depend on the initial resistant allele frequency and it is known that resistance development in pest organisms can occur within 5–100 generations [31]. The relatively high initial frequency estimated explains the immediate, dramatic increase in frequencies of Ile1,016 from the late 1990s to 2006–2008 [6] neglecting genetic drift. house spraying from 1950–1960 and was still used in some locations up until 1998. Malathion was later used for ultra-low volume space spraying of wide areas from 1981 to 1999. In 2000, programs switched to permethrin based insecticides [32]. The spread of resistance genes in a treated region will depend on the initial resistant allele frequency and it is known that resistance development in pest organisms can occur within 5–100 generations [31]. The relatively high initial frequency estimated explains the immediate, dramatic increase in frequencies of Ile1,016 from the late 1990s to 2006–2008 [6] neglecting genetic drift. generations per year will be approximately the same in a timescale of 20 years. Most mutations encoding insecticide resistance are expected to incur a fitness penalty, compared to unmutated genes, in the absence of insecticide. There is some field evidence of reduced fitness of Ile1,016 mutations in Aedes aegypti in permethrin free environments [6] which leads us to make two technical points. Estimating Selection from Mosquito Field Data Firstly, that the selection and dominance coefficients reported here are overall values that combine the mutations benefit when encountering insecticide and any fitness effect in insecticide-free areas. Secondly, the method can equally be applied to measure negative selection pressures, (i.e., when a mutation is being lost from a population) from field data on the mutation after insecticide is withdrawn. As expected, the strength of selection increased as the number of generations per year decreased, whereas there was less time to get to the same frequency of resistance allele. Selection coefficients ranging from 0.042 to 0.053 (assuming 20 and 16 generations per year) are similar to the selection coefficients of DDT and dieldrin resistant phenotypes in Anopheles mosquitoes that have been previously estimated to be on the order of 0.013–0.061 [16]. The values for 0.071 and 0.097 (12 and 9 generations per year) are in the range of what was estimated for antimalarial drug resistance: 0.05–0.1 [33], however the value of 0.147 for 6 generations was higher than any previous estimates. This is the first time selection for insecticide resistance has been quantified in this species and should be seen as a preliminary estimate. Two factors are of particular relevance to field work. Firstly, that surveillance needs to be continuous so that a full dataset covering the whole period of resistance spread becomes available upon which to base these estimates. This may mean monitoring sentinel sites for long periods when resistance is rare or absent, but a continuous dataset is a prerequisite for accurately estimating the dynamics underlying the spread of resistance. Note that a continuous dataset does not necessarily mean collecting samples every generation. The reason the analysis could fail to recover the true parameters (Table 2) was because of large gaps in the survey: simulations of semi-dominant mutations lacked samples from periods of intermediate frequency, while simulations of semi-recessive mutations only contained data from the early stages (Figure 2). Operationally, this suggests that regular, rather than intensive but periodic, sampling is the best strategy. As an example, we re-analysed the semi-dominant dataset but just incorporated samples every 10 generation, i.e., at generations 1, 10, 20, 30:120. Acknowledgments The authors would like to thank Hilary Ranson and Tiago Anta˜o for providing useful comments on earlier versions of the manuscript, and three anonymous reviewers for helpful comments and discussion regarding the manuscript. The number of generations under natural conditions for this species was estimated at 20 or more among strains in field conditions in Brazil, this leads us to consider the results with the highest number of generations as the most likely, but because Aedes aegypti eggs can survive desiccation for months and hatch once submerged in water [39], the number of generations is variable. Nevertheless, the predicted resistance frequency trajectory using equation 1 and the different estimates obtained assuming different Estimating Selection from Mosquito Field Data Our results show rapid selection of mutations at a single locus. www.plosntds.org References Fisher R, Bennett J (1999) The genetical theory of natural selection: a complete variorum edition Oxford University Press. 318 p. 8. Smith JM (1998) Evolutionary genetics Oxford University Press. 354 p. 8. Smith JM (1998) Evolutionary genetics Oxford Un 29. Solano P, Ravel S, Bouyer J, Camara M, Kagbadouno MS, et al. (2009) The population structure of glossina palpalis gambiensis from island and continental locations in coastal guinea. PLoS Negl Trop Dis 3: e392. 9. R Development Core Team (2010) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org. ISBN 3-900051-07-0. 30. 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https://openalex.org/W2997706414	https://online-journal.unja.ac.id/chp/article/download/7936/9790	Indonesian	null	Sintesis dan karakterisasi fotokatalis ZnO/karbon aktif dan aplikasinya pada degradasi rhodamin B	Chempublish journal	2,019	cc-by	3,661	Chempublish Journal Vol. 4 No. 2 (2019) 101-113 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 Sintesis dan karakterisasi fotokatalis ZnO/Karbon Aktif (KA) dan aplikasinya pada degradasi rhodamin B Restina Bemis, Nelson, Ngatijo, Siti Nurjanah, Nur Aini Maghviroh Program Studi Kimia, Fakultas Sains dan Teknologi, Universias Jambi, Jambi. Jl. Jambi-Ma. Bulian KM 15 Mendalo Indah, Jambi, 36361 e-mail: bemisrestina@gmail.com Diterima: 30 Oktober 2019 / Disetujui: 23 Desember 2019 / Dipublikasi online: 31 Desember 2019 DOI: https://doi.org/10.22437/chp.v4i2.7936 ABSTRAK Telah dilakukan sintesis dan karakterisasi ZnO/KA sebagai fotokatalis untuk mendegradasi rhodamin B. Fotokatalis ZnO/KA disintesis menggunakan motode solid state, Zn(NO3)2.4H2O sebagai prekursor dan karbon aktif dari tempurung kelapa. Pola difraksi XRD menunjukkan bahwa fotokatalis ZnO/KA memiliki struktur heksagonal wurzite dengan ukuran kristal 0,12 µm. Hasil UV-Vis menunjukkan ZnO memiliki nilai energi celah pita (band gap energy, Eg) sebesar 3,12 eV dan hasil SEM menunjukkan morfologi permukaan tidak beraturan dan mangalami aglomerasi. Analisis EDS pada ZnO dan ZnO/KA memberikan persentase massa elemen Zn = 59,4%; O = 38,6% dan Zn= 66,9%; O=23,6%; C=7,8%. Hasil uji aktivitas fotokatalitik menunjukkan bahwa kondisi optimum degradasi 10 ppm rhodamin B memerlukan 200 mg fotokatalis ZnO/KA dan waktu radiasi sinar UV selama 90 menit, persentase degradasi sebesar 86,84%. Dari data analisis LC-MS menunjukkan terjadi degradasi molekul rhodamin B (m/z= 442,88) membentuk senyawa intermediet dengan perbandingan rasio massa dan muatan sebesar 387,30; 359,01; 331,12; 132,79 dan 117,03 m/z. Kata kunci: fotokatalis ZnO/KA, fotodegradasi, rhodamin B, solid state Kata kunci: fotokatalis ZnO/KA, fotodegradasi, rhodamin B, solid state ABSTRACT Synthesis and characterization of ZnO/activated carbon as photocatalyst has been carried out to degrade rhodamine B. ZnO/activated carbon photocatalyst has been synthesized using solid state method, Zn(NO)2.4H2O as precursor and activated carbon from coconut shell. The XRD diffraction pattern shows that the ZnO/ activated carbon photocatalyst has a hexagonal wurzite structure with a crystal size of 0.12 µm. UV-Vis results showed that ZnO had a band gap energy (Eg) value of 3.12 eV and SEM results showed irregular surface morphology and experienced agglomeration. The EDS analysis of ZnO and ZnO / activated carbon gives the mass percentage of element Zn = 59.4%; O = 38.6% and Zn = 66.9%; O = 23.6%; C = 7.8%. The results of the photocatalytic activity test showed that the optimum condition for rhodamine B 10 ppm degradation requires 200 mg of ZnO/activated carbon photocatalyst and 90 minutes of UV light radiation, the percentage of degradation was 86.84%. From the LC-MS analysis data showed that the degradation of rhodamine B (m/z = 442.88) molecules formed an intermediate compound with a ratio of mass and charge ratio of 387.30; 359.01; 331.12; 132.79 and 117.03 m/ z. Keywords: fotocatalys ZnO/KA, fotodegradation, rhodamin B, solid state PENDAHULUAN 101 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 Air limbah industri tekstil dapat menimbulkan masalah serius terhadap lingkungan karena terbuat dari senyawa organik azo yang terikat dengan gugus benzene dan bersifat non- biodegradabel, beracun serta stabil sehingga sulit terdegradasi secara alami (bakteri dan panas) (Manurung, et al., 2004; Rashed, et al., 2007). Rhodamin B merupakan salah satu jenis zat warna sintesis yang biasa digunakan dalam industri tekstil. Rhodamin B termasuk pewarna sintetis kationik golongan xanthene bersifat toksik, reaktif, karsinogenik dan dapat terdekomposisi menjadi senyawa yang lebih berbahaya (Kurnia, 2011). Bahaya serius dapat timbulkan bila terakumulasi dalam tubuh yaitu dapat menyebabkan gangguan fungsi hati, ginjal, bahkan menimbulkan kanker hati (Agusriyanti, 2014). Fotodegradasi merupakan metode yang saat ini banyak digunakan karena praktis dan ekonomis, memiliki kemampuan menghasilkan oksidator kuat dengan daya oksidasi tinggi, mampu mendegradasi hingga tingkat mineralisasi, mampu mendegradasi senyawa yang tidak dapat didegradasi secara biologi, dapat digunakan pada senyawa organik maupun anorganik, dan menggunakan energi rendah (energi cahaya) sebagai pengaktif (He, et al. 2009). Fotodegradasi berprinsip pada penggunaan fotokatalis berbahan semikonduktor. ZnO memiliki energi celah pita (band gap/Eg) sebesar 3,2 eV sehingga dapat digunakan sebagai fotokatalis. Beberapa hasil penelitian mengenai potensi ZnO sebagai fotokatalis telah dilaporkan diantaranya munujukkan bahwa ZnO merupakan fotokatalis yang aktif (Sakthivel, et al., 2003) dan mampu mendegradasi polutan hingga 82,18 % (Kristriya, 2013). ZnO sebagai fotokatalis memiliki keterbatasan dalam menyerap zat yang akan didegradasi, sehingga perlu dilakukan modifikasi permukaan. Karbon aktif dapat digunakan sebagai pengemban karena mempunyai luas area permukaan yang spesifik dengan struktur berpori sehingga diharapkan dapat meningkatkan daya adsorpsi dan aktivitas fotokatalitik ZnO. Alat dan Bahan Peralatan yang digunakan adalah alat-alat gelas, pipet tetes, spatula, batang pengaduk, corong Buchner, timbangan analitik, ayakan 100 mesh, indikator universal, pH meter, pengaduk magnetik, pemanas, cawan porselen, mortal dan pastle, seperangkat alat refluks, kertas saring, aluminium foil, oven, furnace, 102 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 kotak radiasi, lampu in-Lite TUV 30W/G T8 dengan panjang gelombang 253,7 nm, fourier transformation inrfa-red (FTIR), X- ray Diffraction (XRD), Scanning Electron Microscopy (SEM-EDS), Spektrofotometer UV-Vis dan liquid chromatography-mass spectroscopy (LC-MS). Bahan yang digunakan adalah Zn(NO3)2.4H2O, etanol absolute, trietanolamin, NaOH, HCl, karbon tempurung kelapa. Sintesis ZnO Sebanyak 8,17 gram Zn(NO3)2.4H2O dilarutkan dalam etanol dan diaduk menggunakan magnetic stirer selama 30 menit. 12,5 mL Triethanolamine (TEA) ditambahkan secara perlahan disertai pengadukan konstan dan pemanasan pada suhu 80˚C selama 30 menit, sehingga diperoleh koloid-gel berwarna putih. Koloid- gel dioven pada suhu 50˚C selama 2 jam kemudian di diamkan selama 24 jam pada suhu ruang (aging). Koloid-gel disaring menggunakan corong Buchner. Residu dikeringkan dengan pemanasan oven suhu 60˚C selama 14 jam dan kemudian dikalsinasi pada suhu 750˚C selama 2 jam (Shokuhfar, et al., 2008). Serbuk padatan yang dihasilkan dikarakterisasi menggunakan XRD, FTIR, SEM- EDS dan spektrofotometer UV-Vis. Aktivasi Karbon Sebanyak 50 gram serbuk karbon tempurung kelapa ditambahkan larutan NaOH 1M. Larutan diaduk menggunakan pengaduk magnetik selama 4 jam disertai pemanasan suhu 85˚C. Larutan disaring dan dicuci dengan air destilasi hingga pH netral. Residu dikeringkan mengunakan oven pada suhu 110˚C selama 1 jam dan dilanjutkan dengan kalsinasi pada suhu 600˚C selama 1 jam sehingga dihasilkan karbon aktif. Sintesis ZnO/KA Sebanyak 1,6 gram ZnO sintesis ditambah 50 mL air destilat untuk menghasilkan larutan ZnO. Sebanyak 0,4 gram karbon aktif (KA) ditambahkan dalam larutan ZnO kemudian direfluks dengan pemanasan 90˚C selama 5 jam disertai pengadukkan menggunakan pengaduk magnetik. Larutan disaring dengan penyaring Buchner dan dikeringkan dalam oven pada suhu 120˚C selama ±30 menit. ZnO/KA kemudian dikalsinasi pada suhu 400˚C selama 4 jam (Septiani, et al., 2014). Fotokatalis ZnO/KA yang dihasilkan dikarakterisasi menggunakan XRD, FTIR dan SEM-EDS. Uji Aktivitas Fotokatalitik ZnO/KA 103 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 Penentuan massa optimum fotokatalis ZnO/KA Enam buah gelas kimia 100 mL diisi dengan 50 mL larutan rhodamin B 10 ppm pada pH 11, dan ditambahkan fotokatalis ZnO/KA sebanyak 0, 50, 100, 150, 200 dan 250 mg. Larutan diradiasi dibawah lampu UV sambil diaduk menggunakan pengaduk magnetik dengan kecepatan konstan selama 30 menit. Suspensi disaring dan filtrat diukur absorbansinya dengan spektrofotometer UV-Vis pada panjang gelombang maksimum rhodamin B. Penentuan waktu radiasi optimum fotokatalis ZnO/KA. Empat buah gelas kimia 100 mL diisi 50 mL larutan rhodamin B 10 ppm pada pH 11, dan ditambahkan fotokatalis ZnO/KA dengan jumlah optimum yang diperoleh pada prosedur sebelumnya. Campuran lalu diradiasi dibawah lampu UV selama 30, 60, 90 dan 120 menit sambil diaduk menggunakan pengaduk magnetik dengan kecepatan konstan. Masing- masing filtrat diukur absorbansinya dengan spektrofotometer UV-Vis pada panjang gelombang maksimum rhodamin B. larutan dengan nilai persentase degradasi optimum dianalisis menggunakan LC- MS. Spektrofotometer FTIR (Fourier Transformation Infra-Red) Analisis spektrum FTIR ZnO dan ZnO/KA bertujuan untuk mengidentifikasi gugus fungsi. Hasil spektrum FTIR ZnO dan ZnO/KA dapat dilihat pada gambar Gambar 1. Spektrum FTIR a.) ZnO Standar b.) ZnO Sintesis c.) ZnO/KA O S d b Gambar 1. Spektrum FTIR a.) ZnO Standar b.) ZnO Sintesis c.) ZnO/KA Tabel 1. Interpretasi serapan FTIR ZnO dan ZnO/KA Tabel 1. Interpretasi serapan FTIR ZnO dan ZnO/KA Tabel 1. Interpretasi serapan FTIR ZnO dan ZnO/KA 104 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 Bilangan Gelombang (cm-1) Gugus fungsi ZnO ZnO/ KA Jayaram an et al (2014) Yulianti (2012) Septiani et al ( 2014) Rentang 420 437 452,7 480 420 346-550 Zn-O Stretching - 1384,21 - - 1384 1333-1453 C-O Stretching 1637,06 1618,87 - 1639 1637 1600an O-H Bending 3454 3450,05 3425,5 3433 3400 3000-3500 O-H Stretching XRD (X-ray Diffraction) Karakterisasi XRD dilakukan pada ZnO dan ZnO/KA dengan tujuan untuk mengidentifikasi fasa Kristal, struktur Kristal dan mengetahui ukuran kristal. Gambar 2. Difraktogram XRD a) Standar ZnO b) ZnO sintesis c) ZnO/KA 2Theta Intensitas Gambar 2. Difraktogram XRD a) Standar ZnO b) ZnO sintesis c) ZnO/KA Gambar 2(a) merupakan pola difraksi ZnO standar COD-inorg N0.96-900- 4180 yang puncak dengan sudut 2θ pada 31,80˚ , 34,47˚ dan 36,29˚ dengan bentuk heksagonal wurzite (Kihara and Donnay, 1985). Gambar 2(b) menunjukkan pola difraksi ZnO sintesis pada 2θ yaitu 31,747˚, 34,426˚ dan 36,241˚. Kristal ZnO yang mempunyai bentuk struktur heksagonal wurzite. Hal yang sama juga diperoleh Shokuhfar et al (2008) dan Septiani et al (2014) yang memperoleh posisi sudut puncak difraksi pada daerah 2θ = 31,75˚, 34,40˚; dan 36,22˚ dengan bentuk struktur heksagonal wurzite. Gambar 2(c) merupakan pola difraksi sinar-X dari ZnO/KA. Berdasarkan difragtogram ZnO/KA teramati dengan jelas bahwa adanya pola yang sama antara ketiga pola puncak difragtogram 105 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 (gambar 2), dimana hal tersebut didukung pula dengan data sudut difraksi dari difragtogram ZnO/KA yang muncul pada kisaran daerah 2θ= 31,790˚; 34,449˚; dan 36,284˚ dengan kristal berukuran 0,12 µm. Dari hasil analisis difraksi tidak teramati kehadiran pola difraksi khas dari karbon aktif yang memiliki pola difraksi amorf. Hal ini diduga karena sedikitnya jumlah karbon aktif yang ditambahkan pada ZnO sehingga tidak mempengaruhi pola difraksi ZnO. SEM-EDS (Scanning Electron Miscroscopy-Energy Dispersive X-ray Spectroscopy) Morfologi permukaan dan unsur-unsur komposisi ZnO dan ZnO/KA dapat diidentifikasi menggunakan SEM-EDS. Gambar 3. Morfologi a) ZnO b) ZnO/KA a b a b Gambar 3. Morfologi a) ZnO b) ZnO/KA Berdasarkan gambar 3(a) memperllihatkan bentuk permukaan ZnO tidak beraturan dan terjadi aglomerasi. Menurut Kanade et al (2006), penggumpalan partikel ini terjadi karena adanya proses penyimpanan (aging) pada saat sintesis ZnO sehingga menambah waktu reaksi pembentukkan ZnO. Selanjutnya gambar 3(b) menunjukkan bahwa ZnO dengan bentuk tidak beraturan menyisip dan menyebar secara merata, penyebaran ini sebagian teramati berada pada permukaan karbon aktif yang memiliki rongga atau pori pada permukaannya. Menurut Cazetta et al (2011) ciri-ciri khas dari karbon aktif ditandai dengan adanya rongga atau pori pada bagian permukaannya. Dari rongga atau pori yang dimiliki karbon aktif dapat membantu kinerja proses adsorpsi ZnO sehingga meningkatan aktivitas fotokatalitik ZnO dalam mendegradasi zat warna. Selain itu, karbon aktif berfungsi sebagai penghalang terjadinya penumpukan partikel ZnO sehingga dihasilkan luas permukaan ZnO yang besar, semakin luas 106 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 permukaan fotokatalis mampu meningkatkan aktivitas fotokatalitik (Septiani et al., 2014). Berdasarkan hasil analisis menggunakan softwere Image-J diperoleh ukuran partikel ZnO dan ZnO/KA sebesar 5,4 µm. Berikut adalah spektrum EDS dari ZnO dan ZnO/KA dapat dilihat pada gambar 4. Gambar 4. Spektrum EDS a) ZnO dan b) ZnO/KA b. ZnO a. ZnO/karbon aktif Energi (keV) counts/sec counts/sec Energi (keV) counts/sec Gambar 4. Spektrum EDS a) ZnO dan b) ZnO/KA Tabel 2. Hasil komposisi elemen ZnO dan ZnO/KA menggunakan EDS Sampel Elemen Zn O Al C ZnO 59,4% 38,6% 2,0% - ZnO/KA 66,9% 23,6% 1,7% 7,8% Tabel 2. Hasil komposisi elemen ZnO dan ZnO/KA menggunakan EDS Berdasarkan tabel 4 terlihat bahwa dari kedua sampel berupa ZnO dan ZnO/KA memiliki komposisi 2 unsur yang dominan yaitu Zn dan O serta adanya elemen C pada ZnO/KA yang berasal dari karbon aktif. Dari persentase komposisi elemen terlihat bahwa persentase O mengalami penurunan. Hal ini dikarenakan adanya pengaruh temperatur kalsinasi yang cukup tinggi pada sintesis ZnO/KA sehingga menyebabkan semua kandungan O yang bukan merupakan ikatan Zn- O pada ZnO sintesis mengalami penguapan (Sirenden, 2012). 107 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 Penentuan Massa Optimum Fotokatalis ZnO/KA Penentuan massa optimum dilakukan dengan menambahkan fotokatalis ZnO/KA dari berbagai variasi massa yaitu 50; 100; 150; 200 dan 250 mg kedalam 50 mL larutan rhodamin B 10 ppm serta larutan dikondisikan pada pH 11 dan diradiasi selama 30 menit dibawah lampu UV. Selama proses radiasi UV, dilakukan pengadukan dengan pengaduk magnetik agar semua fotokatalis ZnO/KA terkena radiasi sinar UV (bersumber dari lampu UV Merck In-Lite TUV T8 Germicidal 30W) dan reaksi dapat berlangsung secara merata. Pada penelitian ini dilakukkan hal serupa pada larutan kontrol berupa larutan rhodamin B tanpa adanya penambahan fotokatalis ZnO/KA. Data variasi massa fotokatalis ZnO/KA terhadap degradasi rhodamin B dapat dilihat pada grafi di bawah ini. Gambar 5. Grafik penentuan massa optimum fotokatalis ZnO/KA. 0 22.02 45.065 52.033 65.052 62.779 0 10 20 30 40 50 60 70 0 50 100 150 200 250 300 Degradasi (%) Massa fotokatalis ZnO/karbon aktif (mg) Gambar 5. Grafik penentuan massa optimum fotokatalis ZnO/KA. Gambar 5 terlihat bahwa persentase degradasi meningkat seiring dengan meningkatnya penambahan jumlah fotokatalis ZnO/KA. Hal tersebut dikarenakan fotokatalis ZnO/KA sebagai fotokatalis membantu proses degradasi rhodamin B dan menyebabkan degradasi berlangsung lebih cepat. Namun seiring meningkatnya jumlah massa fotokatalis ZnO/KA yang ditambahkan (250 mg) menyebabkan terjadinya penurunan %D yang disebabkan tidak seimbangnya antara jumlah fotokatalis ZnO/KA dengan energi yang diberikan sebesar 30W. Energi sebesar 30 W yang diberikan pada fotokatalis ZnO/KA menyebabkan tidak maksimalnya proses eksitasi elektron dari pita valensi ke pita konduksi sehingga berpengaruh pada berkurangnya jumlah produk berupa radikal hidroksil (OH•) yang berperan sebagai agen pendegradasi (Wu, 1998). Penambahan fotokatalis ZnO/KA yang memberikan hasil persentase degradasi paling besar adalah pada penambahan fotokatalis sebanyak 200 mg dengan persentase degradasi sebesar 108 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 65,05%. Berikut adalah larutan yang telah diradiasi sinar UV dengan variasi massa fotokatalis. Gambar 1. Rhodamin B, a) sebelum b) setelah diradiasi sinar UV dengan variasi massa fotokatalis ZnO/KA a). b). b). a). Gambar 1. Rhodamin B, a) sebelum b) setelah diradiasi sinar UV dengan variasi massa fotokatalis ZnO/KA Penentuan Waktu Optimum Fotokatalis ZnO/KA Penentuan waktu optimum dilakukan dengan memvariasikan waktu radiasi sinar UV dan dilakukan penambahan massa fotokatalis ZnO/KA optimum yang diperoleh dari uji sebelumnya yaitu 200 mg. Penentuan waktu optimum dilakukan pada 30, 60, 90, dan 120 menit. Gambar 8. Grafik penentuan waktu optimum fotokatalis ZnO/KA. 0 65.052 79.929 86.838 85.832 0 20 40 60 80 100 0 20 40 60 80 100 120 140 Degradasi (%) Waktu (menit) Gambar 8. Grafik penentuan waktu optimum fotokatalis ZnO/KA. Dari gambar 8 terlihat bahwa semakin bertambahnya waktu radiasi menyebabkan persentase degradasi zat warna rhodamin B semakin meningkat. Hal ini dikarenakan semakin lama radiasi menyebabkan semakin lama waktu kontak antara foton dengan fotokatalis ZnO/KA sehingga semakin banyak elektron yang mengalami eksitasi dari pita valensi ke pita konduksi, sehingga akan lebih banyak terbentuknya superoksida (O2•-) dan radikal hidroksil (OH•) yang berfungsi sebagai oksidator dalam proses degradasi molekul Rhodamin B (Melysa, 2017). Pada penelitian ini penentuan waktu optimum dihentikan setelah dilakukan variasi waktu radiasi 120 menit. Penurunan % degradasi yang terjadi disebabkan karena fotokatalis yang digunakan telah melewati kemampuan 109 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 optimum untuk menghasilkan radikal hidroksil (OH•) yang berperan sebagai agen pendegradasi molekul rhodamin B. Menurunnya kemampuan tersebut dapat disebabkan akibat terjadinya proses rekombinasi elektron yaitu proses kembalinya elektron yang telah mengalami loncatan ke pita konduksi kembali ke pita valensi sehingga tidak dihasilkan spesies (pasangan h+ dan e-) pemicu reaksi pembentukkan OH• (Wu, 1998). Dengan demikian gambar 28 menunjukkan bahwa radiasi optimum adalah pada waktu 90 menit yaitu dengan persentase degradasi 86,84%. Secara visual terlihat bahwa saat dilakukan variasi waktu radiasi UV pada larutan rhodamin B 10 ppm yang telah ditambahkan fotokatalis ZnO/KA optimum, larutan tersebut mengalami perubahan warna dari merah muda terang menjadi sedikit merah muda. Gambar 9. Rhodamin B a). sebelum b). setelah diradiasi sinar UV dengan variasi waktu 30, 60, 90, dan 120 menit a). b). b). a). b). a). Gambar 9. Rhodamin B a). sebelum b). setelah diradiasi sinar UV dengan variasi waktu 30, 60, 90, dan 120 menit Dari data persentase degradasi yang diperoleh malalui analisis menggunakan spektrofotometer UV-Vis menujukkan terjadinya penurunan konsentrasi rhodamin B diakibatkan terjadinya degradasi molekul rhodamin B. Untuk mengetahui senyawa yang terbentuk setelah dilakukan degradasi menggunakan fotokatalis ZnO/KA maka dilakukan analisis menggunakan LC-MS (Liquid Chromatography- Mass Spectroscopy). Pada analisis LC-MS ini digunakan sampel rhodamin B optimum dengan persentase degradasi sebesar 86,84%. KESIMPULAN Berdasarkan penelitian yang telah dilakukan, maka dapat disimpulkan bahwa sintesis ZnO/KA telah berhasil disintesis dengan menggunakan metode solid state. Hasil XRD menunjukkan ZnO dengan struktur Kristal heksagonal wurzite dan polikristalin. Ukuran Kristal ZnO dan ZnO/KA adalah sebesar 0,12 µm. Hasil spektrum FTIR menunjukkan adanya serapan vibrasi ulur (stretching) Zn-O pada bilangan gelombang 420-437,56 cm-1 dan serapan stretching C-O yang diasumsikan berasal dari karbon aktif. Hasil SEM- EDS menunjukkan permukaan ZnO berbentuk tidak beraturan dan menggumpal. Nilai band gap ZnO yang diperoleh dengan metode Kubelka-Munk adalah sebesar 3,12 eV. Massa optimum fotokatalis ZnO/KA untuk mendegradasi rhodamin B 10 ppm adalah 200 mg dengan persentase degradasi sebesar 65,052%. Waktu optimum fotokatalis ZnO/KA untuk fotodegrdasi rhodamin B 10 ppm adalah selama 90 menit dengan persentase degradasi sebesar 86,838%. Hasil LC-MS menunjukkan terjadinya degradasi molekul rhodamin B dengan senyawa intermediet m/z= 387,30; 359,01; 331,12; 132,79 dan 117,03. Penentuan Waktu Optimum Fotokatalis ZnO/KA 4 No. 2 (2019) 101-113 yaitu proses perubahan senyawa intermediet menjadi senyawa target dengan massa yang relatif sederhana yaitu berupa CO2 dan H2O. Penentuan Waktu Optimum Fotokatalis ZnO/KA Berdasarkan kromatogram LC di peroleh beberapa waktu retensi yaitu 10,65; 13,20 ; 14,91 ; 15,93 ; 17,46 dan 18,48. Analisis lebih lanjut dilakukan pada ke enam waktu retensi yang dihasilkan dan diperoleh data senyawa Use the "Insert Citation" button to add citations to this document. intermediet berupa m/z massa molekul pada waktu retensi 10,65; 13,20; 15,93 dan 18,48 yang disajikan dalam tabel 3. 110 Chempublish Journal Vol. 4 No. 2 (2019) 101-113 Tabel 3. Hasil identifikasi senyawa intermediet rhodamin B Ret. Time (menit) Penelitian (m/z) Chen. et al., 2003 (m/z) He. et al., 2009 (m/z) Senyawa intermediet rhodamin B 15,93 331,12 331,1 331,2 Rhodamine (R) 13,20 359,01 359,1 359,3 N-ethylrhodamine (ER) 10,65 387,30 387,1 387,3 N,N-diethylrhodamine (DR) 10,65 387,30 387,1 387,3 N-ethyl-N’-ethylrhodamine (EER) - 415,1 415,3 N,N-diethyl-N’-ethyl-rhodamine (DER) 18,48 442,88 443,1 443,4 Rhodamine B (RhB) 15,93 132,79 - 132 2-Hydroxipentanedioic acid 3,20 117,03 - 118 Succinic acid Tabel 3. Hasil identifikasi senyawa intermediet rhodamin B Berdasarkan identifikasi menggunakan LC-MS diketahui terbentuknya beberapa senyawa intermediet diantaranya N-ethyl-N’-ethylrhodamine (EER), N,N- diethylrhodamine (DR), N-ethylrhodamine (ER), dan Rhodamine (R) yang terbentuk akibat adanya reaksi oleh radikal OH• yang menyebabkan terjadinya pemecahan molekul rhodamin B. Selain itu, pada penelitian ini diperoleh senyawa intermediet yang telah mengalami pembukaan cincin aromatik berupa 2-Hydroxipentanedioic acid dan Succinic acid. Senyawa intermediet dengan struktur ikatan terbuka ini nantinya dapat mengalami proses lebih lanjut menghasilkan senyawa yang lebih sederhana (senyawa target) diantaranya berupa CO2 dan H2O. Menurut Chen et al (2003) dan He et al (2009) proses degradasi rhodamin B terjadi secara bertahap yaitu terdiri dari empat proses yang diawali dengan proses N-de-ethylation. Proses N-de-ethylation yaitu proses pemutusan gugus alkil menghasilkan senyawa intermediet dengan m/z= 415,387; 359; 387 dan 331, namun pada penelitian ini senyawa intermediet yang dihasilkan berupa senyawa intermediet dengan m/z= 359; 387 dan 331. Proses selanjutnya adalah tahapan chromophore cleavage berupa pemecahan struktur konjugasi dari senyawa intermediet hasil proses N-de-ethylation membentuk senyawa dengan m/z= 166; 138 dan 122 (He et al., 2009) yang terbentuk akibat terjadinya reaksi antara radikal OH• dengan atom rhodamin B yang tidak stabil. Namun pada penelitian ini, senyawa dari hasil chromophore cleavage tidak ditemukan melainkan ditemukan munculannya senyawa dengan m/z = 132,79 dan 117,03 yang merupakan senyawa intermediet yang telah mengalami tahapan proses pembukaan ikatan cincin aromatik (opening ring) yang terjadi akibat adanya delokalisasi elektron dan reaksi dengan radikal hidroksil. Pemecahan atau degradasi molekul (produk opening-ring) terus berlanjut ketahap mineralisasi 111 Chempublish Journal Vol. DAFTAR PUSTAKA Agusriyanti S. 2014. Pemanfaatan Zeolit Alam Ciamis Sebagai Pengemban Fotokatalis TiO2 Untuk Fotodegradasi Zat Warna Rhodamin B. Skripsi. Yogyakarta: Universitas Negeri Sunan Kalijaga. 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https://openalex.org/W4205226484	https://toknowpress.net/submission/index.php/ijmkl/article/view/48/32	English	null	Work-Life Balance, Upward Career Mobility and Further Education: The Case for Working Mothers	International journal of management, knowledge and learning	2,021	cc-by-sa	11,263	Work-Life Balance, Upward Career Mobility and Further Education: The Case for Working Mothers Marisabelle Camilleri Pegaso International and MCAST, Malta marisabelle.camilleri@mcast.edu.mt Damian Spiteri University of Malta, Malta damian.spiteri@um.edu.mt Damian Spiteri University of Malta, Malta damian.spiteri@um.edu.mt Purpose: This paper aims to explore how working mothers make sense and respond to their professional desires, responsibilities and expectations within the context of motherhood. It also seeks to explore what it takes for mothers to live up to their potential at work, what it takes for mothers to reach top positions in their field of work and the challenges they face. All of this with the ultimate aim of pooling opinions as to which incentives, strategies or measures can help reduce the conflict between family life and work. Study design/methodology/approach: Via a phenomenological approach and through the lens of social constructivism, this qualitative study is based on interviews with seven mothers holding executive or managerial positions in Malta. Findings: Balancing family and work proves to be extremely difficult, especially when the mother holds executive or managerial roles, and this comes with much stress and requires constant planning and organisation, time management, perseverance, resilience, spousal and parental support, and completely sacrificing one's time for hobbies or similar interests. Mothers' innate drive to prioritise their families caused all of the interviewees to shape their careers or studies around family responsibilities, with all but one opting for career breaks when their children were still young, yet such a decision was never regretted. All the interviewees chose to pursue self-actualising roles at work at some point or another. Such an intrinsic drive to reach their full potential caused almost all of the interviewees to further their studies up to a certain extent. Originality/value: This paper provides raw insights into the struggles working mothers face when balancing work and family life, primarily if the mother seeks to actualise herself via a fulfilling career. The paper presents recommendations for policymakers, employers and society as a whole on how mothers' potential in the workforce can be cultivated. Volume 10 (2021) 305–321 \| https://www.doi.org/10.53615/2232-5697.10.305-321 International Journal of Management, Knowledge and Learning \| ISSN 2232-5697 Other more dated studies also convey that it is challenging for mothers in different fields of work to progress to top positions (Belkin, 2003; Zemon and Harrison Bahr, 2005; Ranson, Introduction Right up to the early decades in the post-war era, members of the teaching profession, who were females, were forced to choose between either getting married or working; they could not do both. In 2018, in Malta, 57.1% of mothers with children up to 17 years participated in gainful employment 2016, with 61.8% of them engaged in full-time employment. The rate of employed fathers, on the other hand, amounted to 95.6% in 2016 (NSO, 2018). Add to this discrepancy, female underrepresentation in senior positions in Malta ensues, with only 7% female managers compared to 11% male ones in 2015 in Malta (NSO, 2015). Hence, this research aims to shed light on the challenges mothers face in participating in the world of work and possibly, developing their careers further and advancing in their careers. According to a study by KMPG (2015) issued by the Centre for Labour Studies at the University of Malta, 50% of female managers in Malta are childless. Furthermore, the statement "women generally have less freedom to pursue their career due to family/children responsibilities" was the most significant for male and female respondents. The issue of "work-life balance" and "coping with responsibilities outside work" was cited as the main culprit for the female under- representation in managerial positions in Malta. "Getting the work-life balance right" was also cited as the main challenge of occupying CEO positions (Pace, 2017). In light of this, this paper aims to explore how mothers make sense and respond to their professional desires, responsibilities and expectations within the context of motherhood by, first and foremost, delving deeper into the compromises, if any, these mothers are constrained to do, in order to strike a balance between work and family life. Another aim of this paper is to explore if working mothers forfeit developing their careers further for motherhood responsibilities. What does it take for mothers to live up to their potential at work? Is there any lost potential in this regard? Are there any ways by which such a situation can be mitigated? Thirdly, this paper shall also identify what it takes for mothers to reach top positions in their field of work, the challenges they face and how easy or difficult it is to attain and maintain such positions. The ultimate aim is to pool opinions on which incentives, strategies or measures can help reduce any conflict between family life and work. Introduction Various studies (Aveling, 2002; Baber and Monaghan, 1988; Bielby and Bielby, 1984; Bhattacharyya, 2009; 2016; Granrose and Caplan, 1996; Herman and Lewis, 2012; Hoffnung, 2004, 2011; Lahiri-Dutt and Sil, 2014) claim that it is tough for women to "have it all" in terms of financial stability, marriage, children, and career. This refers not only to those working in STEM (science, technology, engineering, mathematics) sectors (Herman and Lewis, 2012) but also in other areas of economic activity too (Bhattacharyya, 2016). In a 2021 report by McKinsey and Co., it is stated that "women continue to face a "broken rung" at the first step up to manager: for every 100 men promoted to manager, only 86 women are promoted. As a result, men significantly outnumber women at the managerial level, which means there are far fewer women to promote to higher levels. The broken rung likely explains why the representation of women at the senior manager, director, and VP levels has improved more slowly than the pipeline overall". Other more dated studies also convey that it is challenging for mothers in different fields of work to progress to top positions (Belkin, 2003; Zemon and Harrison Bahr, 2005; Ranson, Volume 10 (2021) 305–321 \| https://www.doi.org/10.53615/2232-5697.10.305-321 306 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education 2005). Some women may delay becoming mothers altogether (Miller, 2008) or become self- employed (Duberley and Carrigan, 2012) as an alternative. Herman and Lewis (2012) and McIntosh et al. (2012) also portray how benefitting from family-friendly measures at work often forfeits chances for career progression in various sectors, whilst Brown (2010) shows that great conflict between home life and work still ensues irrespective of any adopted family-friendly measures. Going back in time, women did not have many labour market opportunities. Upon reaching motherhood, most women used to leave the labour market due to family responsibilities or opt for part-time employment. The present government's goal is to raise the female employment rate, as can be seen by the opening of new childcare facility services and reducing national insurance and taxes to women bearing children. Historically, gender discrimination in the working environment was common practice in Malta; and it was commonplace for male candidates to be preferred since females were destined to take a long time off work due to pregnancy and related childcare matters. Introduction With this in mind, employers would have a clearer insight into how to feasibly help harmonise the balance between the two and adopt ways that foster self-growth and facilitate mothers' self-actualisation at work. This generates greater fulfilment at work for the working mother, in turn maximising her value and input in the organisation. Literature review A central tenet of this paper is that motherhood is socially and culturally constructed. Up to a few years ago - perhaps even to this day - in several societies, including Malta, intensive mothering with its whole emotionally demanding and time-consuming child-centricity (Hays, Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education 307 1996), has been presented as the predominant women's discourse (Hays, 1996; Hilbrecht et al., 2008). This discourse often leads to setting standards that mothers feel obliged to reach to be seen as performing optimally in society. The child-caring role is seen as central to mothering, and it is given a dominant position in a woman's life. 1996), has been presented as the predominant women's discourse (Hays, 1996; Hilbrecht et al., 2008). This discourse often leads to setting standards that mothers feel obliged to reach to be seen as performing optimally in society. The child-caring role is seen as central to mothering, and it is given a dominant position in a woman's life. From a social constructionist perspective, the assigning of this dominant position to mothering comes about since the discourses and constructions of motherhood that are embedded in society are internalised by individual women, that utilise them as standards against which they tally their own experiences and practices to form their own ideologies (Elvin-Nowak and Thomsson 2001). This prevailing discourse of being a mother over being a worker can only be understood concerning other prevailing discourses about what it means to be a good mother. Ferdinand de Saussure, the father of structuralism, explained this when he stated that the meaning of a text is influenced by the way a language is structured. A sign can only convey meaning if it is differentiated from other signs in the system of signs. Likewise, from a social constructionist viewpoint, what it means to be a good mother cannot be objectified, as there is never a 'signified.' The notion of being a good mother is constructed by discourse participants in their various discourse communities as a collective. For this reason, there is no one way to be a good mother and combine this with being a working mother. Instead, there are likely many ways in which society can see them as 'good' mothers to their children and combine work with this discourse. Literature review Nightingale and Cromby (2002) explain that there is “a play of linguistic meaning and signification in force that is shaped and 308 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education constrained by embodiment, materiality, socio-cultural institutions, interpersonal practices and their historical trajectories (all of these structured by, and reproducing structures of, power) - such that language does not independently and thoroughly constitute our world” (p. 706). Therefore while it is legitimate to say that the birth and upbringing of a child marks a significant change in family dynamics; it also needs to be pointed out that the child's birth often leads to the formation of a 'motherhood constellation', which places the mother in a position to mentally give importance to all issues related to motherhood, with new boundaries are set as the family composition changes (Borghini et al., 2006). This expectation reinforces the symbolic dimension of the different roles of fathers and mothers, and in this respect, language forms the basic building blocks of a social system (Luckmann 2008, 277). This becomes entrenched in society as it leads to the communication construction of a collective memory where expectations about mothers are seen as taken-for-granted realities and makes these realities seem to be an invention passed down over time "consciously designed to deal with present problems" (Schwartz 1991, p. 221). Indeed, in the context of working mothers, numerous studies (e.g. Betz and Fitzgerald, 1987 in Whitmarsh, 2007; Ezzedeen and Ritchey, 2009; Brown, 2010) show that many mothers choose to interrupt their careers by choice in order to dedicate more time to caring for their children; yet if fathers do so, it may be assumed that such a decision would be taken for a set of reasons different to those of working mothers. It is likely for this reason that in many societies, mothers often find themselves constrained to modify their career aspirations to enable a more compatible match with their family responsibilities (Jackson and Scharman, 2002; Whitmarsh, 2007; Ezzedeen and Ritchey, 2009; Brown, 2010; Pas et al., 2011; Mirick and Wladkowski, 2018). This is primarily in the face of the shifting of the traditional motherhood ideology, so "women are expected to work at least a few days per week, yet they are also expected to mother intensively when at home" (SCP, 2008 in Pas et al., 2011). Literature review However, there are competing dominant discourses, such as a prevailing belief that "a woman's place is in the home". In that case, working mothers would likely divest themselves of their own self-identity to be 'good' mothers to their children (Hays, 1996). For instance, 'working mother guilt' may hinder a mother from engaging in work. This may arise, for instance, when responsibilities are not equally shared between working mothers and their partners (Bailey, 2000; Borg, 2011). Working mother guilt may also be augmented by what has been described as a "historical bias against motherhood" in the sense that in universities, working mothers are believed to drop out more and to progress academically at a slower rate and thereby perform less well than male or childless female counterparts (Brus, 2006; Kmec, 2013; Mason and Goulden, 2004; Mason et al., 2013). This is likely to follow the belief that since mothers tend to assume most caregiving roles, academic mothers experience greater "stress and pressure" than fathers, especially in managing the dual roles of being a parent and an academic (Wilton and Ross, 2017). This belief that working mothers are less likely to perform optimally in academic tasks is summed up by Young and Wright (2001, p.560) when stating that (for working mothers) "the balance of family and work (is likely to be) … exhausting and overwhelming under the best of circumstances". When considering Maltese couples with children, only 49% of women compared to 91% of men are employed full-time. This gap is higher when compared to the percentages of couples without children. Couples with children report having 96% of women regularly carry out cooking and housework, whereas only 31% of men carry out these tasks on the same frequency (European Institute for Gender Equality, 2018). Spousal positive reinforcement and support are crucial for mothers to maintain a career (Whitmarsh, 2007; Wingfield, 2011). Shared family and household responsibilities are also important (Borg, 2011) and may be influenced by language. From a social constructionist perspective, language does not only describe reality but co-constitutes it. Why is it acceptable to speak about a working mother, but had we to speak about a working father, this would be likely to raise eyebrows, at least in certain cultures? Literature review It may, however, be argued, based on the work of anthropologists like Sherry Ortner (see Ortner, 1974), that women's physiology and reproductive functions make her appear closer to nature whilst men look to adapt to their environment and therefore appear to be closer to broader cultural participation. Women's activities are consequently restricted to the domestic sphere, while men are licenced to go wider afield, becoming more prominently inserted in public spaces rather than private ones. The mother is also biologically better equipped to be the person rearing the children, implying that, particularly from a social constructionist perspective, culture, and society itself, may even lead to the generation of an 'internal conflict' for working mothers, since having a demanding career may be perceived as incompatible with being a 'good' mother. However, this does not negate that, particularly from contemporary Western societies' perspective, there are several advantages to working mothers retaining their work and their career. Besides the financial benefits derived from salaries, a working mother can be an inspiration for her children (Borg, 2011). Pas et al. (2011) show that a group of female doctors, who were also mothers, saw their work to enable them to be more self-resilient and independent, thereby serving as effective role models to their children. On the other hand, conflicts between work and family life have been associated with several negative consequences, both individually and for organisations themselves. These include higher levels of dissatisfaction at work, work distress and adverse reactions to the extra- organisational stressors if a personally satisfying work-life balance is not achieved (Mirick and Wladkowski, 2018). To make matters worse, in some countries, women tend to avoid availing themselves of family-friendly policies out of fear of losing their jobs (Cabrera, 2009). It is also possible that people do not avail themselves of the resources available out of shame in some cultural contexts, although this is not necessarily universal. For instance, Herman and Lewis (2012) explain that making extensive use of a childcare centre is still frowned upon in countries 309 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education such as Germany, even though it is common practice in France. A possible interpretation of this is that French mothers take career continuation upon their transition to motherhood for granted. Literature review The distinction that Herman and Lewis are drawing between Germany and France accentuates that the dilemmas that some working mothers may face about availing themselves of childcare facilities are not their personal issues, thereby requiring self-confidence to deal with, but rather may be more widely located in social contexts that may be oppressive, and thereby are political (Hanisch, 1970). Within the Maltese context, the demand for childcare facilities is increasing. However, this is not necessarily indicative of working mothers being in a better position to give priority to their careers, since it could be triggered by the rise of immigrant populations in Malta and also the economic necessities of certain families who may use childcare facilities because they feel constrained to do so as both parents have to work in order to make ends meet. Previous studies in Malta on occupational issues do not focus specifically on the issues of working mothers, even though countering gender discrimination has been the subject of attention from both academic and policy perspectives. The National Council for the Promotion of Equality (ncpe.gov.mt) has lobbied for further gender equality. Its efforts have likely borne fruit since even though Malta is seen as lagging the EU average in gender equality, it is one of the countries with the largest improvement over a decade - based on the results of the 2020 Gender Equality Index results. The University of Malta runs a Centre for Labour Studies, which, as stated on the university website, has as one its aims that of promoting social dialogue, active involvement and the effective participation of both male and female workers, and their representatives, in specific workplaces, and labour policy more generally. The approach adopted One's position can be more than one position, each competing with the other to be given attention, thereby pointing to the notion of multiple selves. Moustakas (1994) recommends that phenomenological research carefully follow a series of procedures, and an adaptation of Moustakas' approach has been adopted in this paper. First, Moustakas suggests commencing from developing a topic or problem that is liable to have deep personal meaning for those experiencing the phenomenon, as would be the case for working mothers, who must deal with the different demands of their homes and their workplaces. Moustakas then suggests an extensive literature review that would enable researchers to advance their knowledge on the subject they are studying and offer insights from different social, cultural, and temporal perspectives. Once the literature review has been completed, the researcher would then need to construct a set of interview questions designed to guide the interview process, using meta-reflection to understand one's different positions about what being a working mother infers. In the case of this research, this process was further enriched by discussions between the two researchers that also took into consideration their different gender and that their families were in different stages of development. They also used bracketing in assembling the questions to reduce bias. When brainstorming potential questions that they could ask, the researchers examined how their personal awareness might enter the research. Because of this meta-awareness, they put in efforts to suspend their own understanding so that the participants' voices would be elicited - as far as possible. Finally, Moustakas suggests that the schedule of questions would be flexibly put to interviewees when conducting the interviews to ensure that their perspective is captured as far as possible; and allow them to add further data that they considered relevant, thereby enabling them to a thick representation of reality to ensue. In other words, besides having "the purpose of obtaining descriptions of the life world of the interviewee in order to interpret the meaning of the described phenomena" (Kvale and Brinkmann, 2008, p. 3), semi-structured interviews have the additional advantage of making greater use of "knowledge-producing potentials of dialogues" by allowing room for the interviewees to follow-up on whatever points they wish to (Brinkmann, 2013, p.21). In the final stage of the research, Moustakas suggests that the researcher analyses the data, synthesising the discovered meanings to determine the essence of the experience. The approach adopted This paper aims to provide insights into working mothers' lives in Malta. It promotes an understanding of working mothers combining the commitments associated with family life with working life. The paper adopts a phenomenological approach that blends with its social constructionist orientation. Moustakas (1994) believes that the phenomenological approach captures the essence of people's individual experiences by synthesising them into a collective description of the phenomenon being studied. From a social constructionist paradigm, a study focused on how women construct their identities as family members, mothers, workers, colleagues, and people with a possible position in workplace hierarchies is a highly subjective one. As Gergen and Gergen (2003) explain, "social constructionist inquiry is principally concerned with explicating the processes by which people come to describe, explain, or otherwise account for the world (including themselves) in which they live" (p.15). This implies that, from a social constructionist paradigm, different people are liable to experience these roles and how they combine these roles differently. However, the same person will define them and redefine them differently in different phases in life. In addition, these ways of defining roles are bound in political discourse. Burr (2003) writes that "the popular representation of the good mother as one who spends time with her children when they are young and who sacrifices her own needs to theirs (effectively) helps to keep women out of full-time employment and ensures their economic dependency on men" (p. 123). In the light of this, as Creswell (2013, 2014) explains, a phenomenological study looks beyond how individuals construct meaning of their own experiences, focusing on how people report how they experience events differently within the extenuating circumstances in which they find themselves. Historically, Edmund Husserl is believed to have founded phenomenological research (Moustakas, 1994). Husserl believed that through actively engaging with a small number of individual research participants, who are actively associated with a phenomenon of interest, it would be possible to understand their lived experiences. McNamee and Gergen (1999) share 310 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education this aspiration to understand people's experiences using dialogue. One aspires to understand the other by broadening one's understanding of reality without losing sight of oneself. McNamee explains that "dialogue is a process of holding firmly to one's position while maintaining a curiosity and respect for another's very different position". The approach adopted This called on the researchers to explore the subjective understanding of an experience and any social, cultural, and historical ties to it, thereby enhancing its phenomenological validity. In the case of the research undertaken, this implied that the researchers needed to be vigilant to prevailing gender discourses and outlooks and political factors that may have enabled or impeded working mothers from achieving their aspirations. In this way, it was more likely that the research would reflect the "fluidity and complexity of ways of performing mothering practices alongside professional work …. where there are deeply embedded structural constraints but (there is) also some space for a personal agency" (Herman et al., 2012, p.3). According to Ponterotto (2005), the interaction between the interviewer and interviewee is crucial to uncover 'meaning' which needs to be brought forward through deep reflection. This lends itself to the adoption of a qualitative research design that gives rise to an analysis of how the participants’ account is embedded in the inter-relationship between language, culture, and society; and how it is this embeddedness that enables it to have meaning. Adopting a qualitative research design enables an in-depth understanding of the participants' personal lives to be rendered. Hakim (1987) defines qualitative research as "a direct window on the lives of the participants … (that) offers richly descriptive reports of individuals' perceptions, attitudes, 311 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education beliefs, views and feelings, the meaning and interpretations given to events and things, as well as their behaviour" (p.211). From a social constructionist point of view, where social reality is seen as emanating from the interactions between social actors and their historical and cultural contexts (Burr, 2003), qualitative research makes it possible for explorations to be undertaken; thereby, in the case of the current research undertaking, allowing the analysis to be focused on the women's subjective appreciation of their own lived experiences, including their studies and careers, as well as their personal lives and corresponding expectations. Field notes, later utilised to inform the analysis, referred to the tone of voice, facial expressions, and the researcher's thoughts. Each interview was recorded and transcribed verbatim to enhance confirmation and credibility (Lincoln and Guba, 1985; Guba and Lincoln, 1994) whilst preserving interviewee anonymity. Family support All the interviewees expressed satisfaction and fulfilment in their role at work, and all of them felt that they were living up to their potential. Nevertheless, the participants reported that things have not always been so. Most said they had to find ways of actualising themselves at work, without, as much as possible, failing to look after their families. Astrid said she waited for her children to grow up and be independent and said that now that she was free of certain obligations since her children were older, she could give more importance to her job and derive satisfaction from it. Likewise, Rose speaks about her need to contribute and derives a heightened sense of self-worth from doing so. She said, "I like the challenges my work gives me, the fact that I am contributing to improving people's lives, even though I am in the background, but what I do indirectly helps people's lives and my personal growth." Similar to what Ezzedeen and Ritchey (2009) found in a study on working mothers, having another role apart from that of a mother makes it possible for them to say that they feel 'complete'. Ritienne explains this by saying that "being a career woman is part of who I am, a part of my identity. It has helped me become more confident, assertive, and more aware of the realities around us. It has provided me with opportunities not only to improve myself personally and professionally but also to improve the quality of the life of my children", Rose, who likes to think she is on the brink of retirement, yet, says that she does not cease to take on new projects, talks about her yearning to put her abilities and experience to practice: "Look, I always say that if society did not want me to use my potential and push through and get involved in a thousand things, all that it was to do was to deprive me of education. The fact that I had an education, sitting back was just not good enough for me. I felt that my contribution had value. If I had not contributed and involved myself the way I did […], I would probably be somewhere banging my head against the wall […]. I would be a very depressed person […]. The approach adopted To ensure that consistency prevailed throughout the research collection part of the analysis, it was decided that all research interviews would be conducted by the female researcher only. It was presumed that the participants, who were all female, might have answered questions differently had they been interviewed by the male researcher. The participants were selected utilising a snowball sample, and seven interviewees participated. It so happens that all the participants live with their partner (male), which may reflect that most families with children in Malta are heterosexual couples. The following table provides some demographic information on the interviewees: Figure 1 Demographic data of the interviewees. Figure 1 Demographic data of the interviewees. Thematic analysis was used to analyse the data generated by the interviews. Based on previous research that the researchers had undertaken, thematic analysis was ideal for identifying, analysing, and eliciting patterns (themes) within data. In other words, the meaning that emerges comes from the data; any patterns in the responses given emerge out of the data rather than being imposed on it prior to the analysis (or collection) of the data (Strauss and Corbin, 1990). The analysis involved several steps, including reading and re-reading the transcripts, comparing, and sorting data and coding any observations and annotations comprised in the field notes. The codes were then examined, paying close attention to context so that common patterns and trends were accumulated. After a vigorous process of checking, rechecking, reflecting, and 312 and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education revising the links between the knowledge brought forward in the literature review and the data generated through the data sets, assertions and generalisations were then drawn out as theories or constructs were examined in light of relevant bodies of theory (Miles and Huberman, 1994; Miles, Huberman and Saldana, 2014; Roulston, 2014), following on from intensive discussions between the researchers. Family support The same can be said for Mary, who waited until her children were "old enough with my daughter at sixth form" to do her Masters' degree. She also went for an online course rather than one based on face-to-face learning. Some of the participants described more intense negative feelings even though they evidently put these aside to fulfil their career ambitions. Rachel described her heartbreak and feelings of guilt when she had to go abroad for five days for a meeting relating to her studies toward her doctorate. She vividly describes saying goodbye to her son, who was still a baby. However, some of the participants decided to turn down opportunities for career advancement. Rose, for example, refused two scholarships for her doctorate to be present for her daughter, who was doing her end-of-school exams. She said, "and yes, I regret it […]. I regret it because it was a difficult, crucial period in my daughter's life, in her studies, and I felt that I should not impose my further hurdles against her advancement. It was a crucial time. My place was here in Malta [not abroad]. I reasoned that my daughter did not ask me to come to this world. It was my doing, and so, my responsibility to look after […], but I cannot say that I am not hurt, that I am not hurt still, since I did not pursue my studies at a doctoral level then, and I find it almost impossible to start now." Most of the interviewed women have taken a career break or slowed down their careers either to attend to their children full-time or at some crucial period in their children's lives. Carol explains that "I stopped for two years to take a break because my child needed me, and I stopped my career for a while. In the end, I stopped; the father remained where he was. It is always the mother who has to give up". Rachel explains that in the first five or six years of her son's life, she opted not to go abroad on work-related trips, which may have negatively impacted her reputation in terms of publications. Family support My work gives me great satisfaction." On the issue of balancing work life and family life, Ritienne said: "my children are proud of me and look up to me…It gives me personal satisfaction as I thrive when there is a challenge, and thus I am able to feel happy in my everyday life. This impacts positively on me as a parent as the children can see that I love what I do. It sets a good example for them, too – they have seen me working hard and know that hard work pays off. They can also see that despite the pressures of work, they always come first…" However, balancing work and family life does not come without costs, as Carol explains when she says, "you have to make a choice. If you want to satisfy all the demands of the family, frankly, one should not go for a high-flying career. I know very well it does not work”. Ritienne also cautions against over-stressing herself by working, even though she believes she has "found the right balance" for herself. She says, "My suggestion to all women is: if you want a career and you have a family, know what it will cost you. If a woman feels that it will be too demanding and will create lots of stress and conflict with family life, my strong advice does not to go for a career. In the end, both will suffer because the psychological stress that you and those around you will have to endure will affect you dearly". All the interviewees mentioned studies as part of their working life. They all appeared keen to make studying as non-disruptive as possible to their roles as mothers and their family life. Astrid explained that she had delayed starting her master's degree for some time. She explained that 313 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education not furthering her education earlier is her most profound regret, but "it was simply not possible to be in a management position, with those hours, and study simultaneously, with the kids. She said that the turning point had come about last year when she had said to herself, "the children are growing," which led her to permit herself to enrol for her degree via distance learning not to disrupt her family. Family support Astrid recounts how, in addition to parental leave, she held a position as a Director for almost five years just because the hours were “convenient…for my children… it was convenient as it was more structured, with more fixed hours” despite “being desk-based and not really what I like doing… deep down I knew that I did not belong there” while Christabelle refused the position of a Head of School twice because of family responsibilities and only finally accepted it as she was guaranteed the possibility of stepping down at any time if she wanted to. Similarly, Mary also spent a few years at home to raise her kids and later waited until her children "were old enough to be a little independent, so if anything crops up, they will manage to do things on their own" until she opens up her own Childcare Centre. At the same time, Ritienne stopped working completely for four years when her two kids were young. Furthermore, she worked in the same school as her children for many years until she "could afford to work different hours when they started growing up". Spousal and parental support One of the managers interviewed in this study said "We should be pushing for partners to do more and take equal responsibilities so that you get the two partners devoting [time to care] […] sacrificing less on both sides of the parenting or care partnership". Christabelle and Carol firmly believe that it is the work commitment they went in for that caused them to jeopardise their duties as mothers, with Christabelle saying, "I have guilt feelings all the time […] a lot […] all the time […] everyday…that I am not doing my job well as a mother due to this job […] every day […] all the time […] huge guilt feelings every day […] and I am very worried about this." Carol confirms the existence of this struggle claiming that "Of course it does [clash]. Those who say that it does not are not true to themselves. Senior leadership roles are very demanding, and one has to be very clear of the demands they make and the toll they take on our lives. Many sacrifices and compromises have to be made daily to be successful at work and to juggle with work-life and work-home balance successfully”. Astrid recounts how on certain days, work takes over, so she makes up for this by dedicating more time to her family on days when the workload is lighter. Similar concerns about work commitment have been raised in a study by Brown (2010), with mothers declaring having missed out on promotions due to moving to part-time status, hence, losing access to career advancement opportunities or because "they [promotions] would have involved more time commitment than I felt comfortable giving while being a mother to my children." Christabelle said that her husband would feel that "he would be less of a man if he gave up his full-time job to dedicate more time to the home, bringing up financial reasons for not doing so, even if I had to earn more than he does." A further factor that was intertwined with spousal support was support from extended family members. All the interviewed mothers said that they got support from their immediate family members, particularly their parents. For example, Carol describes her parents as "the driving force behind me", whilst Rose explains that "If I had not my mum who was available and supported me, I would not have done it". Spousal and parental support The participants also mentioned the support of their partners and spouses as a vital feature of their working lives and careers. Without that support, they all admitted that, as Ritienne put it, "the road would have been distinctively harder since I would have felt all alone." When asked if the daily struggle to maintain a healthy work-life balance was the same for fathers, Carol explains, "No, it is not the same. "Fathers still do not get it. For them, going to work and spending the whole day there and letting mothers deal with the rest is something I have seen all over the world. I believe it will take another century to see real change in this, should it ever Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education 314 happen." Using similar reasoning, Rose says: "No, obviously not! Rather we praise fathers for coming to work! No. We, socially, still have this major distinction where we expect this from mothers and expect something else from fathers. For example, if my husband had had to spend some time at home with our daughter while I was abroad, he would almost be seen as someone to be pitied! His own daughter! They would think, "he should have his wife, and his wife should be more present…". Ritienne reasons out that the struggle for fathers depends on the extent to which they desire to be present in their children's lives, whilst Astrid and Mary believe that it all has to do with the arrangement struck between the parents themselves. Both Astrid and Mary have had the support of their spouses in their career-related and family-related decisions, with Astrid depicting that "he [husband] helps me in my work and the decisions we make and with the kids, but deep down I still think that the load is on me. I still remain the person responsible for the children." Astrid explained further that “he [my husband] helps me in my work and the decisions we make and with the kids, but deep down I still think that the load is on me, even though if a meeting cropped up and I ask him to pick them up, he finds a way out, so I always remain the person responsible for the children”. These situations are analogous to those of the female managers interviewed in the study by McKie and Jyrkinen (2018). Spousal and parental support Ritienne says that “my family helps a lot – the kids go to my parents’ house after school – so, in that way, they get a good meal and a safe place to do their homework until I pick them up”. Support at work The participants mentioned various ways in which they felt supported at work. Carol explained that she received a degree of practical empowerment, rather than sole empathy, from her managers at work. She says, "I am lucky that I have a CEO who is mindful of family commitments, and he encourages me and allows me much flexibility. He knows I will deliver. 315 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education However, I must run a department of 20 people, and I need to be there to drive things. However, had it not been for my flexibility, I would not manage to do the things I do." Astrid says that she owes a lot to her colleagues, who found a way to step in for her to maintain her position as a director, particularly when she had taken a year off as parental leave. This motivated her further to invest time and energy into her work. Rose also enjoyed support and encouragement from her managers and colleagues; and spoke about how she was involved in decisions that enabled her to meet work and family commitments. She said that her managers had asked her questions like "what would be the best way to do this? What would suit you best?" Gravitating against this type of empathy were gender stereotypes and associated stigmas. The associated adverse perceptions about women in leadership positions have been seen to prevail in many societies and investigated in various studies (example; Ching-Yim and Harris Bond, 2002; Mostafa, 2003; Cortis and Cassar, 2005). The participants in this study did not believe that they had been harassed at work; however, Carol said that initially, she thought she was not trusted at the workplace. She said she used to avoid mentioning motherhood and family at first but only mentioned it once her colleagues at work had got to know her. Having said this, Mirick and Wladkowski (2018) point out that there may be an unspoken consensus among male co- workers that one "should not emphasise motherhood and family, talk about it, or let it interfere with their work," showing the possible existence of a cultural script separating work life from family life. Support at work Conversely, Ritienne explained how "taking up a managerial position was highly contested by people in my immediate family – the reason being that there is always judgement against a mother who seems to value her career." This made it important for her to feel comfortable when working, and she mentioned that she was glad she was not put down at work because she was a woman. Even though eliminating discrimination and eliminating inequality could be a boost for economic growth and to diminish inequality (Wayne, 2017), some of the participants mentioned that they were presented with several challenges at the workplace simply because they were working mothers, but then took it upon themselves that they had the onus to adapt. True to this line of reasoning about adapting to workplace situations, Gavin et al. (2003) suggests that one's character is key to the effectiveness and health of an executive. All the interviewed mothers displayed various skills that convey an ability to have good relations with colleagues. Discussion The participants' narratives were driven by the fact that they were working mothers and people who prioritised work and family. However, the emphasis on feeling fulfilled and the heightened sense of self-worth that they derived from having an education shows that not only the women's lived experience is constituted, as a construction, with a specific cultural and historical background that reinforces high education levels but also as something that was somewhat expected of them if they were to self-actualise themselves and thereby independent of their volition, at least to a certain degree. Conrad and Baker (2010) observe that “the meanings of phenomena do not necessarily inhere in the phenomena themselves but develop through interaction in a social context” (p.65). Likewise, while most participants managed to balance work, studies and being mothers, others decided not to pursue their studies. However, advancing in their studies reflects a particular social context where advancement in studies may open the doors to potential career progressions. Thereby the regret at being unable to further studies may betray a gendered discourse where an implicit reference is being made to the domination of one gender over another; since men may find achieving career goals far easier since they are less constrained by family commitments, and thereby this sense of regret is a socio-cultural construction linked to the power structures of society. In effect, almost all the interviewees mentioned understanding husbands or partners. 316 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education Nevertheless, they (as mothers) still undertake the major load of responsibility for the children and the household chores, including all the planning and organising activities associated with raising a family. As Astrid put it, "motherhood and work clash mostly because as a society we are still far behind in terms of work expectations, work ethic and demands – for men, being a workaholic is still thought to be positive, whereas I strongly believe that society would be much better if more family-friendly measures were in place. More than the kind of job responsibilities, the lack of flexibility sometimes negatively impacts my role as a mother – I do my best to juggle and find a good balance; however, it is never enough. Discussion Men are somehow expected to juggle differently." When the interviewees mentioned that they derived continuous support from their parents, who in turn volunteered to take care of their grandchildren while their daughters tended to work responsibilities, it seems that this further cemented the discourse that holds mothers responsible for their children’s well-being, development, and successful adaptation in life. The participants were more likely to reach out to their own mothers for support than their husbands. This could reflect guilt and shame that they sometimes reported experiencing because of working and studying, rather than simply feeling more comfortable leaving the children with their own parents. If so, the disabling experiences of dominant discourses around working mothers may never have been addressed or challenged since they may not have been brought into conscious awareness. It transpires that, in this study, all the participants had children who did not have disabilities and thereby did not show the signs of distress that parents of disabled children experience. Also, none of the mothers was disadvantaged socio-economically, and this is likely to have had a positive bearing on family functioning. Support and empowerment from the participants' superiors and, in some cases, colleagues at work impacted the interviewees significantly. The participants believed that balancing family and work proves to be extremely difficult and requires constant planning and organisation, time management, perseverance, and resilience. A particular point worthy of mention is that none of the interviewees ever mention sending their children to any formal Childcare Centres, even though there has been a recent surge of provision of this facility both by the state and by private providers in Malta. When the researcher purposely brought up childcare provision toward the end of the interview, the participants did not consider childcare as a feasible option, this possibly showing that they had their parents and support network to turn to. Christabelle believes that childcare is not an answer since "children miss out a lot when the mother is not present". However, both Astrid and Rose mentioned personal shame as the reason for not sending their children to childcare, explaining that they believe that society does not always approve of a mother who sends her children to childcare. Rose explains that "social judgement is still there. If a mother had a baby and returned to work, we still have that big question mark. Discussion It seems like we do not have the space to say, "it is her choice.” Making it clear that she was referring to other people, she then added that "an unhappy mother is not the best for any child. So, we need to understand that if a mother feels self-actualised at the place of work, picks up her children from childcare, and takes care of them in that way, then this is fine. However, society is not there yet". What Rose was saying is that for there to be a greater sensitivity to the concerns of working mothers, society must simultaneously acknowledge a mother's contribution towards society through motherhood itself, through her potential contribution to organisations and the labour force as a whole, and for her need to actualise herself in her place of work – while taking into consideration her personal circumstances and the wider societal influences that have the potential to leave an impact on how she perceives and frames the resources that are available to empower her to reach her personal goals. 317 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education Main inferences Across the interviews, the mothers' innate drive to prioritise their children was evident. All of the interviewees seem to have shaped their careers or studies around family responsibilities, with all but one opting for career breaks when their children were still young. Despite the possible negative repercussions in their careers, such a decision was never regretted; somewhat, it was perceived as an investment in their children. Nevertheless, all of the interviewees chose to pursue self-actualising roles at work at some point or another, and all of the interviewees found ways to engage in further studies. Almost all interviewees mention understanding husbands or partners, yet, it seems that the mothers still undertake full responsibility for the children and the household chores, including all the planning and organising. Issues were also raised as to why the mother has to jeopardise her career through career breaks or similar arrangements rather than the father. The patriarchal culture and the social judgement towards mothers who decide to pursue their careers have also featured prominently. All interviewees derived continuous support from their parents, particularly in taking care of their grandchildren, while their daughters tended to work responsibilities. Support and empowerment from their superiors and, to a lesser extent, colleagues at work left a considerable impact on the interviewees. Whilst the majority also mention hurdles and challenges posed by their superiors or colleagues, these did little to belittle their quest to live up to their potential at work. In terms of the latter, all Interviewees express pride and fulfilment from their role at work; however, this does not come without many sacrifices and much stress, especially since they all seem keen to fulfil both work and motherhood duties. Alas, balancing family and work proves to be extremely difficult and requires constant planning and organisation, time management, perseverance, resilience, and completely sacrificing one's time for hobbies or similar interests. In fact, a participant primarily questions whether assuming such a role at work is worth the negative impact it is leaving on her family, whilst another claims that she has made a conscious decision when pursuing such a high-flying career, that it may compromise her motherhood responsibilities. Recommendations Whilst attaining a work-family balance is very difficult, if not next to impossible for some, family-friendly measures at work, such as flexible hours or teleworking, do ease this plight. However, such measures need to be standardised, regulated, and monitored by sound policies, which measure work by 'output' rather than the number of hours spent. A further consideration is that young working mothers are millennials born between 1980 and 2000. Their name derives from that they are closest to a new millennium and live in a "digital age". Briffa (2020), while focusing mainly on the hospitality industry, points out as young employees, Maltese Millennials prefer a work-leisure balance from the start and demand different working conditions and remunerations than those that older generations were likely to be ready to settle for. In Horizon (2020), gender is a cross-cutting issue. It is mainstreamed in each of the different parts of the Work Programme, thereby reinforcing a mentality where women and men's needs and behaviours are considered. Again, such measures would, perhaps, attract greater female engagement in the workforce, yet would do little to promote mothers' self-actualisation at work. As pointed out in the report by McKinsey and Co. (2021), companies should communicate what is expected of employees and 318 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education what it means to have an inclusive culture. The report points out that building this thinking into company values is a good start. However, organisations would benefit from articulating the specific behaviours and actions that promote inclusion. This implies that more supportive measures and structures need to be set up and a degree of flexibility afforded to mothers who wish to move further in their careers. These need to be coupled with mentoring, role models and empowerment initiatives that facilitate mothers to succeed both at their role at work and motherhood, without compromising any of the two. Juggling between such roles is undoubtedly not a "walk in the park", but mothers are more likely to succeed given the necessary support, the space, and the time (beyond the rigid office hours). Engaging in roles of responsibility and giving their best in them whilst the children are growing up tend to infringe upon family responsibilities, especially when the parents do not see formal childcare services as the answer or in situations where a lack of support from family members prevail. Recommendations What might help mitigate the situation is the provision of such jobs on a reduced or part-time basis, although these might not always be feasible. Tailoring office hours at par with school hours may also facilitate this. What would make a difference for mothers, in general, in terms of their work-family balance, is the empathy and support their workplaces afford, and these do include not only organisational policies but also the organisational culture and the attitudes of the superiors themselves (Ezzedeen, 2009; Brown, 2010). Finally, society must simultaneously acknowledge a mother's contribution towards society through motherhood itself, her potential contribution in organisations and the labour force, and her need to actualise herself in her place of work and give opportunities and support her decision to do so just like society does for fathers. Limitations and opportunities for further research The limited contact time between the researcher and the interviewee may have translated into less data being gathered than had more time been available. Furthermore, the anecdotes of the children, partners or spouses, and other immediate family members of the interviewees would have provided a complete picture of their lives and produced more reliable results, particularly in terms of triangulation. The interview responses cannot be extrapolated to all mothers in executive positions. In other words, the small sample size of the participants taking part in the study hinders the ability to generalise and reproduce the findings beyond the constraints of the sample. On the other hand, the complexity of women's family balance and career success has been recognised but inadequately studied (Ezzedeen, 2009; Brown, 2010). Hence, similar studies, preferably on a larger scale, would undoubtedly yield valuable data upon which policymakers and organisations can act. Such studies may take different forms, including cohort or longitudinal studies. Some variables to consider when investigating mothers' work-family balance may include; the different stages of the children's lives, the varying degrees of support available to the mother, a changing of job for the mother or father and/or a changing of marital status. Other studies may focus on single mothers as another cohort. Comparative studies may also be held with fathers, possibly yielding fruitful data to inform national policies aimed at both parents and not only mothers. Finally, an equally exciting study would entail exploring the experiences of women who dismissed motherhood altogether to pursue their careers and/or studies exclusively. Valuable data may also be obtained from ‘mumpreneurs’ who take pride in a “sense of control they had over their work which enabled them to fit hours around other responsibilities”, yet experience “stressful times trying to combine the business with motherhood” and “tension when 319 Camilleri and Spitteri \| Work-Life Balance, Upward Career Mobility and Further Education their espoused reasons for setting up a business – to be available for their children – conflicted with how they were allocating their time” (Pace Frendo, 2017). 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https://openalex.org/W2790608696	https://www.intechopen.com/citation-pdf-url/58308	English	null	Climate Control in Mediterranean Greenhouses	InTech eBooks	2,018	cc-by	6,960	Chapter 9 Chapter 9 Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.72367 http://dx.doi.org/10.5772/intechopen.72367 Abstract As climate control in greenhouses directly affects crop yields, there is an increasing trend for advancements in environmentally controlled agricultural-production techniques. In the Mediterranean region, the temperatures during the period from December to Febru- ary are below 12C when the daily total radiation 8.4 MJ/m2day. Based on the region’s climate data, greenhouses require heating during the period from November to March, ventilation and shading from February to May and cooling from June to September. In order to maintain day and night temperatures of 18/16C, annual heat energy requirement of PE greenhouses is 95-256 kWh/m2. In view of environment and production costs, conservation of heating energy is as important as heating itself. Heat energy saving is about 37% when energy curtains are used. Greenhouse temperature can be increased by 8C in palliative non-heated greenhouses where energy curtains and water mattresses are used in addition to passively used solar energy. Ventilation openings at the roofs of these greenhouses should adequately be 20-25%. When outside noon-time temperature is above 30C in June, evaporative cooling of greenhouse is essential. Depending on outside humidity and volume of exchanged air for cooling, a temperature difference of 6C can be achieved with evaporative cooling of greenhouses in August. Keywords: greenhouse heating, energy saving, ventilation, cooling A. Nafi Baytorun and Zeynep Zaimoglu A. Nafi Baytorun and Zeynep Zaimoglu Additional information is available at the end of the chapter 1. Introduction Countries need to increase the efficiency and quality of their agricultural production in order to meet their future requirements in line with population increase. In our country, it has become necessary to take particular measures due to rapid population increase and globaliza- tion of trade. Growing fruits and vegetables in controlled environments with low production costs is among these measures to be taken. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Climate Resilient Agriculture - Strategies and Perspectives 168 Controlling environmental conditions in agricultural production has direct influence on effi- ciency. For this reason, environmentally controlled agricultural techniques have developed at an increasing rate. In environmentally controlled plant production systems, it is aimed to change natural environmental factors according to the optimum requirements of plants. The most common and effective implementation of environmentally controlled plant production takes place in greenhouses. The objective of innovative technologies in greenhouses is to improve “Quality of Life Cycles.” Therefore, in order to achieve sustainability, it is highly important to correctly analyze the inputs and outputs required for the efficiency to be obtained from a unit area in greenhouses [1]. Sustainable greenhouse systems should be equipped with resource conserving, socially supported, commercial, competitive, environmentally friendly, reliable production technolo- gies, and they should aim to reduce energy, water and chemical pesticide requirements besides avoiding waste production [2]. Greenhouse practices in Turkey started first in the 1940s in the Mediterranean region, particu- larly in Antalya, and then spread to the Aegean and Marmara regions depending on ecological conditions. The Mediterranean region has 84% of the country’s total greenhouse area, followed by the Aegean, Black Sea and Marmara regions with 9.4, 4.8 and 1.7%, respectively. With 22,000 ha, Antalya has 37% of the country’s total greenhouse area. A total of 32,000 ha of the 61,500 ha greenhouse area in the country consists of greenhouses, which are defined as high greenhouse systems [3, 4]. As big investment groups entered the sector, modern greenhousing has rapidly developed and reached a level of 1000 ha. This figure increases by 150–200 ha every year. Nowadays, modern greenhousing is practiced in 3% of our total greenhouse area, and in the following decade, this share is expected to reach 15% [5]. In terms of equipment and technology, greenhouses in the Mediterranean region can be divided into two groups: In terms of equipment and technology, greenhouses in the Mediterranean region can be divided into two groups: 1. Greenhouses with low technology: These greenhouses have simple iron structures. They are covered with PE plastic or mixed PE plastic. Starting from 1987, galvanized pipes have been used in greenhouses built with government support provided within the framework of Resource Utilization Support Fund (RUSF). These greenhouses with ventilation open- ings only on the sidewalls have been built as blocks consisting of four sections. In all greenhouses installed with RUSF support, drop irrigation systems are used, and these greenhouses are heated on a regular basis. 2. Modern greenhouses with high technology: These greenhouses require quite high initial invest- ment costs, which are considered as big businesses. They are generally built with galvanized steel and aluminum materials. As cover material glass, mixed PE plastic or double pane PC is used. They are projected as high volume structures. In these types of greenhouses, modern production techniques like soilless agriculture are used. Their irrigation systems are projected as computer-aided closed systems, and they have central heating systems. In addition to natural ventilation, these greenhouses have fans that provide air circulation. Greenhouses, in which ornamental plants are grown, have evaporative cooling systems. During hot periods, besides cooling, moving shading systems are activated. Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 169 2. Climatic values in the Mediterranean region The most important climatic parameters in greenhouse cultivation are solar radiation, day length and temperature values. Solar radiation is one of the most important climatic parameters to be taken into consideration in a location where a greenhouse is going to be built. Total daily solar radiation in a greenhouse location should be minimum 8.5 MJ/m2 day (2.34 kWh/m2day) [6]. However, insolation time is as important as solar radiation. Products grown in greenhouses require an average of 6 h of day length. In other words, during months with short day length (e.g., November, December and January), total day length should be minimum of 500–550 h [6]. Total annual insolation time and intensity of radiation in Turkey are 2.640 h (7.2 h/day) and 1.311 kWh/m2year (3.6 kWh/m2day), respectively. In Tables 1 and 2, long-year average total daily radiation values and insolation time for some cities on the Mediterranean coastline are given. As can be seen from the tables, insolation time and total daily solar radiation values in the Mediterranean region are above average values in whole Turkey. Vegetables grown in greenhouses have adapted to an average temperature of 17–28C. Prod- ucts grown in greenhouses undergo stress at temperatures below 12C and above 32C. At low temperature values like frost, irreversible harms occur. Greenhouses should be heated when the outside temperature falls below 12C. When outside temperature is between 7 and 12C, heating is necessary only during night hours. For desirable plant growth, the difference between night and day temperatures should be 5–7C [6]. City January February March April May June July August September October November December Adana 1.98 2.42 4.12 4.98 6.07 6.68 6.46 5.91 4.90 3.78 2.33 1.81 Antalya 2.12 2.57 4.37 5.47 6.36 6.93 6.65 6.14 5.16 3.93 2.51 1.92 Hatay 1.99 2.42 4.01 4.87 5.96 6.63 6.31 5.82 4.75 3.63 2.35 1.79 İçel 2.11 2.65 4.27 5.24 6.28 6.86 6.66 6.08 5.04 3.84 2.47 1.91 Muğla 2.11 2.42 4.24 5.40 6.22 6.81 6.47 6.05 5.05 3.96 2.56 1.88 Turkey 1.79 2.50 3.87 4.93 6.14 6.57 6.50 5.81 4.81 3.46 2.14 1.59 Table 1. Total daily radiation values in different cities in the Mediterranean region (kWh/m2day). 2. Climatic values in the Mediterranean region City January February March April May June July August September October November December Adana 4.67 5.65 6.97 7.84 9.72 11.29 11.17 11.22 10.15 7.78 5.86 4.21 Antalya 4.95 6.10 7.24 8.29 9.70 11.55 11.84 11.29 9.80 7.68 5.97 4.55 Hatay 5.09 6.22 7.17 8.28 10.23 11.14 10.89 10.47 9.80 7.86 6.37 4.99 İçel 4.99 6.04 7.35 8.38 9.94 11.18 11.45 11.03 10.02 7.91 6.15 4.64 Muğla 5.13 6.20 7.12 8.18 9.91 11.73 11.90 11.31 9.92 7.85 6.01 4.67 Turkey 4.11 5.22 6.27 7.46 9.10 10.81 11.31 10.70 9.23 6.87 5.15 3.75 Table 2. Insolation time in different cities in the Mediterranean region (h). City January February March April May June July August September October November December Adana 1.98 2.42 4.12 4.98 6.07 6.68 6.46 5.91 4.90 3.78 2.33 1.81 Antalya 2.12 2.57 4.37 5.47 6.36 6.93 6.65 6.14 5.16 3.93 2.51 1.92 Hatay 1.99 2.42 4.01 4.87 5.96 6.63 6.31 5.82 4.75 3.63 2.35 1.79 İçel 2.11 2.65 4.27 5.24 6.28 6.86 6.66 6.08 5.04 3.84 2.47 1.91 Muğla 2.11 2.42 4.24 5.40 6.22 6.81 6.47 6.05 5.05 3.96 2.56 1.88 Turkey 1.79 2.50 3.87 4.93 6.14 6.57 6.50 5.81 4.81 3.46 2.14 1.59 Table 1. Total daily radiation values in different cities in the Mediterranean region (kWh/m2day). City January February March April May June July August September October November December Adana 4.67 5.65 6.97 7.84 9.72 11.29 11.17 11.22 10.15 7.78 5.86 4.21 Antalya 4.95 6.10 7.24 8.29 9.70 11.55 11.84 11.29 9.80 7.68 5.97 4.55 Hatay 5.09 6.22 7.17 8.28 10.23 11.14 10.89 10.47 9.80 7.86 6.37 4.99 İçel 4.99 6.04 7.35 8.38 9.94 11.18 11.45 11.03 10.02 7.91 6.15 4.64 Muğla 5.13 6.20 7.12 8.18 9.91 11.73 11.90 11.31 9.92 7.85 6.01 4.67 Turkey 4.11 5.22 6.27 7.46 9.10 10.81 11.31 10.70 9.23 6.87 5.15 3.75 Table 2. Insolation time in different cities in the Mediterranean region (h). 170 Climate Resilient Agriculture - Strategies and Perspectives At temperatures between 12 and 22C, by using passive acclimatization (natural ventilation), it is possible to arrange the greenhouse environment according to the values required by plants. When outside temperature exceeds 27C, it is necessary to install highly expensive cooling systems in greenhouses. The greenhouses on the Mediterranean region should be left idle during the specified periods. 2. Climatic values in the Mediterranean region The graphical representation of long year average temperatures and total daily radiation values of Mediterranean cities Antalya (36:530 N), Mersin (36:480 N) and Hatay (36:140 N) is given in Figure 1. As seen in the figure, total daily radiation in all these three cities on the Mediterranean coastline is below 2.3 kWh/m2day (8.4 MJ/m2 day) during December and January. This indicates that solar radiation is insufficient for plant growth during these 2 months. In order to allow more solar radiation into the greenhouse in December and January, greenhouse roofs should be covered using a material with high impermeability. The 1% decrease in intensity of light reaching the greenhouse results in the same decrease in efficiency. Another factor that affects plant growth is day length. Total day length in Antalya during November–January in Antalya is 474 h. This value is close to the limit value, which is accepted as 500–550 h. An overview of the temperature values of cities in the Mediterranean region (Table 3) shows that average daily temperature during the period between December and February is below 12C. However, as average temperature during these months does not fall below 7C, pro- ducers in this region prefer cold greenhousing and take simple heating measures in order to continue production during very cold days. One of the main problems in unheated PE plastic greenhouses in the Mediterranean region is that greenhouse temperature falls below outside temperature on nights when the sky is clear. This results from the fact that PE plastic transmits long-wave heat rays on a specific band. In their measurements taken in glass and PE plastic greenhouses in the Mediterranean region Figure 1. Average daily temperatures and total radiation values in three cities in the Mediterranean region: Antalya (36:530 N), Mersin (36:480 N) and Hatay (36:140 N). Figure 1. Average daily temperatures and total radiation values in three cities in the Mediterranean region: Antalya (36:530 N), Mersin (36:480 N) and Hatay (36:140 N). 2. Climatic values in the Mediterranean region Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 171 City January February March April May June July August September October November December Adana 9.6 10.6 13.6 17.6 21.7 25.6 28.2 28.6 26.1 21.8 15.7 11.1 Hatay 8.1 9.8 13.2 17.1 21.1 24.8 27.2 27.8 25.7 20.8 14.2 9.5 Antalya 9.9 10.4 12.8 16.1 20.3 25.1 28.2 28.0 24.9 20.3 15.1 11.4 Mersin 10.3 11.1 13.9 17.4 21.1 24.8 27.7 28.2 25.7 21.5 16.1 11.9 Muğla 5.3 5.9 8.6 12.5 17.7 22.9 26.3 26.1 21.8 16.1 10.5 6.8 Table 3. Average daily temperature values of different cities in the Mediterranean region (C). Cover material Outside temperature (C) Inside temperature (C) (tito) (C) Plastic 9.3 9.0 0.3 10.3 9.0 1.3 8.1 8.1 0.0 Glass 7.3 9.1 1.8 6.8 7.7 0.9 8.3 10.1 1.8 Table 4. Inside and outside temperature values recorded on different days in an unheated PE plastic greenhouse (time 17:00–07:00 Average values). Table 4. Inside and outside temperature values recorded on different days in an unheated PE plastic greenhouse (time 17:00–07:00 Average values). (Adana), it is observed that the temperature falls below outside temperature in PE greenhouses without thermal curtains [7]. This situation was not seen in unheated glass greenhouses (Table 4). In order to achieve year-long production in greenhouses in the Mediterranean region, green- houses should be heated during night hours in the winter, ventilated and shaded in transition periods and cooled during hot periods. As cooling is a very costly acclimatization measure, it is not a preferable method for greenhouses, which are usually left idle during such periods. 2.1. Heating Months January February March April May June July August September October November December Qsun 2.12 2.57 4.37 5.47 3.37 6.93 6.65 6.0 5.17 3.93 2.52 1.92 PE plastic greenhouse Q(16) 1.06 0.94 0.57 0.19 0.00 0.00 0.00 0.00 0.00 0.00 0.34 0.79 Q(17/18) 1.27 1.13 0.73 0.31 0.00 0.00 0.00 0.00 0.00 0.04 0.49 0.98 Glass greenhouse Q(16) 1.04 0.92 0.53 0.14 0.00 0.00 0.00 0.00 0.00 0.00 0.28 0.75 Q(17/18) 1.27 1.13 0.69 0.26 0.00 0.00 0.00 0.00 0.00 0.00 0.44 0.96 Table 5. Daily solar energy reaching the greenhouse in Antalya climatic conditions required daily heat energy values for PE plastic and glass greenhouses for different temperatures (kWh/m2day). Greenhouse requirement Heat energy requirement kWh/m2 a Antalya Adana Mersin Hatay Muğla Single pane plastic 126.6 113.6 95.5 140.2 256.3 Roof single, side wall double pane PE plastic 118.3 106.3 89.3 130.6 239.2 Roof single, side wall double pane PE plastic + Thermal curtain moderately insulated 95.1 87.4 72.7 107.7 198.3 Table 6. Heat energy requirements during the production year for different PE plastic greenhouses in some cities in the Mediterranean region when night/day temperature is kept at 16/18C [8]. Heat energy requirement kWh/m2 a Daily heat energy values based on climatic conditions of Antalya for weeks of the year when greenhouse temperature is kept at 18/16C, 18/14C and 16/12C are given in Figure 2. As can be seen from the figure, daily heat energy requirement varies between 0 and 1.3 kWh/m2day depending on the desired greenhouse temperature. A similar change is observed during hours of the day. Heat power requirement in plastic greenhouses installed in the Mediterranean region from 04:00 to 07:00 in January is 85 W/m2, while after 08:00 this value drops to 0 W/m2 [8]. Heat power iterations required for a single pane PE plastic greenhouse in Antalya climatic conditions with temperature kept at 14, 16 and 18C are given in Figure 3. As can be seen from the figure, there is need for high heat power only during a very short period of the year. In Mediterranean climatic conditions, 3012 h of heating is required when day/night temperature is kept at 18C. When temperature is dropped to 16C, 2567 h of heating is required. Heat energy values required throughout the production period for greenhouses in different cities in the Mediterranean region are given in Table 7. 2.1. Heating Total daily solar energy values for Antalya (36:530 N) in different months and required daily heat energy values for PE plastic and glass greenhouses for specific temperatures are given in Table 5. As can be seen from Table 6, total daily solar energy reaching the greenhouse in Mediterranean climatic condition exceedingly meets the heat energy requirement in all months of the year. However, only some of the solar energy reaching the greenhouse during day hours can be stored. Heat losses that occur through the cover material immediately after sunset lead to rapid decreases in greenhouse temperature. Heating in greenhouses has a significant effect on production costs. Heat energy requirements of plastic greenhouses installed in the Mediterranean region vary depending on climate of the location, greenhouse type and greenhouse equipment. Climate Resilient Agriculture - Strategies and Perspectives 172 Greenhouse requirement Heat energy requirement kWh/m2 a Antalya Adana Mersin Hatay Muğla Single pane plastic 126.6 113.6 95.5 140.2 256.3 Roof single, side wall double pane PE plastic 118.3 106.3 89.3 130.6 239.2 Roof single, side wall double pane PE plastic + Thermal curtain moderately insulated 95.1 87.4 72.7 107.7 198.3 Table 6. Heat energy requirements during the production year for different PE plastic greenhouses in some cities in the Mediterranean region when night/day temperature is kept at 16/18C [8]. Months January February March April May June July August September October November December Qsun 2.12 2.57 4.37 5.47 3.37 6.93 6.65 6.0 5.17 3.93 2.52 1.92 PE plastic greenhouse Q(16) 1.06 0.94 0.57 0.19 0.00 0.00 0.00 0.00 0.00 0.00 0.34 0.79 Q(17/18) 1.27 1.13 0.73 0.31 0.00 0.00 0.00 0.00 0.00 0.04 0.49 0.98 Glass greenhouse Q(16) 1.04 0.92 0.53 0.14 0.00 0.00 0.00 0.00 0.00 0.00 0.28 0.75 Q(17/18) 1.27 1.13 0.69 0.26 0.00 0.00 0.00 0.00 0.00 0.00 0.44 0.96 Table 5. Daily solar energy reaching the greenhouse in Antalya climatic conditions required daily heat energy values for PE plastic and glass greenhouses for different temperatures (kWh/m2day). 2.1. Heating These values are for greenhouses with different equipment and when day/night temperature is kept at 16/18C and ventilation temperature is kept at 25C [8]. As can be seen from the table, for each type of greenhouse equipment, the lowest heat energy requirement is observed in Mersin (36:480 N) with 72.7 kWh/m2a, while the highest heat energy requirement is observed in Muğla with 198.3 kWh/m2a. In the Mediterranean region, greenhouses with low technology are not heated. On days when the temperature is low, plants are protected from frost with the help of simple heating stoves. Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 173 Figure 2. Daily heat energy requirements depending on different day/night temperature values for a single pane PE plastic-covered greenhouse in Antalya climatic conditions (kWh/m2day). Figure 2. Daily heat energy requirements depending on different day/night temperature values for a single pane PE plastic-covered greenhouse in Antalya climatic conditions (kWh/m2day). Figure 3. Heat power iterations for different greenhouse temperatures in Antalya climatic conditions. Cover material Outside temperature (C) Under curtain temperature (C) Over curtain temperature (C) (ti-paltto) (C) (ti-püstto) (C) Plastic 9.1 10.7 8.8 1.6 0.3 5.0 5.6 3.3 0.6 1.7 8.5 10.0 7.8 1.5 0.7 Glass 9.0 12.7 9.5 3.6 0.5 8.2 12.0 8.6 3.7 0.4 10.1 13.6 10.7 3.4 0.6 Table 7. Under curtain, over curtain and outside temperature values in unheated plastic and glass greenhouses with thermal curtains opened and closed (time 17:00–07:00 Average values). Figure 3. Heat power iterations for different greenhouse temperatures in Antalya climatic conditions. Figure 3. Heat power iterations for different greenhouse temperatures in Antalya climatic conditions. gure 3. Heat power iterations for different greenhouse temperatures in Antalya climatic conditions. Figure 3. Heat power iterations for different greenhouse temperatures in Antalya climatic conditions. Figure 3. Heat power iterations for different greenhouse temperatures in Antalya climatic conditions. Cover material Outside temperature (C) Under curtain temperature (C) Over curtain temperature (C) (ti-paltto) (C) (ti-püstto) (C) Plastic 9.1 10.7 8.8 1.6 0.3 5.0 5.6 3.3 0.6 1.7 8.5 10.0 7.8 1.5 0.7 Glass 9.0 12.7 9.5 3.6 0.5 8.2 12.0 8.6 3.7 0.4 10.1 13.6 10.7 3.4 0.6 Table 7. Under curtain, over curtain and outside temperature values in unheated plastic and glass greenhouses with thermal curtains opened and closed (time 17:00–07:00 Average values). Table 7. Table 7. Under curtain, over curtain and outside temperature values in unheated plastic and glass greenhouses with thermal curtains opened and closed (time 17:00–07:00 Average values). 2.1. Heating Under curtain, over curtain and outside temperature values in unheated plastic and glass greenhouses with thermal curtains opened and closed (time 17:00–07:00 Average values). Climate Resilient Agriculture - Strategies and Perspectives 174 However, with this kind of heating, heat energy is not distributed properly in the greenhouse, and plants close to the heating stove are harmed. In small family businesses, pipe heating systems are not economical due to greenhouse sizes and initial investment costs. In these businesses, instead of pipe heating, low-cost direct-fire air blast heating systems are preferred. 2.2. Heat energy conservation As much as greenhouse heating, conservation of heat energy has great importance due to increasing energy prices and CO2 releases of energy sources. In order to conserve heat energy in greenhouses, multipane cover materials may be used. However, besides temperature and humidity values, light (PAR) in the greenhouse should be kept at the highest levels as it is one of the most significant factors for plant growth. For these reasons, it is suggested to cover the side walls of greenhouse in the Mediterranean region with multipane cover material for heat conservation, while the roof area should be covered with single pane material in order to allow sufficient light into the greenhouse. Thermal curtains are used for heat conservation in greenhouses. Average under curtain, over curtain and outside temperatures measured in unheated glass and PE plastic Mediterranean greenhouses with open and closed thermal curtains between 17:00 and 7:00 are given in Table 7. As can be seen from the table, under curtain temperature in the PE plastic greenhouse with thermal curtains closed is above outside temperature, while over curtain temperature values are below outside temperature. In the glass greenhouse, both under curtain and over curtain temperature values recorded are above outside [7, 9]. Depending on the properties of thermal curtains, heat conservation in heated greenhouses can be achieved at different ratios. In Figure 4, fuel quantities based on temperature differences (∆T) in a PE plastic greenhouse with and without thermal curtains are given [7]. When the thermal curtain is open, the amount of fuel (diesel) required for a 7 K temperature difference is Figure 4. Fuel consumption based on temperature differences (∆T) in a PE plastic greenhouse heated with direct-fired air blast heating system, with thermal curtains opened and closed. Figure 4. Fuel consumption based on temperature differences (∆T) in a PE plastic greenhouse heated with direct-fired air blast heating system, with thermal curtains opened and closed. Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 175 0.118 l/m2. The fuel requirement for the same temperature difference with the thermal curtain closed is 0.074 l/m2. This is equivalent to a 37% energy saving in a greenhouse with a direct- fired air blast heating system. Impermeability of thermal curtains used in greenhouses is very important in terms of energy savings. Edges where thermal curtains meet the side walls and facades should be leakproof. 2.2. Heat energy conservation Otherwise, transfer of heated and rising air through the roof cover material will be unavoid- able. Annual heat energy and saving ratios calculated based on the impermeability of thermal curtains in PE plastic greenhouses under Mediterranean climatic conditions with night/day temperature 16/18C and ventilation temperature 25C are given in Table 8 [9]. As can be seen from the table, there will be a 27% difference in energy savings between thermal curtains with perfect insulation and those with poor insulation. 2.3. Passively benefiting from solar energy in unheated greenhouses In Mediterranean climatic conditions, there is no need for heating during day hours as solar energy reaching the greenhouse exceedingly meets the daily energy requirement of the green- house. However, after sunset, greenhouse temperature drops rapidly depending on the ther- mal properties of the cover material. In a study aiming to passively benefit from solar energy in greenhouses with low technology, water mattresses consisting of transparent PE tubes (with a diameter of 31.8 cm, width of 150 μm and water capacity of 80 l/m) were placed between plant rows. Measurements showed that in the case of using water mattresses in a glass greenhouse, the temperature difference is 2.8–3.4C, while in a greenhouse without water mattresses, this value is 1.2–2.7C (Table 9) [7]. While the temperature difference in a glass greenhouse with thermal curtains and water mattresses ranges from 6.3 to 8.1K, the temperature difference in a greenhouse with thermal curtains but without water mattresses were recorded as 1.6 to 2.2K (Table 10) [7]. 2.4. Ventilation In the evaluations based on long year hourly temperature data for Antalya (36:530 N), where greenhousing is a common agricultural practice, it is seen that the temperature is above 26C in 1628 h of the year (Table 11). Temperature iterations for temperatures above 26C based on the ratio of ventilation openings to greenhouse floor area are given in Table 10. As can be seen from the table, as the ratio of ventilation openings to greenhouse floor area increases, iterations for temperatures above 26C decrease. In Antalya climatic conditions, when the ratio of ventilation openings to greenhouse floor area is 20%, during 206 h of the total 744 h of May, the greenhouse temper- ature is above 26C. Taking into consideration the long year hourly climatic values of Antalya, hourly temperature values calculated for greenhouses with different ventilation openings are given in Table 12. As can be seen from the table, average outside temperature values obtained from long year climatic values in May vary between 16 and 26C. The simulation calculations show that when the outside temperature is 25.7C at 12:00 in May, temperature in a greenhouse with 5% ventilation opening is 30.4C and temperature in a greenhouse with 10% ventilation opening is 28.5C. 2.4. Ventilation In the Mediterranean region, it is necessary to ventilate greenhouses during day hours of the winter months. Ventilation in winter months is done more to regulate CO2 concentration than Greenhouse equipment Heat energy requirement [kWh/m2a] Savings ratio [%] Impermeability Good Average Poor Good-Average Good-Poor Without thermal curtain 118.3 With thermal curtain 80.5 95.1 109.6 15.4 26.6 Savings ratio 32.0 19.6 7.4 Table 8. Heat energy requirements based on the impermeability of thermal curtains in PE plastic greenhouses with single pane roof and double pane side walls under Antalya climatic conditions with night/day temperature 16/18C [7]. Table 8. Heat energy requirements based on the impermeability of thermal curtains in PE plastic greenhouses with single pane roof and double pane side walls under Antalya climatic conditions with night/day temperature 16/18C [7]. Climate Resilient Agriculture - Strategies and Perspectives 176 to,min(C) ti,min-tube With water mattresses (C) ti,min-tubeless Without water mattresses (C) ΔT With water mattresses (C) ΔT Without water mattresses(C) ttubettubeless (C) 2.9 6.1 4.1 3.2 1.2 2.0 3.0 5.8 4.7 2.8 1.7 1.1 4.1 7.5 6.8 3.4 2.7 1.7 Table 9. Minimum temperature differences obtained in an unheated Mediterranean greenhouse with water mattresses (C). to,min(C) ti,min-tube With water mattresses (C) ti,min-tubeless With water mattresses (C) ΔT With water mattresses (C) ΔT Without water mattresses (C) ttubettubeless (C) 0.0 6.8 2.2 6.8 2.2 4.6 1.8 8.1 3.5 6.3 1.7 4.6 5.2 11.6 6.8 6.4 1.6 4.8 0.1 8.0 2.0 8.1 2.1 6.0 Table 10. Minimum temperature differences obtained in an unheated Mediterranean greenhouse with thermal curtains and water mattresses (C). Table 10. Minimum temperature differences obtained in an unheated Mediterranean greenhouse with thermal curtains and water mattresses (C). to send away high temperatures. It is only possible to obtain the temperatures that plants have adapted to (17–27C) by regular ventilation from mid-February until the first week of May [5]. In the evaluations based on long year hourly temperature data for Antalya (36:530 N), where greenhousing is a common agricultural practice, it is seen that the temperature is above 26C in 1628 h of the year (Table 11). to send away high temperatures. It is only possible to obtain the temperatures that plants have adapted to (17–27C) by regular ventilation from mid-February until the first week of May [5]. 2.5. Ventilation and shading Starting from the first week of May, greenhouses in the Mediterranean region are shaded with clay or whitewash. With shading, greenhouse temperature rises are prevented by reducing solar radiation that reaches the greenhouse. In greenhouses installed in recent years, solar Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 177 radiation is reduced by partially opening thermal curtains. In June, under Antalya climatic conditions, when radiation reaching the greenhouse is reduced by 50% with shading and when the ratio of ventilation openings is 20%, greenhouse temperature is above 26C for 96 h. AV/AG January February Mach April May June July Augusts September October November December Total Number of hours when outside temperature is above 26C 0 0 0 0 0 311 487 466 298 66 0 0 1628 Number of hours when greenhouse temperature is above 26 0.01 102 145 237 290 366 405 518 488 357 288 200 105 3501 0.05 0 0 78 205 335 389 504 480 336 254 98 0 2679 0.10 0 0 0 82 278 382 496 479 329 222 40 0 2308 0.15 0 0 0 18 246 375 489 477 323 201 10 0 2139 0.20 0 0 0 5 206 373 488 476 321 177 2 0 2048 0.25 0 0 0 0 160 370 488 474 320 163 0 0 1975 0.30 0 0 0 0 130 367 488 474 319 148 0 0 1926 0.35 0 0 0 0 113 362 487 474 318 142 0 0 1896 0.40 0 0 0 0 99 357 487 474 317 134 0 0 1868 Table 11. Temperature iterations for temperatures above 26C outside the greenhouse and in a plastic greenhouse with different ventilation opening ratios under Antalya climatic conditions in May (h). nuary February Mach April May June July Augusts September October November December Total Table 11. Temperature iterations for temperatures above 26C outside the greenhouse and in a plastic greenhouse with different ventilation opening ratios under Antalya climatic conditions in May (h). ble 12. Inside temperature values calculated for a plastic greenhouse with different ventilation opening ratios (A der Antalya climatic conditions in May Table 12. Inside temperature values calculated for a plastic greenhouse with different ventilation opening ratios (AV/AG) under Antalya climatic conditions in May. ble 12. Inside temperature values calculated for a plastic greenhouse with different ventilation opening ratios (A der Antalya climatic conditions in May. talya climatic conditions in May. Inside temperature values calculated for a plastic greenhouse with different ventilation opening ratios (AV/AG) l l d 2.5. Ventilation and shading Time Ratio of ventilation opening ratio to greenhouse floor area (AV AG) (%) Outside temperature ta (C) 1 5 10 15 25 Temperature in the greenhouse ti (C) 7 23.1 21.7 21.1 20.8 20.6 20.0 8 28.0 25.5 24.4 23.9 23.4 22.4 9 32.0 28.4 26.9 26.2 25.6 24.2 10 34.6 30.0 28.2 27.4 26.6 25.3 11 35.7 30.4 28.5 27.7 27.0 25.6 12 36.1 30.4 28.5 27.7 27.0 25.7 13 35.7 30.1 28.3 27.5 26.8 25.6 14 34.8 29.6 27.9 27.2 26.6 25.5 15 33.1 28.7 27.2 26.6 26.1 25.1 16 30.5 27.3 26.2 25.7 25.3 24.5 17 28.0 25.9 25.1 24.8 24.5 23.9 18 25.0 24.0 23.6 23.4 23.2 22.9 19 22.0 21.8 21.8 21.7 21.7 21.6 Table 12. Inside temperature values calculated for a plastic greenhouse with different ventilation opening ratios (AV/AG) under Antalya climatic conditions in May. Ratio of ventilation opening ratio to greenhouse floor area (AV AG) (%) Outside temperature ta (C) Climate Resilient Agriculture - Strategies and Perspectives 178 Time Ratio of ventilation opening ratio to greenhouse floor area (AV AG) (%) Outside temperature ta (C) 1 10 15 20 25 Temperature in the greenhouse ti (C) 7 27.0 25.8 25.6 25.5 25.5 25.1 8 30.3 28.3 28.0 27.9 27.8 27.3 9 32.8 30.1 29.7 29.5 29.4 28.8 10 34.5 31.1 30.7 30.5 30.3 29.6 11 35.4 31.5 31.1 30.8 30.6 29.9 12 35.6 31.6 31.2 31.0 30.8 30.1 13 35.1 31.2 30.8 30.6 30.4 29.8 14 34.5 30.8 30.5 30.3 30.1 29.6 15 33.6 30.5 30.2 30.0 29.9 29.4 16 32.3 29.9 29.6 29.5 29.4 29 17 30.7 29.0 28.8 28.7 28.7 28.3 18 28.9 27.9 27.8 27.7 27.7 27.5 19 26.5 26.3 26.2 26.2 26.2 26.1 Table 13. Temperature values calculated for different ratios of ventilation openings in a plastic greenhouse where 50% shading is done in June, under Antalya climatic conditions. Ratio of ventilation opening ratio to greenhouse floor area (AV AG) (%) Outside temperature ta (C) Table 13. Temperature values calculated for different ratios of ventilation openings in a plastic greenhouse where 50% shading is done in June, under Antalya climatic conditions. Although solar radiation causing increases in greenhouse temperatures under Mediterranean climatic conditions can be reduced to a certain degree with shading, it is not possible to obtain the environment temperature that can be tolerated by plants using shading in certain months of the year. 2.5. Ventilation and shading Temperature values based on hours of the day and ratios of ventilation openings for a plastic greenhouse where 50% shading is done during June are given in Table 13. As can be seen from the table, even when the ratio of ventilation openings to greenhouse floor area is 25%, temperature values in a shaded greenhouse are above 30C at 10.00–14.00. Under these conditions, evaporative cooling becomes necessary for continuation of plant growth. 2.6. Cooling When average daily temperature is above 22C and the maximum temperature is 27C, active cooling in the greenhouse is necessary [10]. When average daily temperature values of the Mediterranean climate zone are reviewed, it is seen that starting from June average tempera- ture values are above 22C (Table 3). Temperature and humidity values calculated in August for a glass greenhouse in Adana (37:010 N) with shading and evaporative (Fan&Pad) system is given in Figure 5. As can be seen from the figure, in the Mediterranean region, outside temperature values during the day can be as high as 35.2C in August. Despite shading and evaporative cooling, temperature in the greenhouse reached 29.1C at 15:00. As can be seen from the figure, humidity values throughout the day varied between 90 and 98%. In Adana climatic conditions, using shading and active cooling in August resulted in a temperature difference (∆T) of 6.1 K [11]. Figure 5. Temperature values calculated in August for a glass greenhouse obtained with shading and evaporative cooling. Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 179 Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 179 Figure 5. Temperature values calculated in August for a glass greenhouse obtained with shading and evaporative cooling. 3. Result and evaluation In order to be able to achieve high-quality production and efficiency in the Mediterranean climate zone, greenhouses should be heated during November–March, ventilated and shaded during February–May and cooled during June–September. On the Mediterranean coastline, simple greenhouses are not heated regularly as average daily temperature does not fall below 7C. In such businesses, when the temperature is very low, plants in the greenhouse are protected against frost using simple methods. Depending on the production (single produc- tion, spring or fall production), a 9–12 kg/m2 efficiency can be obtained in tomato production. In simple plastic greenhouses, it is not economical to install pipe heating systems due to greenhouse dimensions and cost of heating systems. In these greenhouses, instead of pipe heating systems, direct-fire air blast heating systems could be used. However, in this case, it is necessary to choose cheap fuel as well. In greenhouses built on the Mediterranean coastline in recent years, central heating systems are installed, and coal is used as fuel. Heat requirement of a single pane PE plastic greenhouse on the Mediterranean coastline, in which there is no heat conservation and day/night temper- ature is kept at 18/16C day/night, varies between 95 and 256 kWh/m2 depending on the climatic properties of the greenhouse site. This is equivalent to approximately 18–47 kg/m2 year imported coal. As much as greenhouse heating, heat conservation in heated greenhouses has great impor- tance. Since total solar radiation in the Mediterranean region is lower than 2.34 kWh/m2day during December and January, greenhouse roofs are covered with single pane and side walls are covered with double pane cover material. In the Mediterranean region, thermal curtains are used in greenhouses for heat conservation. In Antalya climatic conditions, heat require- ment for a PE plastic greenhouse with side walls covered with double pane thermal curtains is 80.5 kWh/m2 area when night/day temperature is kept at 16/18C. In a greenhouse without Climate Resilient Agriculture - Strategies and Perspectives 180 thermal curtains, this value is 118.3 kHz/m2 areas. In other words, 32% heat energy is saved in a greenhouse with thermal curtains. This is equivalent to 7.1 kg/m2 year imported coals. In Mediterranean climatic conditions, 30 kg/m2 truss tomatoes can be produced in heated modern greenhouses which in heated greenhouses where CO2 fertilization is done, the effi- ciency is 40 kg/m2. Approximately 18–28 kg/m2 efficiency increase in heated greenhouses should cover heating expenses. 3. Result and evaluation Depending on the climate of the region, the cost of truss tomato production in heated modern greenhouses varies between 1.29 and 1.69 TL/kg. In the feasibility calculations made for modern greenhouses in the Mediterranean region, return on investment is 14–25%, depending on the production methods of the business [12]. The quality of products obtained from heated greenhouses is higher than the quality of plants grown in unheated greenhouse. Also, due to humidity control, agricultural pesticide use is less. In order to benefit more from solar energy on the Mediterranean climate zone, it is appropriate to use water mattresses. With the help of water mattresses, a 2–3K temperature difference can be obtained, but when water mattresses are used together with thermal curtains, the temper- ature difference obtained becomes 6K. However, using water mattresses, it is not possible to obtain the optimum greenhouse temperature during night hours. In Mediterranean climatic conditions, a 20–25% ratio for ventilation openings in the roof area is sufficient. Increasing the sizes of the ventilation openings on the roof in May has very little impact on temperature difference. However, it should be kept in mind that insect screens placed on ventilation openings in modern greenhouses decrease the effectiveness of ventilation. Greenhouse shading implemented in a way that it does not affect air circulation can help reduce temperature difference in the greenhouse when used together with ventilation. In a greenhouse with 20% ventilation area on the roof, reducing solar radiation by 50% with shading can reduce the temperature difference (∆T) in June up to 1C. However, in June, evaporative cooling is needed as outside temperature around noon is above 30C. In Mediter- ranean climatic conditions, temperature difference obtained with evaporative cooling in August (depending on outside humidity and exchanged air volume) is nearly 6C. However, evaporative cooling is not preferred in production greenhouses because it requires electrical energy and a high quantity of clean water. For this reason, greenhouses on the Mediterranean coastline should be left idle starting from the second week of June. 2 Department of Environmental Engineering, Çukurova University Faculty of Engineering and Architecture, Balcalı-Adana, Turkey Author details A. Nafi Baytorun1 and Zeynep Zaimoglu2* Address all correspondence to: zeynepzaimoglu6@gmail.com Address all correspondence to: zeynepzaimoglu6@gmail.com 1 Department of Agricultural Structures and Irrigation, Çukurova University Faculty of Agriculture, Balcalı-Adana, Turkey 2 Department of Environmental Engineering, Çukurova University Faculty of Engineering and Architecture, Balcalı-Adana, Turkey Climate Control in Mediterranean Greenhouses http://dx.doi.org/10.5772/intechopen.72367 181 References [1] Munoz P, Anton A, Nunez M, Paranjpe A, Arino T, Castells X, Montero JI, Rieradevall J. Comparing the environmental impacts of Greenhouse versus open-field tomato produc- tion in the mediterranean region. Acta Horticulturea. 2008;801(2):1591-1596 [2] Giuliano G, Gordon P, Pan Q, Park JY, Wang LL. Estimating freight flows for metropol- itan area highway networks using secondary data sources. Networks and Spatial Eco- nomics. 2010;10(1):73-91 [3] TÜİK. Tarım istatistikleri özeti. Ankara-Türkiye: Türkiye İstatistik Kurumu; 2014 [4] Zabeltitz Chr.von. Integrated Greenhouse Systems for Mild Climates. Springer: Verlag Berlin Heidelberg. 2011 [5] Eker MM. Jeotermal seralarda hedef, 30 bin hektar. Jeotermal belediyeler dergisi. sayı 6. 2012: s.5-14 [6] Nisen A, Grafiadellis M, Jiménez R, La Malfa G, Martinez-Garcia PF, Monteiro A, Verlodt H, Villele O, Cv Z, Jc D, Baudoin W, Jc G. Cultures protegees en climat mediterranean. Rome: FAO; 1988 [7] Baytorun AN, Abak K, Tokgöz H, Güler Y, Üstün S. Seraların kışın iklimlendirilmesi ve denetimi üzerinde araştırmalar. Türkiye Bilimsel ve Teknik Araştırma Kurumu. Proje no TOAG-993; 1995 [8] Baytorun AN, Akyüz A, Üstün S. Seralarda isıtma sistemlerinin modellemesi ve karar verme aşamasında bilimsel verilere dayalı uzman sistemin geliştirilmesi. TÜBİTAK Proje No: 114O533; 2016 [9] Baytorun AN, Abak K, Daşgan HY, Topçu S. Climatic problems of the plastic green- houses in Turkey. International Congress for Plastics in Agriculture. CIPA Procedings. Tel Aviv. Israel; 1997 [10] Kittas C, Katsoulas N, Bartzanas T, Bakker S. Greenhouse climate control and energy use. Good Agricultural Practices for greenhouse vegetable crops. Principles for Mediterranean Climate Areas. Rome: FAO; 2013 [11] Tekinel O, Baytorun N, Demir Y. Çukurova koşullarında seralarda ıslak yastıklarla soğutma olanakları. Türkish Journal of Agriculture and Forestry. 1989. Cilt:13, Sayı 3b, s: 1284-1293 [12] Baytorun AN. Sera projelerinin hazırlanmasında dikkate alınacak kriterler, ve TR 63 bölgesi için örnek sera fizibiliteleri. DOĞAKA (Basımda); 2016
https://openalex.org/W2904867200	https://www.matec-conferences.org/articles/matecconf/pdf/2018/110/matecconf_ipicse2018_03037.pdf	English	null	Design of interacting wells for optimization of investments and operating costs while constructing water-diverting structures	MATEC web of conferences	2,018	cc-by	3,259	MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 https://doi.org/10.1051/matecconf/201825103037 Design of interacting wells for optimization of investments and operating costs while constructing water-diverting structures Vladimir Shcherbakov 1, Alexander Akulshin 2 , Konstantin Chizhik 3,* and Mikhail Tolstoy4 1Voronezh State Technical University 84, 20-let Oktyabrya st., Voronezh, Russia 2South-West State University 94, 50-let Oktyabrya st., Kursk, Russia 3Moscow State University of Civil Engineering, Yaroslavskoe shosse, 26, Moscow, 129337, Russia 4Irkutsk National Research Technical University, 83, Lermontova st., Irkutsk, Russia Abstract. Constructions for water withdrawal are the constitutive building industry component. The cost of these constructions is rather high. That is why while designing it is necessary to choose optimal design model and operating conditions during design life. Operation experience showed that the wells are desirably placed along one line to provide the most optimal conditions for water withdrawal. While designing the wells interaction is considered as a group operation if the distance between them is less than two radiuses of influence. Such wells disposition allows reducing the area and cutting down the investments for water withdrawal construction and also creating the better conditions for equipment and mains operation. Design of interacting wells consists of finding the tube well number, the distance between them, the discharge and levels (static and dynamic). At operating condition determination, it is necessary to consider the combined action of pure water reservoirs and tube well. * Corresponding author: irkyt-44@yandex.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction Water withdrawal wells consisting of several tube wells are used for settlements with a great amount of drinking water discharge consumption. Operation experience showed that it is desirable to place the wells along one line to provide the most optimal conditions for water withdrawal [1,2,6,10,16]. The wells interaction is designed if the distance between the wells is less than 2R that is they work as group ones. Such wells disposition allows reducing the area and cutting down the investments for water wells construction and creating the better conditions for equipment and mains operation. However, because of their influence on each other the wells discharge comes down. * Corresponding author: irkyt-44@yandex.ru MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 https://doi.org/10.1051/matecconf/201825103037 2 Methods The design of the interacting wells is in determining the number of tube wells, the distance between them, water discharge and levels (static and dynamic). g y The design is done in the following order [3,7,8,12,17]: The design is done in the following order [3,7,8,12,17]: 1) determine the water discharge for one well Qw; g g [ ] 1) determine the water discharge for one well Qw; 2) determine the well radius of influence R (the distance from the center to the point of static level recovery according to the formula at 5.1 R  ; (1) m2/day at 5.1 R  ; (1) w – pressure conductivity factor m2/day. – pressure conductivity factor m2/day. Suitable for: Suitable for: - confined water strata onfined water strata М - confined water strata  М k a f  ; (2)  М k a f  ; (2) - free flow strata  av f h k a  ; (3)  av f h k a  ; (3) (3) h –mean power of waterbearing stratum during the period of pumping, m. hav= 0,8Н; μ –water yield factor; h –mean power of waterbearing stratum during the period of pumping, m. hav= 0,8Н; μ –water yield factor; μ y ; t – standard time of well operation, years (25 years = 9125 days). 3) interacting well discharge is designed by formula standard time of well operation, years (25 years = 9125 days). interacting well discharge is designed by formula ; (4) i l l l i i k di h bl ; (4) (4) where αin – interaction factor, for practical calculation is taken according to the table1 Table 1. Value of interaction factor. Distance between wells l , m 2 R R 0,5 R 0,2 R 0,02 R 0,002 R α 1 0,97 0,9 0,81 0,64 0,53 Table 1. Value of interaction factor. 4) distance between wells is determined according to the table 2. 4) distance between wells is determined according to the table 2. Table 2. Distance between water wells. Water-bearing rock Well productivity, m3/hour Up to 20 20-100 100-500 Fine sand 50 50-70 70-100 Medium sand 70-100 100-150 120-150 Coarse-grained sand 100-120 120-150 150-200 Gravel and fractured ground 120-150 150-200 200-250 Table 2. Distance between water wells. Table 2. Distance between water wells. 2 Methods the number of operating wells is determined by the formula ; (5) ; (5) 3 (5) where Q– the necessary discharge of water intake, m3/day. where Q– the necessary discharge of water intake, m3/day. where Q– the necessary discharge of water intake, m3/day. e number of redundant wells is determined according to the table 3. 6) actual discharge (m3/day) interacting wells is determined according to the number of workers and needed discharge 2 MATEC Web of Conferences 251, 03037 (2018) https://doi.org/10.1051/matecconf/201825103037 IPICSE-2018 MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 https://doi.org/10.1051/matecconf/201825103037 ; (6) 7) level lowing, m - lowing level in each well: ; (7) (6) ; (7) Table 3. The number of redundant wells. Number of operating wells The number of redundant wells on water intake of category I II III From 1 to 4 1 1 1 From 5 to 12 2 1 - 13 and more 20% 10% - Maximal lowing level: Maximal lowing level: ; (8) ; (8) (8) where r0 – well radius, in which the lowing is determined, м; di f ll h f ll i l where r0 – well radius, in which the lowing is determined, м; di t f ll №1 t th f ll i ll where r0 – well radius, in which the lowing is determined, м; r2-1, r3-1… ri-1– distance from well № 1 up to the following we 1– distance from well № 1 up to the following wells, m. r2-1, r3-1… ri-1– distance from well № 1 up to the following wells, m. Thus, the power of water bearing strata and filtration factor are the same on the whole water intake, the productivity of the pump equipment assembled in every tube wells is equal that is well discharges are equal. then the designed maximal lowing is compared hen the designed maximal lowing is compare Smaxwith allowable lowing Sval. At Smax> Sval, Smaxwith allowable lowing Sval. At Smax> Sval, Smaxwith allowable lowing Sval. At Smax> Sval, widen the distance between wells and repeat the calculations. The allowable level lowing depends on the hydrological conditions of water bearing strata, tube well structure (well), place of pumping aggregate and filter position. 2 Methods This lowing is determined depending on the producing strata pressure by the following formula: Sval = Н – [(0,3-0,5)М - ∆hp - ∆hf]; (9) (10) - inpressure water well Sval = Н – [(0,3-0,5)М - ∆hp - ∆hf]; (9) - free flow strata (10) - inpressure water well - inpressure water well (9) (10) (10) - free flow strata where ∆hp – maximal depth of pipe edge immersion under dynamic level in the well, m; ∆hf – in nonpressure well at inlet through the filter, m. where ∆hp – maximal depth of pipe edge immersion under dynamic level in the well, m; ∆hf – in nonpressure well at inlet through the filter, m. where ∆hp maximal depth of pipe edge immersion under dynamic level in the well, m; ∆hf – in nonpressure well at inlet through the filter, m. f p g , 9) in conclusion, the dynamic level position in the well is determined: f p g , 9) in conclusion, the dynamic level position in the well is determined: p Z d Z st- S t (11) (11) Z d Z st- S t where Z d – dynamic level, m; where Z d – dynamic level, m; Z st – static level, m; Z st – static level, m; Z st – static level, m; S t – accepted value of level lowing, m. S t – accepted value of level lowing, m. p g Design of water lifting station consists of some stages: b i i f d i d l Design of water lifting station consists of some stages: Design of water lifting station consists of some stages: - substantiation of design model; - determination of plot size for the water station - determination of plot size for the water station - substantiation of rational scheme of well position inside the plot; p p - substantiation of well operation during the design working period (discharge and dynamic level lowing). - substantiation of well operation during the design working period (discharge and dynamic level lowing). Linear scheme of well connection to the prefabricated water conduit is the simplest and applied during the water conduit stringing into one line [4,5,9,11,13] (Fig. 1). Prefabricated water conduct diameter can increase, at growing of joined wells number and as a result at the discharge increase. 3 https://doi.org/10.1051/matecconf/201825103037 MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 Fig. 1. Linear scheme of well connection to the prefabricated water conduit in one line 1 –water well; 2 –prefabricated water conduit Fig. 1. Linear scheme of well connection to the prefabricated water conduit in one line 1 –water well; 2 –prefabricated water conduit While determining the prefabricated water conduit operation behavior it is necessary to take into consideration the combined work of pure water tanks and tube wells. Thus, the pressure created by submersible drive pump unit at the point of connection to the prefabricated water conduit Н1 should be greater than in water conduit. Fig. 2. Linear scheme of wells connection on water diversion work from underground spring 1 – water receiving structures (wells); 2 pressure pipes; 3 – prefabricated water conduit; 4 – stop control valve; 5 – main water conduit; 6 – pure water tank. Fig. 2. Linear scheme of wells connection on water diversion work from underground spring 1 – water receiving structures (wells); 2 pressure pipes; 3 – prefabricated water conduit; 4 – stop control valve; 5 – main water conduit; 6 – pure water tank. In Fig. 2 there is the scheme with well point systems applied as water diversion works. Water derived from water bearing strata by submersible pumps is placed in wells into pressure water conduits delivered into collection water conduit and then comes into pure water tanks. S t – accepted value of level lowing, m. General scheme of structures disposition on water system presented in draw 2 is the most commonly used in construction practice and underground water-diverting structure operation when wells are used as water receiving structures as the quality of water extracted from water bearing strata before delivery to a consumer needs improving. If on the water-diverting structure the amount of wells are big and they are located along a river and on the distance L from it, it is possible to use formula admitting the substitution of real well raw, by the galleries with 1m of length discharge [4,14, 15,16]: 4 4 4 MATEC Web of Conferences 251, 03037 (2018) https://doi.org/10.1051/matecconf/201825103037 IPICSE 2018 MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 https://doi.org/10.1051/matecconf/201825103037 (12) where  Q - total discharge of wells; Q –one well discharge; l- half the gallery length; - half the distance between wells; 1  n l  ; n –general number of wells. (12) where  Q - total discharge of wells; Q –one well discharge; Q –one well discharge; l- half the gallery length; - half the distance between wells; 1  n l  ; n –general number of wells. If the linear series of wells has a length commensurate with the distance to the river (that is 2l≈L), the equation for a gallery of finite length is used in the calculations. Then, when using a method of mirror displays, we receive the dependence allowing to define decrease in dynamic level at any time, time at the long periods pumping out to any point of layer M (draw. 3). y If the linear raw has the length commensurable with the distance up to a river (that is 2l≈L), the equations for the galleries of final length are applied [17,18] : (13) (13) = 1 2 1 2 ) ( 1 ) ( 1 ln 2 1 2 2 2 2 2 2           L y arctg L L L arctg L y L y    (14) (14) These formulas give the possibility to determine how the dynamic level falls at points remote from the wells located perpendicularly to it. 1. V.I. Shcherbakov, A.A. Akulshin Water intake structures from underground sources (Monograph) LAPLAMBERT Academic Publishing, 192 (2017) 4 Discussion The design of interacting wells group consists of defining the tube wells number, distance between them, discharge and levels (static and dynamic). While determining the operation conditions of water collection conduit it is to consider combined work of pure water tanks and tube wells. The diameter of water collection conduit is to be increased at increasing of connected wells number and consequently the increasing of discharge. S t – accepted value of level lowing, m. g g y p If wells give the constant discharge during long period of operation the expression (17) is      r R S km Q ln 2 (18) (18) R – well radius of influence. If during the water intake the wells are located in one row and parallel to water body bank the calculation can be done by the expression:                  0 ln 2 2 2 1 ln 2 r l L l arctg l L l L S km Q (19) (19)   L – distance between the rows of wells, m. L – distance between the rows of wells, m. L – distance between the rows of wells, m. 3 Results Investments for building and creation of the best conditions for the equipment and mains operation depend on the choice of the scheme of water-diverting structure, its operation performances. The analysis of optimization of interacting wells operation showed that linear scheme of tube wells connection to the collection water conduit water conduit is the simplest one and is used while water conduit placing in one row. S t – accepted value of level lowing, m. If it is demanded to determine the dynamic level lowering in the very tube wells the function is expressed as following: These formulas give the possibility to determine how the dynamic level falls at points remote from the wells located perpendicularly to it. These formulas give the possibility to determine how the dynamic level falls at points remote from the wells located perpendicularly to it. remote from the wells located perpendicularly to it. If it is demanded to determine the dynamic level lowering in the very tube wells the function is expressed as following: = ) (ln 2 1 21 ) 2 1( 1 ln 2 1 0           r L L arctg L L (15) (15) Simply transforming the formula (12) and putting formula (14) into it we receive the dependence with the help of which we define the water discharge of one well: Simply transforming the formula (12) and putting formula (14) into it we receive the dependence with the help of which we define the water discharge of one well: l km S Q   2  )) (ln 2 1 21 ) 2 1( 1 ln 2 1 ( 0           r L L arctg L L (16) (16) (16) Fig. 3. Scheme of the design of linear row of wells locating near the river. Fig. 3. Scheme of the design of linear row of wells locating near the river. 5 5 MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 https://doi.org/10.1051/matecconf/201825103037 As a result, we receive the general dependence of discharge for one well: As a result, we receive the general dependence of discharge for one well:                   at r E S km Q i 4 4 2 (17) (17)   k- filtration factor, m/day; m–water bearing horizon power, m; m–water bearing horizon power, m; g p , ; of water pumping from one well t - duration of water pumping from one well t - duration of water pumping from one well R – well radius, m; d i i f R – well radius, m; а – pressure conductivity factor Еi– integral exponential function; g p ε – factor considering filter resistance, considering hydraulic uplift. References 6 6 https://doi.org/10.1051/matecconf/201825103037 MATEC Web of Conferences 251, 03037 (2018) IPICSE-2018 2. S.A. Nozdratenko, A.A. Akulshin, A.A. Akulshin Preventive measures to extend the life of water wells University-book, Kursk, 140 (2015) 3. V.S. Pereverzeva, A.A. Akulshin Determination of the optimal duty cycle of wire filters depending on the parameters of tubular wells and soil University-book, Kursk, 438 (2014) 4. V.I. Shcherbakov, I.Y. Purusova Methods of calculating interacting water intake wells Sat. sci. Art. / RAASN, TSTU. - Voronezh-Tambov: Publishing house of Tamb. state. tech. U-ta, 281-284 (2009) 5. G.N. Kamensky Fundamentals of the dynamics of underground water Gosgeolizdat, 248 (1943) 6. S.M Chudnovsky, A.V. Zenkov Designing, construction and operation of water intake wells: training manual Vologda: VSTU, 134 (2008) 7. TR 31.13330.2012. Water supply. External networks and facilities. Updated version. SNiP 2.04.02-84* - M .: Ministry of Regional Development of Russia, 128 (2012) 8. N.A. Plotnikov, V.S. Alekseev Designing and operation of water intakes of underground waters M., Stroiizdat,, 256 (1990) 9. F. M. Bochever. Designing of water intakes of underground waters M., Stroiizdat, 292 (1976) 10. M.Y..Panov, V.I. Shcherbakov, I.Y. Purusova Modeling of flow distribution and management of water-lifting stations Bulletin of Voronezh Technical University, series Energetika, 3/6 182-185 (2007) 11. MG Zhurba, LI Sokolov, Z.M. Govorova Water supply. Design of systems and structures. In 3 volumes. Volume 1. Water supply systems, water intake facilities. - М .: Publishing house of the Association of Construction Universities, 400 (2010) 12. A.S. Belitsky, V.V. Dubrovsky Designing of exploration and production wells for water supply - 3rd ed. M .: Nedra, 256 (1974) 13. D.N. Bashkatov, V.L. Rogovoy Drilling of wells for water M., Kolos, 208 (1976) 14. V.V. Ivashechkin Improvement of water intake well designs and methods of their overhaul Energy. Izv. supreme. of educational institutions and energy. associations of the CIS, 2, 175-186 (2016) 15. A.M. Krygina On the issue of technical operation of water intake wells Industrial and Civil Engineering, 8 40-41 (2011) 16. А. Akulshin A method of calculating filtration rate of an infiltration water intake in Kursk region Journal of Applied Engineering Science, 3 208-211 (2017) 17. V.I. Shcherbakov, K.I. Chizhik, N.P. Konovalov, I.Y. Purusova Energy efficiency of functioning of water reservoir wells and collecting conduit Construction. The formation of living Environement. IOP Conference series: Materials Science and Engineering 365 art. 022060 (2018) 18. V.I. Shcherbakov, A.A. Akulshin, N.V. References Bredikhina The calculation of interacting wells in order to optimize the capital and operating costs for the construction of water intakes Izvestiya South-Western State University, 2 (77) 44-51 (2018) 7
https://openalex.org/W4285024835	https://zenodo.org/records/2282667/files/Plant_Introd_73_011-020.pdf	Ukrainian	null	Evaluating the results of introduction of aromatic plants from the Lamiaceae Lindl. family in Ukrainian Polissya	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	6,519	ОЦІНКА УСПІШНОСТІ ІНТРОДУКЦІЇ АРОМАТИЧНИХ РОСЛИН РОДИНИ LAMIACEAE LINDL. В УМОВАХ ПОЛІССЯ УКРАЇНИ Мета роботи — оцінити результати успішності інтродукції та визначити перспективи культ олісся України однорічних і багаторічних малопоширених видів ароматичних рослин родини Lam Матеріал та методи. Досліджено 17 однорічних і багаторічних видів нетрадиційних ароматичних рослин родини Lamiaceae, які було інтродуковано впродовж 2008—2016 рр. у Ботанічному саду Житомирського національного агро- екологічного університету. Використано лабораторні, польові та інтродукційні методи. Оцінено загальний стан рос- лин, особливості насінного і вегетативного розмноження, їх зимо-, морозо- та посухостійкість, а також стійкість до хвороб і шкідників. Результати. Серед культивованих ароматичних рослин родини Lamiaceae 3 однорічних та 8 багаторічних видів (67,4 % від загальної кількості досліджених) виявились особливо перспективними і високостійкими. В умовах Полісся України вони добре ростуть і нормально розвиваються, зберігаючи притаманну їм життєву форму, плодоносять, майже не зазнають суттєвих ушкоджень від морозів, посухи та патогенних організмів. Висновки. Dracocephalum moldavica L., Satureja hortensis L., S. montana L., Elsholzia cristata Willd., Hyssopus officinalis L., H. аngustifolius M. Bieb., Origanum vulgare L., Nepeta transcaucasica Grossh., Salvia officinalis L., S. sclarea L. і S. aethio- pis L. — екологічно пластичні види, придатні для введення у промислову культуру на Поліссі України. Як високопер- спективні інтродуценти вони можуть бути цінним джерелом поповнення рослинних ресурсів та вихідним матеріа- лом для селекційних досліджень і створення високоадаптивних сортів. лючові слова: ароматичні рослини, Lamiaceae Lindl., успішність інтродукції, Полісся Україн Культивування рослин, які мають значний біологічний та господарський потенціал, у нових умовах зростання сприяє розширенню асортименту лікарських, пряносмакових, аро- матичних, ефіроолійних, медоносних і деко- ративних видів [22]. Досить цінними у цьому відношенні є представники родини Lamiaceae Lindl. За інформацією сайту The Plant List (2013) до складу родини Lamiaceae входять 245 родів і 7850 видів [26]. Поширення представ- ників родини — космополітне, але найбільше видове їх розмаїття зосереджено в країнах Серед земномор’я, Передньої Азії, на Далекому Сході, Кавказі та в Європейсько-Азіатсько-Си- бір сько му регіоні [5, 12, 24]. У флорі України трапляється близько 250 видів. Однак при- родні запаси дикорослих рослин родини по- вністю або частково вичерпані, тому інтро- дукція і культивування цінних видів збагатить біотичне різноманіття місцевої флори [21]. Інтродукція рослин — важливий чинник зба- гачення рослинних ресурсів, а також збіль- шення біотичного різноманіття культурфіто- ценозів. Екологічний підхід в інтродукційних дослідженнях потребує вивчення сукупності умов та впливу чинників, які діють на орга- нізм рослин у нових природних умовах чи культурі [9, 20]. Інтродукційна діяльність пе- редбачає насамперед мобілізацію природних ресурсів для пошуку перспективних цінних видів рослин, які є джерелом біологічно ак- тивних сполук. УДК 582. 929.4:581.5 (477.42) УДК 582. 929.4:581.5 (477.42) Матеріал та методи Предмет досліджень — 17 видів рослин родини Lamiaceae, які культивували впродовж 2008— 2016 рр. у Ботанічному саду Житомирського національного агроекологічного університету: однорічні — змієголовник молдавський (Dra- cocephalum moldavica L.), чабер садовий (Satu- reja hortensis L.), ельшольція гребінчаста (El- sholzia cristata Willd.), васильки звичайні (Oci- mum basilicum L.), в. священні (O. sanctum L.), монарда лимонна (Мonarda citriodora Cerv.); багаторічні — гісоп лікарський (Hyssopus officina- lis L.), г. вузьколистий (H. аngustifolius M. Bieb.), чабер гірський (Satureja montana L.), лаванда справжня (Lavandula vera D. C.), лофант гану- совий (Lophanthus anisatus Adans.), монарда двійчаста (Monarda didyma L.), материнка зви- чайна (Origanum vulgare L.), шавлія лікарська (Salvia officinalis L.), непета закавказька (Nepe- ta transcaucasica Grossh.); дво- або трирічні — шавлія ефіопська (Salvia aethiopis L.) і ш. мус- катна (S. sclarea L.). При оцінюванні інтродукційної стійкості ви- дів родини Lamiaceae виділено чотири групи рослин [7, 14, 23]: I — нестійкі, II — слабко- стійкі, III — стійкі, IV — високостійкі рослини. ОЦІНКА УСПІШНОСТІ ІНТРОДУКЦІЇ АРОМАТИЧНИХ РОСЛИН РОДИНИ LAMIACEAE LINDL. В УМОВАХ ПОЛІССЯ УКРАЇНИ Успішність інтродукції визна- чається адаптаційними можливостями рос- лин, важливими з яких є проходження всіх етапів індивідуального та сезонного розвитку рослин, їх здатність до розмноження, стій- кість і невибагливість у нових умовах вирощу- вання [9, 11]. © Л.А. КОТЮК, Д.Б. РАХМЕТОВ, Т.В. ПІНКІНА, 2017 © Л.А. КОТЮК, Д.Б. РАХМЕТОВ, Т.В. ПІНКІНА, 2017 11 ISSN 1605-6574. Інтродукція рослин, 2017, № 1 Л.А. Котюк, Д.Б. Рахметов, Т.В. Пінкіна радою та ін. (2012). Особливо перспективні (ОП) багаторічні види розмножуються насін- ням та вегетативно і не потребують спеціаль- них агротехнічних заходів. Для них характер- на висока адаптивна здатність на всіх етапах онтогенезу, зимо- і посухостійкість. Вони стій- кі до шкідників та хвороб, щорічно цвітуть і плодоносять, дають самосів (45—54 бали). Пер- спективні види (П) розмножуються насінням або вегетативно, потребують поливу, підбору світ ло вих або тіньових ділянок, зрідка підмер- за ють, щорічно цвітуть і плодоносять (у не- сприят ли ві роки репродуктивна здатність цих видів ослаблена), дають самосів (35—44 бали). Малопер спективні види (МП) слабкозимостій- кі, насіння утворюють нещорічно (20—34 бали). Для оцінки однорічних рослин у зв’язку з тим, що їх вегетативне розмноження в умовах від- критого ґрунту виявилося неможливим, вико- рис товували модифіковану нами шкалу: 36— 45 балів — особливо перспективний вид, 26— 35 балів — перспективний, 16—25 балів — ма ло- пер спек тивний [10]. Нині досліджено незначну кількість видів ароматичних рослин родини Lamiaceae, недо- статньо відомостей про їх адаптивні особли- вості. Мета досліджень — оцінити результати ус- піш ності інтродукції та визначити перспекти- ви культивування в умовах Полісся України однорічних і багаторічних малопоширених видів ароматичних рослин родини Lamiaceae. Результати та обговорення Важливим показником адаптації інтродуцен- тів до нових умов зростання є їх здатність до репродуктивного і вегетативного розмножен- ня. В умовах досліджень однорічні види фор- мували плоди та насіння, проте їх вегетативне розмноження виявилося неможливим. Під час культивування ароматичних рослин в умовах Полісся України оцінювали їх життє- вість та успішність інтродукції. Оцінку загаль- ного стану рослин здійснювали у фазу цвітін- ня і перед збором урожаю. Використовували рекомендації [1, 7, 10, 14—16, 18,19, 23, 25]. Найкращу здатність до насінного роз мно- жен ня (9 балів) відзначено у рослин D. molda vica, S. hortensis, E. cristata, здатність рослин O. sanc- tum і O. basilicum оцінено 7 балами, а М. citriodo- ra — 5 балами. При сівбі насіння у відкритий ґрунт у O. basilicum і М. citriodora порівняно з іншими видами рослин сходи з’являлися на 7—14 діб пізніше, рослини розвивалися менш інтенсивно і не завжди формували насіння до настання осінніх заморозків. Для визначення стійкості рослин до шкід- ників і хвороб застосовували 9-бальну шка лу, запропоновану О.А. Порадою (2007) [18], для встановлення видового складу шкодочинних організмів — загальноприйняті методики і визначники [2, 6, 16, 17]. Оцінку успішності інтродукції однорічних та багаторічних видів проведено за комплек- сом характеристик, запропонованих О.А. По- Серед багаторічних видів найкращу здат- ність до насінного розмноження (9 балів) ви- ISSN 1605-6574. Інтродукція рослин, 2017, № 1 12 Оцінка успішності інтродукції ароматичних рослин родини Lamiaceae Lindl. в умовах Полісся України шності інтродукції ароматичних рослин родини Lamiaceae Lindl. в умовах Полісся України і високих температур влітку, життєвий цикл інт родуцентів подовжувався, не завжди фор- мувалось або не визрівало насіння. Загальний стан цих видів оцінено 5 балами. явлено у рослин H. officinalis, H. angustifolius. За цим критерієм рослини L. anisatus, S. mon- tana, N. transcaucasica, S. sclarea і S. aethiopis оці- нено 7 балами, а M. didyma, S. officinalis, O. vul- gare і L. vera — 5 балами [12]. явлено у рослин H. officinalis, H. angustifolius. За цим критерієм рослини L. anisatus, S. mon- tana, N. transcaucasica, S. sclarea і S. aethiopis оці- нено 7 балами, а M. didyma, S. officinalis, O. vul- gare і L. vera — 5 балами [12]. Відмінний стан рослин (9 балів) відзначено у більшості багаторічних видів (H. officinalis, H. аngustifolius, L. anisatus, S. montana, O. vulgare, N. transcaucasica, S. officinalis, S. sclarea і S. aethio- pis). Ці види добре відновлювалися після пе- резимівлі, рясно цвіли і плодоносили, були високопродуктивними. Загальний стан рос- лин M. didyma та L. Результати та обговорення vera оцінено 7 балами, ос- кільки рослини за роки досліджень були менш продуктивними і більше залежними від еко- логічних чинників. Відзначено, що в умовах інтродукції сіянці N. transcaucasica формували повноцінне на- сіння в перший рік життя. Рослини H. officina- lis і L. anisatus також формували насіння у пер- ший рік вегетації рослин, хоча за повідомлен- нями Е.П. Вороніна (2001) і А.А. Аутко зі спів авт. (2003) вони плодоносять на другий рік життя [3, 8]. На другий рік життя спостерігали пло- доношення у рослин H. аngustifolius, S. sclarea, S. aethiopis і M. didyma, на третій — у рослин S. montana, O. vulgare і L. vera. Холодостійкість і морозостійкість досліджу- ваних однорічних видів рослин характери зу ва- лися широкою амплітудою. Найнижчу хо ло до - стій кість відзначено у рослин O. basilicum, ріст яких сповільнювався за температури +10…5 °С, а весняні сходи гинули. Ці рослини — неморо- зостійкі, при зниженні температури восени до 0…–2 °С відзначали повну загибель рослин (відмирання стебел, листків, суцвіть). Дещо вищу морозо- і холодостійкість установлено у рослин O. sanctum і М. citriodora, які сповіль- нювали свій ріст за температури +8…2 °С, пов ну їх загибель спостерігали при зниженні темпе- ратури до –5 °С. За критеріями холодо- і моро- зостійкості рослин O. basilicum оцінено 3 ба ла- ми, а O. sanctum та М. citriodora — 5 балами. Найкращу здатність до вегетативного роз- мно ження (9 балів) виявлено у рослин S. officina- lis при поділі куща або здерев’янілими живцями трирічних або п’ятирічних рослин у ранньовес- няний період. За вегетативного розмноження приживалися понад 90 і 80 % саджанців, вони формували незначну кількість су цвіть і насіння. Рослини H. officinalis, H. аn gus tifolius, L. anisatus, S. montana, L. vera на чет вертому—п’ятому році життя, а рослини S. aethi o pis і S. sclarea — на дру- гому році розмножували поділом куща на част- ки, що спрощувала партикуляція кореневої системи. Рослини N. transcaucasica, M. didyma та O. vulgare на тре тьому—п’ятому році життя розмножували шляхом поділу кореневища із бруньками відновлення на частки. Рослини E. cristata характеризувалися ви- щою холодо- і морозостійкістю. Весняне по- холодання спричинило не загибель сходів, а лише їх пожовтіння і сповільнення росту. Пов- ну загибель рослин спостерігали за темпера- тури –5 °С та нижче. За критеріями холодо- і морозостійкості рослини оцінено 7 балами. Саджанці H. officinalis, H. аngustifolius, L. ani- satus, S. montana, L. vera, S. aethiopis, S. sclarea і O. Результати та обговорення vulgare добре приживалися та плодоносили, тому за критерієм здатності до вегетативного розмноження ці види оцінено 7 балами. Са- джанці M. didyma добре приживались, але не завжди утворювали генеративні органи, тому цей вид оцінено 5 балами. Досить холодо- і морозостійкими виявили- ся рослини D. moldavica і S. hortensis, які за цими критеріями оцінено 9 балами. Сходи під час весняних заморозків виявилися стій- кими до дії низьких температур, а зниження температури у вересні—жовтні до –5 °С не зав- дало шкоди рослинам. Самосів рослин цих видів гинув за умови тривалої дії температу- ри, нижчої за –10 °С. Спостереження показали, що рослини од но- річних видів D. moldavica, S. hortensis та E. cristata зростали і розвивалися нормально, формували повноцінне насіння, тому за загальним станом види оцінено 9 балами. O. basilicum, O. sanctum та М. citriodora давали зріджені сходи, ріст рос- лин дуже сповільнювався за відсутності опадів 13 ISSN 1605-6574. Інтродукція рослин, 2017, № 1 Л.А. Котюк, Д.Б. Рахметов, Т.В. Пінкіна За ступенем холодостійкості і зимостійко- сті досліджувані багаторічні інтродуценти роз- поділили на дві групи [25 ]. До першої групи віднесли види, які успішно зимували без ук- риття впродовж багатьох років і не випадали: H. officinalis, H. аngustifolius, S. montana, M. di- dyma, O. vulgare, N. transcaucasica, S. aethiopis, S. sclarea, S. officinalis. Рослини цих видів ви- тримували вплив осінніх і весняних замороз- ків (–5 …–10 °С) і низькі температури взимку (до –35 °С) без ушкоджень. За критеріями хо- лодо- і зимостійкості зазначені види оцінено 9 балами. nalis, S. montana, M. didyma, O. vulgare, N. trans- caucasica, S. aethiopis, S. sclarea, S. officinalis, які походять із Північної Америки, Далекого Схо ду і Середземномор’я. nalis, S. montana, M. didyma, O. vulgare, N. trans- caucasica, S. aethiopis, S. sclarea, S. officinalis, які походять із Північної Америки, Далекого Схо ду і Середземномор’я. nalis, S. montana, M. didyma, O. vulgare, N. trans- caucasica, S. aethiopis, S. sclarea, S. officinalis, які походять із Північної Америки, Далекого Схо ду і Середземномор’я. Стійкість рослин до посухи — один із важ- ливих критеріїв успішної інтродукції видів в умовах нестійкого зволоження, які спостері- гали впродовж багатьох років досліджень. По- сухостійкість — це здатність рослин витри- мувати посуху без різкого зниження врожай- ності [25]. Спостереження показали, що найбільш ви- могливими до умов зволоження серед одно- річних видів виявилися рослини O. basilicum і O. sanctum. ISSN 1605-6574. Інтродукція рослин, 2017, № 1 Результати та обговорення в умовах Полісся України Для підвищення продуктивності цих видів за відсутності опадів упродовж тривалого періо- ду доцільно здійснювати їх полив. гали знач ної затримки ростових процесів і зниження продуктивності, виявлено лише не- значне пожовтіння нижніх стеблових чи при- кореневих листків. E. cristata, D. moldavica і S. hortensis віднесе- но до середньо посухостійких. Зазначені ви ди також потребували достатнього зволоження для проростання насіння. Поява сходів за відсут- ності дощів затримувалася на 7—10 діб. У до- рослому стані рослини виявилися стійкіши- ми до недостатнього водозабезпечення, але дещо знижувалась їх продуктивність, спосте- рігали процеси завчасного відмирання. Зазна- чені ви ди за критерієм посухостійкості оці- нено 7 балами. Отже, в умовах інтродукції на Поліссі Украї- ни багаторічні види (за винятком M. didyma) виявилися посухостійкими, однорічні види E. cristata, D. moldavica та S. hortensis — середньо- стійкими, М. citriodora і O. sanctum — із низькою стійкістю, O. basilicum — із дуже низькою стій- кістю до недостатнього вологозабезпечення. Крім абіотичних чинників, на ріст і розви- ток інтродукованих рослин значно впливають біотичні, тобто інші живі організми — тварини, гриби, бактерії та віруси. Одні з них корисні для рослин (комахи-запилювачі, ентомофа- ги, ґрунтові гриби), інші — паразити, за вдають значної шкоди, спричиняючи пошкодження тканин і загибель рослин. Тому резистентність рослин до патогенів грибного, бактеріального та вірусного походження і ко мах-фітофагів є важливим показником їх адаптації в умовах інтродукції. Серед багаторічних видів не виявлено росли- ни із дуже низькою (слабкою) посухостійкістю. Середня посухостійкість (5 балів) притаманна рослинам M. didyma, у яких спостерігали суттє- ве зниження продуктивності за від сутності дощів. У рослин L. anisatus незнач но знижува- лась урожайність, вони були посухостійкими і за цим критерієм їх оцінено 7 балами. Рослини H. officinalis, H. аngustifolius, S. mon- tana, O. vulgare, N. transcaucasica, L. vera, S. aethio- pis, S. sclarea, S. officinalis оцінено 9 балами та віднесено до посухостійких видів, оскільки за умов недостатнього зволоження не спостері- Ентомологічні дослідження показали, що хоча посіви ароматичних рослин родини La- miaceae заселені видами комах-фітофагів, во- ни не завдали суттєвої шкоди інтродуцентам. Таблиця 2. Оцінка успішності інтродукції багаторічних видів родини Lamiaceae в умовах Полісся України Table 2. Результати та обговорення За недостатньої кількості вологи у ґрунті сходи зазначених видів з’являлись із за- тримкою на 14—18 діб, розвиток дорослих рослин сповільнювався, спостерігали пожов- тіння листків, сповільнення росту пагонів, нерегулярне формування суцвіть. Менш зале- жали від недостатнього зволоження рослини O. sanctum, у яких наявне густе опушення лист- ків і стебел, що, ймовірно, сприяло меншому випаровуванню води. Низька посухостійкість властива також рослинам М. citriodora. За умов недостатньої зволоженості поява сходів за- тримувалася на 7—14 діб, а у дорослих особин сповільнювалися ростові процеси, що негатив- но впливало на продуктивність рослин. За кри- терієм посухостійкості види оцінено 1 (O. ba si- li cum) та 3 (O. sanctum, М. citriodora) балами. L. vera і L. anisatus — менш стійкі види рос- лин, їх віднесли до другої групи. Сходи і до- рослі особини виявилися стійкими до дії тем- ператур до –10 °С, але за тривалої дії нижчих температур та інших негативних чинників спо- стерігали випадіння — від 2 % (2-3-й рік жит- тя) до 4 % (4—6-й рік життя) рослин L. vera, 5—8 % особин L. anisatus. За критеріями холо- до- і зимостійкості ці види оцінено 7 балами. Спостереження показали, що серед досліджува- них багаторічних видів нестійких до дії від’єм- них температур не виявлено. Таким чином, із однорічних видів найбільш толерантними до дії низьких температур вия- вилися рослини S. hortensis і D. moldavica, цен- тром походження яких є Європейсько-Азіат- сько-Сибірський регіон. Серед багаторічних видів досить холодо- і зимостійкими є H. offici- Таблиця 1. Оцінка успішності інтродукції однорічних видів родини Lamiaceae в умовах Полісся України Table 1. Evaluation of introduction efficiency of annual the Lamiaceae family species in conditions of Ukrainian Polissya Вид Оцінка, бал Сумарна оцінка життєвості / успішність інтродукції насінне розмноження загаль ний стан холодо- і морозо- стійкість посухо- стійкість стійкість до хвороб і шкідни- ків Dracocephalum moldavica 9 9 9 7 9 43 / ОП Satureja hortensis 9 9 9 7 9 43 / ОП Elsholzia cristata 9 9 7 7 9 41 / ОП Ocimum basilicum 7 5 3 1 7 23 / МП O. sanctum 7 5 5 3 7 29 / П Мonarda citriodora 5 5 5 3 5 23 / МП Таблиця 1. Оцінка успішності інтродукції однорічних видів родини Lamiaceae в умовах Полісс шності інтродукції однорічних видів родини Lamiaceae в умовах Полісся України 14 Оцінка успішності інтродукції ароматичних рослин родини Lamiaceae Lindl. в умовах Полісся України шності інтродукції ароматичних рослин родини Lamiaceae Lindl. ISSN 1605-6574. Інтродукція рослин, 2017, № 1 Результати та обговорення До перспективних видів віднесено L. anisatus (44), L. vera (42) і M. didyma (36 балів). Малоперспективні багаторічні види в умовах інтродукції не виявлено (табл. 2). На листках D. moldavica і N. transcaucasica траплялися поодинокі екземпляри T. urticae (родина Tetranychidae), які не спричиняли сут- тєвого негативного впливу на рослини, ймо- вірно, завдяки значній кількості покривних та ефіроолійних трихом на епідермі рослин. Переважну більшість однорічних і багато- річних ароматичних рослин ушкоджували ко- махи, які належать до рядів Homoptera (ро дини Cicadellidae, Aphrophoridae, Membracidae), He- miptera (родини Miridae, Pentatomidae, Pyrr- hocoridae, Scutelleridae), Thysanoptera (родина Thripidae), Coleoptera (родини Curculionidae, Chry- somelidae, Scarabaeidae), Diptera (родина Antho- myiidae) і Lepidоptera (родина Noctuidae). За результатами оцінювання інтродукційної стійкості однорічних рослин до високостій- ких віднесено 3 види (D. moldavica, S. hortensis), до стійких — 1 (O. sanctum), до слабкостійких — 2 (O. вasilicum і М. citriodora). Високостійкими виявилися 8 багаторічних видів (H. аngustifolius, H. officinalis, S. montana, O. vulgare, N. transcau- casica, S. of ficinalis, S. sclarea і S. aethiopis), стій- кими — 3 (L. anisatus, L. vera, M. didyma). y ) p p (р ) На деяких рослинах виявлено ознаки вірус- них і грибних хвороб, які незначною мірою знижували продуктивність рослин та якість си- ровини. Ознаки вірусних хвороб у вигляді кур- чавості, жовтяниці або мозаїки листків відзна- чено у рослин L. anisatus (ураженість рослин становила від 10 до 35 %). Борошнисторосяні гриби із роду Erysiphe (клас Ascomycetes, порядок Erysiphales) у вигляді борошнистих «подуше- чок» конідіального спороношення ура жували листки рослин H. officinalis (до 15 %), причому ступінь ураження зростав в умовах надмірного зволоження і загущення насаджень, а ознаки ураження з’являлися восени. Більшою мірою еризифальні гриби уражували рослини M. di dy- ma і М. citriodora, вкриваючи спочатку поду- шечками, а згодом суцільним бо рошнистим на- льотом листки та пагони рослин, особливо вліт- ку, при недостатньому зволоженні та зниженні резистентності рослин. Частка уражених рос- лин досягала 60 %. Збудників ро ду Septoria (клас Ascomycetes, порядок Erysiphales), які спричиня- ли плямистість і від мирання листків, виявлено на рослинах S. sclarea, S. aethiopis та S. officinalis (ступінь ураження — 5 %). Таким чином, при інтродукції ароматичних рослин родини Lamiaceae на Поліссі України 3 однорічних і 8 багаторічних видів (67,4 % від загальної кількості досліджених) виявилися особливо перспективними і високостійкими. В умовах Полісся України вони добре ростуть і нормально розвиваються, зберігаючи прита- манну їм життєву форму, плодоносять, майже не зазнають ушкоджень від морозів, посухи, патогенних організмів. Інтродуценти D. mol- davica, S. Результати та обговорення Evaluation of introduction efficiency of perennial the Lamiaceae family species in conditions of Ukrainian Polissya Вид Оцінка, бал Сумарна оцінка життєвості / успішність інтродукції розмноження загаль- ний стан холо до- і зимо стій- кість посухо- стій кість стійкість до хвороб і шкідни ків насінне веге та- тивне Hyssopus officinalis 9 9 9 9 9 8 53 / ОП H. аngustifolius 9 7 9 9 9 9 52 / ОП Lophanthus anisatus 7 7 9 7 7 7 44 / П Satureja montana 7 7 9 9 9 9 50 / ОП Lavandula vera 5 7 7 7 9 7 42 / П Monarda didyma 5 5 7 9 5 5 36 / П Origanum vulgare 5 7 9 9 9 7 46 / ОП Nepeta transcaucasica 7 7 9 9 9 9 50 / ОП Salvia officinalis 5 9 9 9 9 7 48 / ОП S. sclarea 7 7 9 9 9 7 48 / ОП S. aethiopis 7 7 9 9 9 7 48 / ОП Таблиця 2. Оцінка успішності інтродукції багаторічних видів родини Lamiaceae в умовах Поліс Table 2. Evaluation of introduction efficiency of perennial the Lamiaceae family species in condition аблиця 2. Оцінка успішності інтродукції багаторічних видів родини Lamiaceae в умовах Полісся bl 2 E al ation of introd ction efficienc of perennial the L i famil species in conditions of ті інтродукції багаторічних видів родини Lamiaceae в умовах Полісся України 2. Оцінка успішності інтродукції багаторічних видів родини Lamiaceae в умовах Полісся Україн ISSN 1605-6574. Інтродукція рослин, 2017, № 1 15 Л.А. Котюк, Д.Б. Рахметов, Т.В. Пінкіна На посівах однорічних і багаторічних арома- тичних рослин виявлено представників класів Arachnida та Insecta. оцінка життєвості яких становила 43 бали, до малоперспективних — O. basilicum і М. citrio- dora, які оцінено 23 балами (табл. 1). Із 11 багаторічних видів як особливо пер- спективні відзначено 8 видів: H. officinalis (53 ба- ли), H. аngustifolius (52), N. transcaucasica і S. mon- tana (50), S. officinalis, S. sclarea і S. aethiopis (48), O. vulgare (46 балів). До перспективних видів віднесено L. anisatus (44), L. vera (42) і M. didyma (36 балів). 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(2011), Teoretychni ta prykladni as- pekty introdukcii roslyn v Ukraini [Theoretical and practical aspects of plant introduction in Ukraine]. Kyiv: Agrar Media Grup, 398 p. Цель работы — оценить результаты успешности ин- тродукции и определить перспективы культивирова- ния в условиях Полесья Украины однолетних и мно- голетних малораспространенных ароматических рас- тений семейства Lamiaceae Lindl. 21. Skybicka, M.I. and Mogyljak, M.G. (2013), Perspekty- vy introdukcii likarskyh ta dekoratyvnyh roslyn z ro- dyny Lamiaceae u Zahidnomu Lisostepu Ukrainy [Prospects for the introduction of medicinal and orna- mental plants in the family Lamiaceae in the Western Steppe of Ukraine]. Naukovyj visnyk NLTU Ukrainy [Scientific bulletin of Ukrainian National Forestry University], vol. 23 (10), pp. 40—45. Материал и методы. Исследованы 17 однолетних и мно голетних видов нетрадиционных ароматических растений семейства Lamiaceae, интродуцированных в течение 2008—2016 гг. в Ботаническом саду Жи то мир- ского национального агроэкологического универси- тета. Использованы лабораторные, полевые и ин тро- дукционные методы. Оценены общее состояние рас- тений, особенности семенного и вегетативного раз- множения, их зимо-, морозо- и засухоустойчивость, а также устойчивость к болезням и вредителям. 22. Torikov, V.E. and Meshkov, I.I. (2006), Tehnologija vozdelyvanija i ispol’zovanija lekarstvennyh rastenij [Technology of medicinal plants cultivation and appli- cation]. Moskva : Feniks, 283 p. Результаты. Среди культивируемых ароматических растений семейства Lamiaceae 3 однолетних и 8 мно- голетних видов (67,4% от общего количества исследо- ванных) оказались особенно перспективными и вы- сокостойкими. В условиях Полесья Украины они хо- рошо растут и нормально развиваются, сохраняя при- сущую им жизненную форму, плодоносят, почти не испытывают существенных повреждений от морозов, засухи, патогенных организмов. 23. Trulevich, N.V. (1991), Jekologo-fitocenoticheskie os- novy introdukcii rastenij [Ecological and phytocenosis principles of plants introduction]. Moskva, 216 p. 24. Cherepanov, S.K. (1995), Sosudistye rastenija Rossii i sopredelnyh gosudarstv (v predelah byvshego SSSR) [Vascular plants of Russia and bordering states (within the boundaries of the former USSR)]. Saint Peters- burg: Mir i semja, 992 p. 25. Chernyh, I.V. (2004), Introdukcija prjano-aromat- icheskih i jefiromaslichnyh rastenij v Lesostepnoj zone Juzhnogo Preduralja i ih ispolzovanie v jekoprotek- tivnoj pomoshhi naseleniju [Introduction of spicy aro- matic and essential oil plants in the Forest-Steppe Zone of the Southern Pre-Urals Region and their use for eco-protective assistance to the population] Doc- tor’s thesis. Ufa, 174 p. Выводы. Интродуценты Dracocephalum moldavica L., Satureja hortensis L., S. montana L., Elsholzia cristata Willd., Hyssopus officinalis L., H. аngustifolius M. Bieb., Origanum vulgare L., Nepeta transcaucasica Grossh., Salvia officinalis L., S. sclarea L. Л.А. Котюк 1, Д.Б. Рахметов 2, Т.В. Пинкина 1 1 Житомирский национальный агроэкологический университет, Украина, г. Житомир 2 Национальный ботанический сад имени Н.Н.Гришко НАН Украины, Украина, г. Киев ОЦЕНКА УСПЕШНОСТИ ИНТРОДУКЦИИ Л.А. Котюк 1, Д.Б. Рахметов 2, Т.В. Пинкина 1 1 Житомирский национальный агроэкологический университет, Украина, г. Житомир 2 Национальный ботанический сад имени Н.Н.Гришко НАН Украины, Украина, г. Киев roslyn, zberezhennja ta zbagachennja bioriznomanit- tja v botanichnyh sadah i dendroparkah : mater. mizhn. nauk. konf., prysvjach. 75-richchju Nac. bot. sadu im. M.M. Gryshka NAN Ukrainy (15—17 veresnja 2010 r.) [Plants introduction, preservation and enrichment of biodiversity in botanical gardens and dendroparks: proceedings of international scientific conference de- voted to the 75-th anniversary of M.M. Gryshko Na- tional Botanical Garden of the NAS of Ukraine (Sep- tember 15—17, 2010)]. Kyiv, pp. 88—90. roslyn, zberezhennja ta zbagachennja bioriznomanit- tja v botanichnyh sadah i dendroparkah : mater. mizhn. nauk. konf., prysvjach. 75-richchju Nac. bot. sadu im. M.M. Gryshka NAN Ukrainy (15—17 veresnja 2010 r.) [Plants introduction, preservation and enrichment of biodiversity in botanical gardens and dendroparks: proceedings of international scientific conference de- voted to the 75-th anniversary of M.M. Gryshko Na- tional Botanical Garden of the NAS of Ukraine (Sep- tember 15—17, 2010)]. Kyiv, pp. 88—90. REFERENCES (1982), Lekarstvennye rastenija i ih primenenie [Medicinal plants and their application]. Kyiv: Zdorovje, 232 p. 14. Majsuradze, N.I., Kiselev, V.P., Cherkasov, O.A. et al. (1984), Metodika issledovanij pri introdukcii lekarst- vennyh rastenij [Methods for research of medicinal plants introduction] Lekarstvennoe rastenievodstvo [Medicinal plants studies]. Moskva: CBNTI., vyp. 3, 33 p. 6. Brygadyrenko, V.V. (2003), Osnovy systematyky komah [Principles of insects systematics]. Dnipropetrovsk: RVV DNU, 204 p. 15. Tkachyk, S.O. (2015), Metodyka provedennja eksper- tyzy sortiv roslyn grupy dekoratyvnyh, likarskyh ta efi- roolijnyh, lisovyh na prydatnist do poshyrennja v Ukraini [Methods of testing decorative, medicinal, es- sential oil forest plants as to their suitability for being cultivated in Ukraine], 2nd ed., rev. Vinnycja: TOV «Nilan-LTD», 130 p. 7. Bylov, V.N. and Karpisonova, A.A. (1978), Principy sozdanija kollekcii malorasprostranennyh dekorativ- nyh mnogoletnikov [The principles of formation of a collection of less common ornamental perrnnials]. Bjulleten Glavnogo Botanicheskogo Sada AN SSSR [Bulletin of the Main Botanical Garden of the Acade- my of Sciences of the USSR], vyp. 107, pp. 77—82. 16. Metody opredelenija boleznej i vreditelej selskohoz- jajstvennyh rastenij. [Methods to identify crop diseases and pests] (1987), (Transl. from German by K.V. Pop- kova, V.A. Schmyglia). Moskva: Agropromizdat, 224 p. 8. Voronina, E.P., Godunov, U.N. and Godunovа, E.O. (2001), Novye aromaticheskie rastenija dlja Necher- nozemja [New aromatic plants for Nechornozemya]. Moskva: Nauka, 173 p. 17. Hohrjakov, M.K., Dobrozrakova, T.L., Stepanov, K.M. and Letova, M.F. (2003), Opredelitel boleznej rastenij [Compendium of plant diseases]. Saint Petersburg: Lan, 592 p. 9. Zvolinskij, V.P., Tjutjuma, N.V. and Rybashlykova, L.P. (2013), Introdukcija lekarstvennyh rastenij kak spo- sob sohranenija bioraznoobrazija Astrahanskoj oblasti [The experience of medicative plants introduction in Astrakhan region]. Izvestija Nizhnevolzhskogo ag ro- uni versitetskogo kompleksa: Nauka i vysshee profes- sionalnoe obrazovanie [Proceedings of Nizhnevolzskiy Agrouniversity Complex: Science and Higher Voca- tional Education], vol. 1 (29), pp. 7—11. 18. Porada, O.A. (2007), Metodyka formuvannja ta veden- nja kolekcii likarskyh roslyn [The metods of forming and maintaining a medicinal plants collection]. Bere- zotocha, 50 p. 19. Porada, O.A. (2010), Ocinka perspektyvnosti introduk- cii likarskyh roslyn rodyny Lamiaceae v Poltavskij ob- lasti [Evaluating introduction perspectives of Lamia- ceae officinal plants in Poltava region]. Introdukcija 10. Porada, O.A., Shevchenko, T.L., Syvoglaz, L.M. and Kalinina, M.A. (2012), Ekologo-biologichna ocinka 18 ISSN 1605-6574. Інтродукція рослин, 2017, № 1 ISSN 1605-6574. Інтродукція рослин, 2017, № 1 Оцінка успішності інтродукції ароматичних рослин родини Lamiaceae Lindl. в умовах Пол ОЦЕНКА УСПЕШНОСТИ ИНТРОДУКЦИИ АРОМАТИЧЕСКИХ РАСТЕНИЙ СЕМЕЙСТВА LAMIACEAE LINDL. В УСЛОВИЯХ ПОЛЕСЬЯ УКРАИНЫ и S. aethiopis L. — эколо ги чески пластичные виды растений, пригодные для введения в промыш- ленную культуру на Полесье Ук раины. Как высоко- перспективные интродуценты они могут быть ценным источником пополнения новых растительных ресур- сов и исходным материалом для селекционных иссле- дований и создания высокоадаптированных сортов. 26. The Plant List. Angiosperms. Lamiaceae. 2013. [Elek- tronnyy resurs ]: http://www.theplantlist.org/1.1/browse/ A/Lamiaceae/ Recommended by M.B. Gaponenko Received 17.10.2016 Recommended by M.B. Gaponenko Received 17.10.2016 Ключевые слова: ароматические растения, Lamiaceae Lindl., успешность интродукции, Полесье Украины. Ключевые слова: ароматические растения, Lamiaceae Lindl., успешность интродукции, Полесье Украины. 19 ISSN 1605-6574. Інтродукція рослин, 2017, № 1 Л.А. Котюк, Д.Б. Рахметов, Т.В. Пінкіна L.А. Kotyuk 1, D.B. Rakhmetov 2, T.V. Pіnkіna 1 1 Zhytomyr National Agroecological University, Ukraine, Zhytomyr 2 M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, Ukraine, Kyiv winter-, frost-, drought-resistance alongside of disease and pest resistance. 1 Zhytomyr National Agroecological University, Ukraine, Zhytomyr Results. Among the cultivated aromatic plants from the Lamiaceae family 3 annual and 8 perennial species which accounts for 67.4 % of the total studied material proved extremely perspective and highly resistant. Under condi- tions of Ukrainian Polissya they demonstrated adequate growth and development performance, preserving innate life form, fruit bearing ability, resistance to frosts, droughts, pathogenic organisms. 2 M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, Ukraine, Kyiv ISSN 1605-6574. Інтродукція рослин, 2017, № 1 Key words: aromatic plants, Lamiaceae Lindl., efficiency of introduction, Ukrainian Polissya. L.А. Kotyuk 1, D.B. Rakhmetov 2, T.V. Pіnkіna 1 1 Zhytomyr National Agroecological University, Ukraine, Zhytomyr 2 M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, Ukraine, Kyiv EVALUATING THE RESULTS OF INTRODUCTION OF AROMATIC PLANTS FROM THE LAMIACEAE LINDL. FAMILY IN UKRAINIAN POLISSYA Conclusions. Іntroduced plants Dracocephalum moldavi- ca L., Satureja hortensis L., S. montana L., Elsholzia cristata Willd., Hyssopus officinalis L., H. аngustifolius M. Bieb., Origa- num vulgare L., Nepeta transcaucasica Grossh., Salvia officina- lis L., S. sclarea L. and S. aethiopis L. are ecologically adaptable species with good prospects to be deployed as industrial plants in Ukrainian Polissya. Being extremely perspective, in trodu ced plants can be a valuable source of enriching new plant resour- ces and may be used as base breeding material for comprehen- sive selection research to create highly adaptive varieties. Objective — to evaluate the efficiency of introduction of non-extensively cultivated annual and perennial aromat- ic plants of the Lamiaceae Lindl. family species and to determine their cultivation perspectives in Ukrainian Polissya. Material and methods. 17 non-traditional annual and perennial Lamiaceae aromatic plants have been studied during 2008—2016 after their introduction in Botanical Garden of Zhytomyr Agroecological University. Labora- tory, field and introductory methods have been used to examine the following properties: plant’s general state, peculiarities of seed and vegetative propagation, their Key words: aromatic plants, Lamiaceae Lindl., efficiency of introduction, Ukrainian Polissya. 20 ISSN 1605-6574. Інтродукція рослин, 2017, № 1
https://openalex.org/W3199470480	https://nzjmath.org/index.php/NZJMATH/article/download/92/51	English	null	Smale 17th Problem: Advances and Open Directions	New Zealand journal of mathematics	2,021	cc-by	14,146	NEW ZEALAND JOURNAL OF MATHEMATICS Volume 52 (2021), 233–257 https://doi.org/10.53733/92 NEW ZEALAND JOURNAL OF MATHEMATICS Volume 52 (2021), 233–257 https://doi.org/10.53733/92 Contents 1. Foreword 233 2. Smale 17th Problem 234 2.1. The background 234 2.2. The B´ezout series 235 2.3. Probabilities and complexity 237 2.4. The problem’s statement 238 3. The Solution 238 3.1. Condition numbers 238 3.2. A probabilistic solution 241 3.3. A near solution and other advances 243 3.4. The solution 245 4. Subsequent Progress 246 4.1. Eigenpair computations 246 4.2. Rigid homotopies 246 4.3. Structured systems 250 4.4. Low-complexity systems 252 4.5. Algebraic branching programs 254 References 255 3.1. Condition numbers 3.2. A probabilistic solution 3.3. A near solution and other advances 3.4. The solution 4. Subsequent Progress 4.1. Eigenpair computations 4.2. Rigid homotopies 4.3. Structured systems 4.4. Low-complexity systems 4.5. Algebraic branching programs Key words and phrases: polynomial equation solving, homotopy methods, approximate zero, com- plexity, polynomial time. (Received 4 March, 2021) Dedicated to Vaughan Jones (1952-2020). In memoriam. 2000 Mathematics Subject Classiﬁcation 65H20, 65Y20. Dedicated to Vaughan Jones (1952-2020). In memoriam. Abstract. We give an overview of Smale’s 17th problem describing the context in which Smale proposed it, the ideas that led to its solution, and the extensions and subsequent progress after this solution. 2000 Mathematics Subject Classiﬁcation 65H20, 65Y20. Key words and phrases: polynomial equation solving, homotopy methods, approximate zero, com- plexity, polynomial time. Partially supported by GRF grant CityU 11302321. Partially supported by GRF grant CityU 11302321. 1. Foreword In June 1999 I received an invitation letter to give a short course on a summer school. The letter was signed by Vaughan Jones, then the chairman of the NZMRI (New Zealand Mathematical Research Institute). New Zealand being in the south, the summer school was going to be in January. I immediately accepted. I had never been in New Zealand, but I knew that it shared some similarities with my home country (Uruguay). My stay at Kaikoura was delightful. The gentle beauty 234 FELIPE CUCKER of the town, the vicinity of Marlborough’s vineyards, the whales in the neighboring waters, the kindness of the local people in whose house I stayed, . . . And the school was no less fascinating. Not only because of the interest of its students and the quality of the short courses I took advantage to attend but also because of its setting: lectures and lunches were held at the local Marae. More than twenty years later I still look at it as a unique experience. of the town, the vicinity of Marlborough’s vineyards, the whales in the neighboring waters, the kindness of the local people in whose house I stayed, . . . And the school was no less fascinating. Not only because of the interest of its students and the quality of the short courses I took advantage to attend but also because of its setting: lectures and lunches were held at the local Marae. More than twenty years later I still look at it as a unique experience. q p January 2000 was also the beginning of the 21st century. So, while we were enjoying the school at Kaikoura, the presses of the American Mathematical Society were at work to print a singular volume [4]. Commissioned by the International Mathematical Union, and to celebrate the year 2000 as the Year of Mathematics, the book acknowledges its inspiration on the list of problems proposed by Hilbert in 1900 for the mathematicians of the 20th century. Thus, along with articles describing the state of mathematics at the end of the century a few articles proposed, in the spirit of Hilbert, lists of problems for the mathematicians of the 21st century. Among the latter one was written by Vaughan [14]; it describes ten open problems related to his research interests. Another was writen by Steve Smale [27]1. 1Smale’s paper had actually been ﬁrst published in the Mathematical Intelligencer [29]. 2Of the eighteen, three (The Riemann Hypothesis, the Poincar´e Conjecture and the question “Is P = NP?”) were well-known at the time; the other ﬁfteen were new. 1. Foreword Similar in character to Vaughan’s it describes eighteen problems related to Smale’s research interests2. The nature of the 17th had a large overlap with the subject of my course at Kaikoura. Our aim in this article is to describe —with a focus on ideas rather than on technical details— what this problem is, what the work of Smale leading to it was, how it was solved a few years ago, and what are the questions left open after this solution. It is a ﬁtting update to the course I gave at Vaughan’s invitation. Can one eﬃciently compute (any) one zero of a given F ∈Hd? (S17) 2.2. The B´ezout series. During the early 1990s, in a series of papers known as “the B´ezout series” [22, 23, 24, 26, 25], Mike Shub and Steve Smale laid down the foundations of an approach to answer this question. We won’t attempt to summarize these papers but limit ourselves to describe some of the main ingredients in this approach. 2.2.1. Approximate zeros. We already mentioned that the solutions of a general quintic equation (in one variable) cannot be expressed by radicals. Obviously, neither can the zeros of a system F ∈Hd. The most common way out of this obstacle is to compute a (suﬃciently good) approximation of such a zero. Arguably the most common deﬁnition of approximation is through a bound on the distance to a true zero. If dP denotes the Riemannian distance in Pn (i.e., the angle), we say that z ∈Pn is an ε-approximate zero of F when there is a zero ζ ∈Pn of F such that dP(z, ζ) ≤ε. A shortcoming of this notion is the dependence on ε: being an approximate zero in this sense is not an absolute notion. The deﬁnition adopted in the B´ezout series, which goes back to [28], is diﬀerent and relies on Newton’s method. The latter can be extended to the projective space. Given z ∈Pn and F ∈Hd, the Newton iterate of F at z is NF (z) := z −DzF\|−1 Tz F(z). (1) (1) Here Tz is the tangent space to Pn at z (i.e., the orthogonal complement z⊥to z in Cn+1) and DzF\|Tz is the restriction to this space of the derivative of F at z. A remarkable feature of Newton’s method is its quadratic convergence. Let ζ be a non-singular (i.e., not multiple) zero of F and z be a point suﬃciently close to ζ. Then, Here Tz is the tangent space to Pn at z (i.e., the orthogonal complement z⊥to z in Cn+1) and DzF\|Tz is the restriction to this space of the derivative of F at z. A remarkable feature of Newton’s method is its quadratic convergence. Let ζ be a non-singular (i.e., not multiple) zero of F and z be a point suﬃciently close to ζ. Then, dP(Nk F (z), ζ) ≤dP(NF (z), ζ) 21−2k (2) (2) where Nk F is the kth iterate of (1). 2. Smale 17th Problem Generically (and by this we mean outside a subset of smaller dimension in Hd) the system F has a ﬁnite number of zeros in Pn. B´ezout Theorem states that in this case, and when counted with multiplicity, this number is D := d1d2 · · · dn. The question underlying Smale’s 17th problem is the following: we denote by [x0 : · · · : xn] its class in Pn. In all what follows we will choose representatives (x0, . . . , xn) of a point [x] ∈Pn satisfying that ∥x∥= 1. This has no loss of generality and simpliﬁes a number of expressions. g y Now let d := (d1, . . . , dn) ∈Nn and Hd be the linear space of the systems F = (f1, . . . , fn) with fi ∈C[X0, . . . , Xn] homogeneous of degree di. Generically (and by this we mean outside a subset of smaller dimension in Hd) the system F has a ﬁnite number of zeros in Pn. B´ezout Theorem states that in this case, and when counted with multiplicity, this number is D := d1d2 · · · dn. The question underlying Smale’s 17th problem is the following: (S17) 2. Smale 17th Problem 2.1. The background. Work on the solution of polynomial equations may be traced back to the Babylonians, 4000 years ago, who solved some quadratic equa- tions arising from problems relating areas and sides of rectangles [13]. The general solution to cubic and quartic equations is generally attributed to Ferrari and Car- dano in the 16th century. Solutions could be written in terms of square and cubic roots and involved complex numbers. It took almost three centuries to prove that a similar result cannot be achieved for the general quintic equation [1]. Nonetheless, the solutions exist. The Fundamental Theorem of Algebra states that a polynomial equation of degree d has exactly d zeros in C when counted with their multiplicity. An equally fundamental result in algebraic geometry, known as the B´ezout Theorem, extends the FTA to (square) systems of polynomials. It needs a caveat though. The system {X1 + X2 = 0, X1 + X2 = 1} has, obviously, no zeros: the two lines are parallel. But if one “adds points at inﬁnity” to the complex plane C2 these two lines do meet at one such point. Recall, the complex projective space Pn is the quotient Cn+1/ ∼where x ∼y if x = λy for some λ ∈C, λ ̸= 0. Also, a polynomial f ∈C[X0, X1, . . . , Xn] is homogeneous of degree d when all its monomials have degree d. In this case, if x ∈Cn+1 is such that f(x) = 0 then, for all λ ∈C, f(λx) = λdf(x) = 0. Hence we can talk about the zeros of f in Pn and, by extension, to the zeros in Pn of a system F = (f1, . . . , fn) of homogeneous polynomials (possibly of diﬀerent degrees). Given a point (x0, . . . , xn) ∈Cn+1 \{0} SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 235 we denote by [x0 : · · · : xn] its class in Pn. In all what follows we will choose representatives (x0, . . . , xn) of a point [x] ∈Pn satisfying that ∥x∥= 1. This has no loss of generality and simpliﬁes a number of expressions. Now let d := (d1, . . . , dn) ∈Nn and Hd be the linear space of the systems F = (f1, . . . , fn) with fi ∈C[X0, . . . , Xn] homogeneous of degree di. Can one eﬃciently compute (any) one zero of a given F ∈Hd? (S17) Shub and Smale call a point z satisfying (2) an approximate zero of F and the point ζ its associated zero. Note, this is a qualitative notion; there is no dependence on a parameter measuring how well the point approximates the zero. It is also a strong property. Once a point ζ is an approximate zero of F, inequality (2) allows one to get an ε-approximation with log2(1 + log2 ε) Newton steps. Two questions are naturally posed. Firstly, what does ‘suﬃciently close’ mean? Can one provide estimates for this notion? The answer goes back (again) to [28]. 236 FELIPE CUCKER For a point ζ ∈Pn deﬁne For a point ζ ∈Pn deﬁne γ(F, ζ) := sup k≥2 DζF\|−1 Tζ Dk ζF k! 1 k−1 (3) (3) where Dk ζF is the kth derivative of F at ζ and the norm is the operator norm. The γ-Theorem of Smale states that if F(ζ) = 0 and dP(z, ζ)γ(F, ζ) ≤1 6 then z is an approximate zero of F with associated zero ζ (see, e.g., [17, Thm. 12]). Secondly, in the absence of a true zero ζ at hand, can one certify that a point z is an approximate zero of F? To answer this question let β(F, z) := DzF\|−1 Tz F(z) be the length of the Newton step in (1) and be the length of the Newton step in (1) and α(F, z) := β(F, z)γ(F, z). Smale’s α-Theorem states that there exists a constant α0 such that if α(F, z) ≤α0 then z is an approximate zero of F. A proof with α0 = 0.02 is in [10, Thm. 19.9]. Smale’s α-Theorem states that there exists a constant α0 such that if α(F, z) ≤α0 then z is an approximate zero of F. A proof with α0 = 0.02 is in [10, Thm. 19.9]. The goal in (S17) is therefore not to compute a true zero but an approximate zero as above. 2.2.2. Linear homotopies. The algorithmic scheme considered in the B´ezout series was a linear homotopy. Its general idea (which goes back at least to Lahaye [15]) can be simply described. Assume you want to compute the zeros of the polynomial F = X3 −X2 −5X + 4 and further assume that you aren’t aware of the formula to do so. Can one eﬃciently compute (any) one zero of a given F ∈Hd? (S17) The curve C , the initial pair (G, ζ0), the target pair (F, ζ1) and the points (Qti, zi) that “follow” C . The bottomline is, linear homotopy, in general, works. But for some unlucky choices of F and G it may go awfully wrong. Consequently, to give a formal meaning to the notion of “eﬃciently compute” we need a framework other than the worst-case scenario. 2.3. Probabilities and complexity. To talk about computational eﬃciency sup- poses a cost measure at hand. As we are considering numerical algorithms, that is, algorithms whose basic operations are arithmetic operations and comparisons between (idealized) real numbers the simplest, and most natural, cost measure for a computation with input F is the number cost(F) of such operations performed along the computation. The complexity of an algorithm is a function relating the cost of the algorithm’s computations to the size of the input data. In our case, the size N of a system F ∈Hd is the number of complex numbers we need to describe the system. That is, the number of coeﬃcients in its polynomials. This gives us This gives us N = n X i=1 Ni where Ni = n + di n . (6) (6) We have mentioned that we need a framework for complexity other than the worst- case scenario. The one proposed by Smale, which is widely used, is that of average complexity. To describe it we need some structure on Hd. We have mentioned that we need a framework for complexity other than the worst- case scenario. The one proposed by Smale, which is widely used, is that of average complexity. To describe it we need some structure on Hd. plexity. To describe it we need some structure on We have mentioned that Hd is a linear space. We can turn it into an inner product space by endowing it with the Weyl inner product. This is a dot product in a scaled monomial basis which has the property of being unitarily invariant. That is, for all F, G ∈Hd and all unitary transformation u ∈U(n + 1), we have ⟨F ◦u, G ◦u⟩= ⟨F, G⟩. See [10, §16.1] for details. In all what follows, for a system F ∈Hd, ∥F∥will denote its Weyl norm ⟨F, F⟩1/2. Can one eﬃciently compute (any) one zero of a given F ∈Hd? (S17) You may try to use the fact that you certainly know the zeros of G = X3−X. To do so, consider the segment (in the space of polynomials of degree at most 3) Qt := (1 −t)G + tF, with t ∈[0, 1]. (4) (4) We observe that its extremities are Q0 = G and Q1 = F. Hence, when t varies from 0 to 1, we may expect the zeros of Qt to vary from the zeros of G (1, 0 and −1) to those of F. Starting with one such zero ζ0 of G (say ζ0 = 1) this will induce a curve C = (Qt, ζt)t∈[0,1] with Qt(ζt) = 0 for all t ∈[0, 1] and if we can “follow” this curve via a ﬁnite sequence {Qti, zi}i=0,...,k such that 0 = t0 < t1 < · · · < tk = 1 and zi is a good approximation of ζti then we will end up with zk, a good approximation to the zero ζ1 of Q1 = F. Figure 1 attempts to convey this idea. A few comments are in order. The ﬁrst is that the same idea applies for systems in Hd; we now have n homogeneous polynomials in n + 1 variables and D zeros in Pn instead of 3 in C but the basic idea is the same. The second is that the “lifting” of the segment [Q0, Q1] to the curve C ⊂Hd ×Pn is well-deﬁned provided DζtQt\|Tζt is invertible for all t ∈[0, 1]. Recall that the discriminant variety in Hd is the set Σ := {F ∈Hd \| ∃ζ ∈Pn s.t. F(ζ) = 0 and rank(DζF\|Tζ) < n}. (5) (5) Systems outside Σ are those having D diﬀerent smooth zeros. Then, as long as the segment [Q0, Q1] does not intersect Σ, the D zeros of G induce D curves in Hd ×Pn which do not intersect one another. Furthermore, it is known that Σ is a complex hypersurface in Hd. Hence, it has real codimension 2 in Hd. This implies that, generically, the segment [Q0, Q1] does not intersect Σ. SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 237 Pn Hd ζ0 ζ1 G = Q0 F = Q1 C Figure 1. The curve C , the initial pair (G, ζ0), the target pair (F, ζ1) and the points (Qti, zi) that “follow” C . Figure 1. Can one eﬃciently compute (any) one zero of a given F ∈Hd? (S17) The Weyl norm induces a unit sphere in Hd, in what follows denoted by S(Hd), which we can endow with the uniform distribution. The latter, in turn, allows one 238 FELIPE CUCKER to deﬁne the average cost of an algorithm (taking inputs in S(Hd)) to be (7) E F ∼S(Hd) cost(F). (7) E F ∼S(Hd) cost(F). (7) 2.4. The problem’s statement. We can now formally state Smale’s 17th prob- 2.4. The problem’s statement. We can now formally state Smale’s 17th prob- lem. This statement relies on the usual understanding of “eﬃcient” as solvable with ( ) lem. This statement relies on the usual understanding of “eﬃcient” as solvable with polynomially bounded average cost. The question in (S17) thus becomes Is there an algorithm that, given an F ∈S(Hd) randomly drawn from the uniform distribution, halts with probability one returning an approximate zero of F, and whose average cost is bounded by a polynomial in N? polynomially bounded average cost. The question in (S17) thus becomes Is there an algorithm that, given an F ∈S(Hd) randomly drawn from the uniform distribution, halts with probability one returning an approximate zero of F, and whose average cost is bounded by a polynomial in N? 3. The Solution To turn the linear homotopy scheme into a bona ﬁde algorithm we need to specify how do we choose the initial pair (G, ζ0) and how do we compute the sequences t0, t1, . . . , tk and z0, . . . , zk. Let us start with the latter. Before doing so, however, we observe that it makes sense to work with the angle dS(Q, H) between systems Q, H ∈Hd instead of the distance ∥Q −H∥. This is so because there are no changes in the zeros of a system when the system is multiplied by a constant. Consequently, we will consider the segment [G, F] parameterized by a fraction τ of the angle dS(G, F) instead of a fraction t of the distance ∥F −G∥as in (4). That is, we will consider {Qτ}τ∈[0,1] where Qτ is the only system in [G, F] satisfying dS(G, Qτ) = τ dS(G, F). 3.1. Condition numbers. Assume, for the time being, that we are given F, G and ζ0 with G(ζ0) = 0. A ﬁrst remark is that we won’t attempt to ﬁnd a universal partition of [0, 1] that we can use for all triples (F, G, ζ0) but rather that we will proceed dynamically: at the ith step, we have computed a pair (Qi, zi) for which we know that zi is an approximate zero of Qi with associated zero ζi (we should write Qτi and ζτi but we simplify the notation for ease of reading). Next assume that dP(zi, ζi) ≤ 1 12γ(Qi, ζi). (8) (8) Note that this is stronger, by a factor of 2, than the condition required in Smale’s γ-Theorem (cf. §2.2.1). Given a ∆τ ≤dS(Qi,F ) dS(G,F ) we let Qi+1 be the only system in [Qi, F] satisfying dS(Qi, Qi+1) = ∆τ dS(G, F). G F Qi Qi+1 dS(G, F) τidS(G, F) ∆τdS(G, F) Figure 2. The system Qi+1 given by Qi and the step-length ∆τ. Figure 2. The system Qi+1 given by Qi and the step-length ∆τ. SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 239 The goal is to compute a bound B(Qi, zi) such that The goal is to compute a bound B(Qi, zi) such that ∆τ ≤B(Qi, zi) =⇒dP(zi, ζi+1) ≤ 1 6γ(Qi+1, ζi+1). 3. The Solution (9) (9) If we manage to ﬁnd B such that (9) holds, then zi is an approximate zero of Qi+1 with associated zero ζi+1 (by Smale’s γ-Theorem) and then, taking zi+1 := NQi+1(zi) we have (by (2)) dP(zi+1, ζi+1) ≤1 2dP(zi, ζi+1) ≤ 1 12γ(Qi+1, ζi+1), i.e., that (8) holds, and we can iterate the process. Note that inequality (8) is trivial for i = 0 as z0 = ζ0 and hence dS(z0, ζ0) = 0. This allows us to begin the process. Figure 3 shows one step of the homotopy. Pn Hd Qi Qi+1 ζi ζi+1 zi zi+1 C Figure 3. Computation of next pair (Qti+1,zi+1). Figure 3. Computation of next pair (Qti+1,zi+1). How do we obtain B satisfying (9)? ( ) Recall, we want dP(zi, ζi+1) ≤ 1 6γ(Qi+1,ζi+1). We begin by noting that ( ) Recall, we want dP(zi, ζi+1) ≤ 1 6γ(Qi+1,ζi+1). We begin by noting that ( + + ) dP(zi, ζi+1) ≤dP(zi, ζi) + dP(ζi, ζi+1). (10) dP(zi, ζi+1) ≤dP(zi, ζi) + dP(ζi, ζi+1). (10) Inequality (8) already bounds the ﬁrst term in the right-hand side. In addition, Lipschitz bounds for γ−1 allow one to show, for Qi+1 suﬃciently close to Qi and ζi+1 suﬃciently close to ζi, that dP(zi, ζi) ≤ 1 12γ(Qi, ζi) ≤ 1 c1γ(Qi+1, ζi+1) for some c1 ∈(6, 12). (11) (11) Let us assume for a while that this holds true and focus on the second term in the right-hand side of (10). To bound this term deﬁne, for F ∈Hd and z ∈Pn, the condition number of F at z to be µnorm(F, z) := ∥F∥ DzF\|−1 Tz diag( p di) . 240 FELIPE CUCKER This is a condition number in the classical sense: if F(ζ) = 0, then µnorm(F, ζ) bounds the ﬁrst-order variation of ζ in terms of the variation of F 3. Hence, we may expect that dP(ζi, ζi+1) ≤(1 + c2)∆τµnorm(Qi, ζi) (12) (12) for some small constant c2. For a given c3, if we want the left-hand side above to be at most 1 c3γ(Qi+1,ζi+1) we should impose for some small constant c2. For a given c3, if we want the left-hand side above to be at most 1 c3γ(Qi+1,ζi+1) we should impose ∆τ ≤ 1 c3(1 + c2)γ(Qi+1, ζi+1)µnorm(Qi, ζi). 3Actually, the true condition number would be µ(F, z) (see [10, Corollary 16.14]) which is deﬁned in the same manner but without the diagonal matrix diag(√di). The fact that µnorm is unitarily invariant (whereas µ isn’t), however, make µnorm(F, z) more convenient to use. 4 3. The Solution (13) (13) At this stage we use a fundamental result, known as the Higher Derivative Estimate, which states that, for all F ∈Hd and z ∈Pn, At this stage we use a fundamental result, known as the Higher Derivative Estimate, which states that, for all F ∈Hd and z ∈Pn, γ(F, z) ≤1 2D3/2µnorm(F, z), (14) (14) where D := max{d1, . . . , dn}, to replace (13) by ∆τ ≤ 2 c3(1 + c2)D3/2µnorm(Qi+1, ζi+1)µnorm(Qi, ζi). The use of Lipschitz bounds, now for µ−1 norm, allows one to obtain The use of Lipschitz bounds, now for µ−1 norm, allows one to obtain (15) max{µnorm(Qi+1, ζi+1), µnorm(Qi, ζi)} ≤(1 + c4)µnorm(Qi, zi) (15) for some small c4. We can conclude that taking max{µnorm(Qi+1, ζi+1), µnorm(Qi, ζi)} ≤(1 + c4)µnorm(Qi, zi) (15) for some small c4. We can conclude that taking ∆τ ≤B(Qi, zi) := 2 c3(1 + c2)(1 + c4)2D3/2µnorm(Qi, zi)2 (16) (16) ∆τ ≤B(Qi, zi) := 2 c3(1 + c2)(1 + c4)2D3/2µnorm(Qi, zi)2 (16) we ensure that dP(ζi, ζi+1) ≤ 1 c3γ(Qi+1, ζi+1). (17) Finally, by chosing c3 := 6c1 c1−6 we obtain from (10), (11), and (17) that dP(zi, ζi+1) ≤ 1 c1γ(Qi+1, ζi+1) + 1 c3γ(Qi+1, ζi+1) = 1 6γ(Qi+1, ζi+1). we ensure that dP(ζi, ζi+1) ≤ 1 c3γ(Qi+1, ζi+1). (17) Finally, by chosing c3 := 6c1 c1−6 we obtain from (10), (11), and (17) that dP(zi, ζi+1) ≤ 1 c1γ(Qi+1, ζi+1) + 1 c3γ(Qi+1, ζi+1) = 1 6γ(Qi+1, ζi+1). (17) c3γ(Qi+1, ζi+1) Finally, by chosing c3 := 6c1 c1−6 we obtain from (10), (11), and (17) that dP(zi, ζi+1) ≤ 1 c1γ(Qi+1, ζi+1) + 1 c3γ(Qi+1, ζi+1) = 1 6γ(Qi+1, ζi+1). This general argument comes from [21] where the value of the constants ci is not worked out4. Detailed proofs are in [8, 9] and [10, §17.1]. In the latter it is shown that one can actually chose them so that both (11) and (15) hold and that with that choice (16) becomes B(Qi, zi) := 0.0085 dS(G, F)D3/2µnorm(Qi, zi) (18) (18) This results in an easy-to-describe algorithm LinHom that takes as input a triple (F, G, ζ0) with F, G ∈Hd and G(ζ0) = 0. This results in an easy-to-describe algorithm LinHom that takes as input a triple (F, G, ζ0) with F, G ∈Hd and G(ζ0) = 0. 3Actually, the true condition number would be µ(F, z) (see [10, Corollary 16.14]) which is deﬁned in the same manner but without the diagonal matrix diag(√di). The fact that µnorm is unitarily invariant (whereas µ isn’t), however, make µnorm(F, z) more convenient to use. 4The author of [21] writes “In previous papers we have paid careful attention to the constants. In this paper we are more cavalier.” 3. The Solution 3Actually, the true condition number would be µ(F, z) (see [10, Corollary 16.14]) which is deﬁned in the same manner but without the diagonal matrix diag(√di). The fact that µnorm is unitarily invariant (whereas µ isn’t), however, make µnorm(F, z) more convenient to use. 4The author of [21] writes “In previous papers we have paid careful attention to the constants. In this paper we are more cavalier.” 241 SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS Algorithm LinHom input F, G ∈Hd and ζ0 ∈Pn such that G(ζ0) = 0 τ := 0, Q := G, z := ζ0 repeat ∆τ := 0.0085 dS(F,G)D3/2µ2norm(Q,z) τ := min{1, τ + ∆τ} Q := Qτ z := NQ(z) until τ = 1 RETURN z input F, G ∈Hd and ζ0 ∈Pn such that G(ζ0) = 0 τ := 0, Q := G, z := ζ0 repeat With some (but not much) additional work the arguments above show the fol- lowing result (Proposition 17.3 in [10]). Proposition 3.1. Suppose that [G, F] does not intersect Σ. Then, the execution of LinHom(F, G, ζ0) stops after at most K steps with K = K(F, G, ζ0) ≤188D3/2dS(G, F) Z 1 0 µ2 norm(Qτ, ζτ)dτ. K = K(F, G, ζ0) ≤188D3/2dS(G, F) Z 1 0 µ2 norm(Qτ, ζτ)dτ. The returned point z is an approximate zero of F. ( , , ζ0) ≤ S( , ) Z 0 µnorm(Qτ, ζτ) The returned point z is an approximate zero of F. 2 2 3.2. A probabilistic solution. The ﬁrst breakthrough towards a solution of Smale’s 17th problem was produced by Carlos Beltr´an and Luis Miguel Pardo [7]. It consisted of a randomized algorithm, in the sequel BP, to generate the initial pair (G, ζ0). In the course of its execution (with input (n, d)) this algorithm draws real numbers from the standard normal distribution and, because of this, its output (G, ζ0) is random as well. 3. The Solution 3.1 188D3/2 E F ∼S(Hd) E (G,ζ0)∼ρstd dS(G, F) Z 1 0 µ2 norm(Qτ, ζτ)dτ = 188D3/2 E F ∼S(Hd) E G∼S(Hd) dS(G, F) Z 1 0 1 D X ζ\|Qτ (ζ)=0 µ2 norm(Qτ, ζ)dτ E F ∼S(Hd) E (G,ζ0)∼ρstd K(F, G, ζ0) ≤ Prop. 3.1 188D3/2 E F ∼S(Hd) E (G,ζ0)∼ρstd dS(G, F) Z 1 0 µ2 norm(Qτ, ζτ)dτ = 188D3/2 E F ∼S(Hd) E G∼S(Hd) dS(G, F) Z 1 0 1 D X ζ\|Qτ (ζ)=0 µ2 norm(Qτ, ζ)dτ ≤ 188 πD3/2 Z 1 0 E F ∼S(Hd) E G∼S(Hd) µ2 avg(Qτ)dτ = 188 πD3/2 Z 1 0 E Q∼S(Hd) µ2 avg(Q)dτ ≤ (21) 188 πD3/2 Z 1 0 nNdτ = 188πD3/2nN. ≤ 188 πD3/2 Z 1 0 E F ∼S(Hd) E G∼S(Hd) µ2 avg(Qτ)dτ = 188 πD3/2 Z 1 0 E Q∼S(Hd) µ2 avg(Q)dτ ≤ (21) 188 πD3/2 Z 1 0 nNdτ = 188πD3/2nN. In addition to this, the cost of each step, which is dominated by the computation of the Newton iteration, is O(N + n3), which is O(N) if we assume that di ≥2 for i = 1, . . . , d. This yields a total average cost of O(nD3/2N 2) for LV (as the cost for the execution of BP is dominated by that of the linear homotopy). This O(nD3/2N 2) average cost was independently obtained in [8] and [9]. A few last remarks are necessary. The average complexity bound above considers two diﬀerent sorts of average. On the one hand, the initial pair (G, ζ0) is produced by a randomized algorithm and hence, the algorithm LV itself is randomized. On the other hand, the input system F is considered random to derive average complexity bounds. Algorithm LV is of Las Vegas type: for a given input F ∈S(Hd), if the algo- rithm halts, it returns an approximate zero of F (and it halts with probability 1 for all F outside a set of measure zero). Yet, its running time (its cost) is a ran- dom variable as it depends on (G, ζ0). The argument above does not provide a bound for the randomized cost randcost(F) of LV on input F, which is deﬁned as E(G,ζ0)∼ρstd K(F, G, ζ0). But it does so for the average of these randomized costs over F ∈S(Hd). Because of the probabilistic nature of LV, the bound above was not a solution to Smale’s 17th problem. 3. The Solution If we deﬁne the solution variety to be VS := {(G, ζ) ∈S(Hd) × Pn \| G(ζ) = 0} (19) (19) then the distribution ρstd induced on VS by the ouputs of BP can be easily described as follows: (i) draw G from the uniform distribution on S(Hd) (ii) draw ζ from the (discrete) uniform distribution (20) among the D zeros of G (20) (ii) A surprising feature of BP is that it constructs a polynomial system along with one of its zeros without ever solving a (nonlinear) polynomial system. Roughly speaking, it ﬁrst draws the “linear part” of the system, then computes a zero of this linear part, and ﬁnally adds a suitable complement of higher degree terms. For a description of BP, its properties and its cost, see [8, 9] or [10, §17.6]. A surprising feature of BP is that it constructs a polynomial system along with one of its zeros without ever solving a (nonlinear) polynomial system. Roughly speaking, it ﬁrst draws the “linear part” of the system, then computes a zero of this linear part, and ﬁnally adds a suitable complement of higher degree terms. For a description of BP, its properties and its cost, see [8, 9] or [10, §17.6]. A zero-ﬁnding algorithm becomes clear, let’s call it LV (from Las Vegas, as ran- domized algorithms as ours are called this way). On input F ∈S(Hd), we ﬁrst draw a pair (G, ζ0) ∈VS from ρstd via a call to BP. We then run the linear homotopy on the triple (F, G, ζ0). This returns an approximate zero of F. To understand its cost we next make two fundamental observations. 242 FELIPE CUCKER Firstly, if F, G are independently drawn from S(Hd) and τ ∈[0, 1] is ﬁxed, then the system Qτ ∈[G, F] given by dS(G, Qτ) = τdS(G, F) dS(G, Qτ) = τdS(G, F) is also uniformly distributed in S(Hd). Secondly, let µ2 avg(F) := 1 D X ζ\|F (ζ)=0 µ2 norm(F, ζ). Then [8, Thm. 23], Then [8, Thm. 23], E Q∼S(Hd) µ2 avg(Q) ≤nN. (21) (21) These facts allow us to bound the average number of steps of LV. Indeed, E F ∼S(Hd) E (G,ζ0)∼ρstd K(F, G, ζ0) ≤ Prop. 3. The Solution The latter asked for a deterministic algorithm. More work would be needed to reach this solution. SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 243 3.3. A near solution and other advances. The arguments above, which follow the exposition in [8], relie on (among other things) the fact that when F and G are uniformly drawn from S(Hd), so is, for any τ ∈[0, 1], the system Gτ. But as soon as these distributions are not identical, technical diﬃculties may arise. This is at the root of the diﬀerent choice of setting taken in [9], where the same O(D3/2nN 2) bound for LV is proved as well. The setting here considers Gaussian distributions on Hd. The Weyl norm induces a standard Gaussian distribution N(0, I) on Hd via the density ρ(F) := 1 (2π)N e−∥F ∥2 2 which in turn induces an average cost of an algorithm (taking inputs in Hd) which in turn induces an average cost of an algorithm (taking inputs in Hd) (22) E F ∼N(0,I) cost(F). (22) This quantity is not diﬀerent to that in (7). Indeed, the standard Gaussian distri- bution on R2N induces, via the bijection This quantity is not diﬀerent to that in (7). Indeed, the standard Gaussian distri- bution on R2N induces, via the bijection R2N \ {0} → S(R2N) × (0, ∞) F 7→ F ∥F∥, ∥F∥2 , the uniform distribution on S(R2N) and a χ2-distribution with 2N degrees of free- dom on (0, ∞). Moreover these two components are independent [10, Prop. 2.19]. Using the fact that the right-hand side in Proposition 3.1 is scale-invariant on F it immediately follows that E F ∼S(Hd) cost(F) = E F ∼N(0,I) cost(F). The fact that it is scale-invariant on G allows one to use a version of BP that returns a pair in The fact that it is scale-invariant on G allows one to use a version of BP that returns a pair in a pair in VH := {(G, ζ) ∈Hd × Pn \| G(ζ) = 0} (23) instead of a pair in VS. Furthermore, if F and G are Gaussians, then so is Qt = tF + (1 −t)G a pair in VH := {(G, ζ) ∈Hd × Pn \| G(ζ) = 0} (23) instead of a pair in VS. 3. The Solution Furthermore, if F and G are Gaussians, then so is (23) Qt = tF + (1 −t)G Qt = tF + (1 −t)G for every t ∈[0, 1]. We cannot however directly use these facts in conjunction with Proposition 3.1 because there τ parameterizes a fraction of the angle dS(G, F) and now t parameterizes a fraction of ∥F −G∥. But it is easy to go from one to the other via the change of variables t = ∥G∥ ∥F∥sin α cot(τα) −∥F∥cos α + ∥G∥ t = ∥G∥ ∥F∥sin α cot(τα) −∥F∥cos α + ∥G∥ where α = dS(G, F). Then Proposition 3.1 takes the following form. Corollary 3.2. Suppose that [G, F] does not intersect Σ. Then the execution of LinHom(F, G, ζ0) stops after at most K steps with Corollary 3.2. Suppose that [G, F] does not intersect Σ. Then the execution of LinHom(F, G, ζ0) stops after at most K steps with K = K(F, G, ζ0) ≤188D3/2∥F∥∥G∥ Z 1 0 µ2 norm(Qt, ζt) ∥Qt∥2 dt. 2 The returned point z is an approximate zero of F. 244 FELIPE CUCKER We won’t describe the derivation of the bound for E F ∼N(0,I) E (G,ζ0)∼ρstd K(F, G, ζ0). Suﬃce it to mention that, although diﬀerent in the details, it is similar to the one described in §3.2 and it yields the same O(D3/2nN) bound. 2 One of the diﬀerences is the need to estimate EQ∼N(0,σ2I) µ2 avg(Q) ∥Q∥2 instead of EQ∼S(Hd) µ2 avg(Q) (cf. (21)). It is here when one notes that if any of F or G is not centered (i.e., have a mean diﬀerent from 0), then Qt is still Gaussian (even though not centered). In particular, this is true when either F or G are ﬁxed. If F, for instance, is ﬁxed, then Qt is distributed as N(F, (1−t)2I). This fact was the trigger to extend (21) not only to the Gaussian setting but also to the non-centered case. The main technical result in [9] (see Theorem 3.6 there or [10, Thm. 18.4]) shows that for all ¯Q ∈Hd and σ > 0 we have E Q∼N( ¯ Q,σ2I) µ2 avg(Q) ∥Q∥2 ≤e(n + 1) 2σ2 . (24) (24) With this smoothed analysis at hand, and some additional work, two diﬀerent com- plexity results followed by ﬁxing, respectively, F and (G, ζ0). 3.3.1. Instance complexity. 3. The Solution But by then Smale’s 17th problem had already been solved. 3.4. The solution. The ﬁnal solution of Smale’s 17th problem by Pierre Lairez [16] relies on a beautifully ingenious idea. Beltr´an and Pardo had avoided constructing a pair (G, ζ0) that would work for all inputs F ∈S(Hd) via a randomized construc- tion: the pair (G, ζ0) depended on random reals drawn from a random number generator. Lairez’s solution uses this construction with a twist: the random num- bers are extracted from F itself. To better understand this idea, let’s consider a situation simpliﬁed to the ex- treme. Take a random number F ∈[0, 1] from the uniform distribution (by glueing the extremes of this interval we can look at it as the circle S1, a simpliﬁed version of S(Hd)). Fix a number ℓ∈N. We can associate to F the numbers F ◦:= 2−ℓ⌊2ℓF⌋ and R◦:= 2ℓ(F −F ◦). The ﬁrst, the truncation of F, approximates F by taking the ﬁrst ℓbits of its base-2 expansion. It satisﬁes F −F ◦≤2−ℓ. The second, the fractional part of F, is uniformly distributed in [0, 1] and independent of F ◦. Lairez devised a similar procedure now for systems F ∈S(Hd) drawn from the uniform distribution. The resulting systems F ◦and R◦are both in S(Hd) and satisfy that F ◦approximates F, and that R◦is nearly uniform and nearly independent from F ◦. The fractional part R◦is then used as random source to produce a pair (G, ζ0) ∼ ρstd by a version of the BP algorithm which replaces the set R of numbers obtained from a random number generator by the coeﬃcients of R◦. Once this done, a linear homotopy is performed with initial pair (G, ζ0) and target system F ◦. Had we have started with F ∼S(Hd) and the set R (the random numbers in the call to BP) independent of F, the systems F and G would be uniform in S(Hd) and independent, and the execution LinHom(F, G, ζ0) would end on an approximate zero of F. As it happens, we are starting with F ◦and R◦nearly independent —but not exactly so— and F ◦close to F —but not exactly there—. The fact we want to rely on is that the larger is ℓthe better are these approxi- mations. And that we can quantify this dependence. 3. The Solution Fixing F ∈Hd and looking at the cost of LV with input F lead us to the quantity randcost(F). The ﬁrst of the two results mentioned above ([9, Thm. 3.7] or [10, Thm. 18.2]) shows that, for algorithm LV and F ∈Hd, 3 2 2 randcost(F) ≤O(D3nN 2µ2 max(F)) randcost(F) ≤O(D3nN 2µ2 max(F)) where µmax(F) = maxζ\|F (ζ)=0 µnorm(F). Reasonably enough, the randomized cost for F depends on how well-conditioned the zeros of F are. 3.3.2. Deterministic algorithms. If we ﬁx the initial pair (G, ζ0) instead, and we take the average of the cost of the linear homotopy for an input F drawn from N(0, I) we obtain our second result, namely E F ∼N(0,I) K(F, G, ζ0) = O(D3nNµ2 max(G)) which gives an average total cost of O(D3nN 2µ2 max(G)). This bound deceptively suggests that the solution to Smale’s 17th problem is near; it suﬃces to construct, for a given pair (n, d), a system G ∈Hd with µmax(G) = N O(1). We say ‘decep- tively’ because this innocent-looking task proved to be remarkably diﬃcult. A ﬁrst attempt to use this bound took as G the system given by Gi := 1 √ 2n(Xdi 0 −Xdi i ), i = 1, . . . , n and as ζ0 the point [(1, . . . , 1)]. One can show that µ2 max(G) ≤2(n + 1)D and, together with a diﬀerent algorithmic approach for high-degree systems (those with D > n), the following result ([9, Thm. 3.9] or [10, Thm. 18.3]). and as ζ0 the point [(1, . . . , 1)]. One can show that µ2 max(G) ≤2(n + 1)D and, together with a diﬀerent algorithmic approach for high-degree systems (those with D > n), the following result ([9, Thm. 3.9] or [10, Thm. 18.3]). Theorem 3.3. There is a deterministic algorithm that on input F ∈Hd computes an approximate zero of F with average cost N O(log log N). Moreover, if we restrict data to systems satisfying D ≤n 1 1+ε or D ≥n1+ε SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 245 for some ﬁxed ε > 0, then the average time of the algorithm is polynomial in the input size N. 2 The quest for a construction of a good initial pair (G, ζ0), raised already in the B´ezout series, would not be closed until very recently [6]. 4. Subsequent Progress The positive answer in Lairez’s paper did not bring to a stop the interest on Smale’s 17th problem. On the contrary, a number of questions naturally arose. 4.1. Eigenpair computations. One such question dealt with applying the ideas for the design and analysis of linear homotopies to speciﬁc systems of equations which do not entirely ﬁt the framework above. A clear example is the computation of eigenpairs. Recall, an eigenpair of a matrix A ∈Cn×n is a pair (λ, v) ∈C×Pn−1 satisfying Av = λv. This equality amounts to the n equations A1v = λv1 ... (26) Anv = λvn (26) where Aj denotes the jth row of A. These equations are linear in the variables v1, . . . , vn and quadratic in the set of all the variables. There are n equations with (generically) a ﬁnite number of of solutions in C × Pn−1 but, in glaring contrast with the general framework, we expect system (26) to have only n solutions, not 2n. A tailor-made approach is necessary. Algorithms computing eigenpairs have existed for long. But their analysis, notwithstanding their practical performance, has been wanting. This moved James Demmel [12, pg. 139] to write So the problem of devising an algorithm [for the eigenvalue problem] that is numerically stable and globally (and quickly!) convergent remains open. is numerically stable and globally (and quickly!) convergent remains open. An answer to this problem was given in [3], where the general lines in the previous sections —devising an appropriate condition number and a step-length computation in terms of it, analysing the number of steps of the linear homotopy as in Proposi- tion 3.1, performing a smoothed analysis of the condition number as in (24), and exhibiting a good initial triple (A0, λ0, v0) (which in this context turns out to be easy!)— allowed one to show an O(n7) bound for the average cost of computing eigenpairs. We note that this algorithm, even though its observed average complex- ity in simulations is O(n3.66), is not eﬃcient when compared with the eigensolvers available in packages such as Matlab or Julia. But it answers Demmel’s question in the sense that it was the ﬁrst eigensolver for which correctness, numerical stability, and a polynomially bounded average cost could be established. 4.2. Rigid homotopies. Probably the most obvious question raised by the solu- tion to Smale’s 17th problem dealt with possible complexity improvements. 3. The Solution Lairez used this fact to devise a simple test that ensures that the curve C ◦associated to the triple (F ◦, G, ζ0) is close enough to the curve C associated to (F, G, ζ0), that the approximate zero of F ◦obtained by following C ◦is also an approximate zero of F, and that the average complexity analysis can still be carried on. It suﬃces to ensure that, at every step of the linear homotopy, D3/2µ2 norm(Qi, zi)ϱ ≤ 1 151 (25) (25) where ϱ is an easy-to-compute quantity satisfying dS(F, F ◦) ≤ϱ. Hence, ϱ (and a fortiori dS(F, F ◦)) must be smaller than (151 D3/2µ2 norm(Q, z))−1 all along (a neighborhood of) C ◦. As we don’t know a priori a bound for the maximum of these µ2 norms, the ﬁnal algorithm starts with an initial value for ℓ, computes the associated F ◦and R◦, computes (Q, ζ0) from R◦, and calls for LinHom(F ◦, G, ζ0). 246 FELIPE CUCKER If condition (25) is satisﬁed at all steps, then the returned point in Pn is an approx- imate zero of F. If at some step (25) is violated then the algorithm replaces ℓby 2ℓ and starts again. The complexity analysis in [16] shows that the average total cost of this deterministic procedure is O(nD3/2N 2). This settles Smale’s 17th problem. 4. Subsequent Progress Because just reading the system F takes time N, a lower bound of Ω(N) is clear. The upper bound for the average cost of LV is O(nD3/2N 2). We observe that in this bound the crucial parameter is N. To see why, think on the case n = D. In this case, N ≥ n+D n = 2n n ∼ 4n √πn. That is, N is exponentially larger than n (and D). We therefore want to understand how small can we make the exponent of N. We 247 SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS already noted that the cost of each iteration of the homotopy is O(N). This cannot be improved. The question then becomes, can one decrease the expected number of iterations (without paying for it in the cost of each iteration)? A ﬁrst answer towards this goal was given in [2] where a more elaborated com- putation of ∆τ, which draw from ideas in [3], allowed for a O(D3/2nN 1/2) bound for the expected number of iterations (and hence an O(D3/2nN 3/2) bound for the average total cost). An optimal answer was provided by Lairez [17]. The involved ideas were new and touched both the algorithm and its analysis. Let us begin with the algorithmic ideas. The innovation here is to perform a homotopy that keeps invariant the shape of the hypersurfaces f1 = 0, . . . , fn = 0 (hence the name rigid) continuously deforming only their position within Pn. To describe how this is done, denote by U(n + 1) the group of unitary matrices of dimension n + 1. This group has an obvious action on Pn; for any u ∈U(n + 1) and z ∈Cn+1 \ {0} we deﬁne u([z]) := [u(z)]. It also acts on the space Hd of homogeneous polynomials of degree d in X0, . . . , Xn. If f ∈Hd and u ∈U(n + 1) then uf := f ◦u−1 is easily seen to be in Hd. Its zero set Z(uf) is just a rotation of Z(f); indeed, Z(uf) = u(Z(f)). This action extends componentwise to systems. Let U := U(n + 1)n. If F ∈Hd and u = (u1, . . . , un) ∈U then uf := (u1f1, . . . , unfn) ∈Hd. 4. Subsequent Progress ( ) Assume next that we are given a pair (v, ζ0) ∈U×Pn such that Q0(ζ0) = 0 where Q0 := vF. We can then start a homotopy with the pair (Q0, ζ0). The breakthrough lies on the fact that the segment we will lift is not the segment [Q0, F] ⊂Hd but the geodesic [v, I] ⊂U. Here v = (v1, . . . , vn) and I = (I, . . . , I). This lifting lives in the solution variety (compare with (19) and (23)) VU := {(u, ζ) ∈U × Pn \| (fi ◦ui)(ζ) = 0, i = 1, . . . , n}. In practice, we don’t need to use the geodesic [u, I]; we can use instead any path {ut}t∈[0,T ] with endpoints v and I and satisfying a few conditions (Lairez imposes being 1-Lipschitz and having length at most 4n). The corresponding systems in the homotopy are then Qt := utF = ((ut)1f1, . . . , (ut)nfn). Figure 4 aims at depicting the process. f1 f2 ζ0 ζt ζ1 Figure 4. A rigid homotopy. Left. The given hypersurfaces. Second. The rotated hypersurfaces with a common zero. Third. An intermediate stage in the homotopy. Right. The ﬁnal stage of the homotopy: the original hypersurfaces with a common zero. f2 Figure 4. A rigid homotopy. Left. The given hypersurfaces. Second. The rotated hypersurfaces with a common zero. Third. An intermediate stage in the homotopy. Right. The ﬁnal stage of the homotopy: the original hypersurfaces with a common zero. 248 FELIPE CUCKER The general arrangement in Lairez’s algorithm is otherwise the same as in LV. We ﬁrst generate the random pair (u0, ζ0) ∈VU. To do so Lairez develops in this context a version of the BP algorithm which draws O(n3) real numbers from the standard Gaussian distribution and performs O(n4 + nD4) arithmetic operations. The distribution ρU in VU induced by this version of BP is similar to ρstd; it just replaces G ∈S(Hd) by u ∈U in (20(i)), and G by uF in (20(ii)). After which, the homotopy proceeds as in the linear case, with steps now in the path {ut}t∈[0,T ] ⊂U instead of in the segment [G, F]. [ , ] The computation of the length of these steps is what brings us to the innovations in the analysis of the homotopy. 4. Subsequent Progress Lairez observed that in the derivation of B leading to (18) two inequalities are coarse. Firstly, (12), which bounds the variation of the zero ζ as a worst-case variation. Secondly, the Higher Derivative Estimate (14) that replaces γ by µnorm. In both cases the use of µnorm(F, z) is computationally practical but leads to coarse bounds. One would like to use measures serving the same purposes, yielding ﬁner bounds, and being also inexpensive to compute. To achieve this, Lairez splits condition at a point into two components. Given F ∈Hd and z ∈Pn, we let Fz := f1 ∥Dzf1∥, . . . , fn ∥Dzfn∥ . The incidence condition number of F at z is then given by The incidence condition number of F at z is then given by κ(F, z) := (DzFz)† . Here † denotes Moore-Penrose inverse. When F(z) = 0 this quantity depends only on the angles made by the tangent spaces at z of the n hypersurfaces Z(fi) := {z ∈ Pn \| fi(z) = 0}. Moreover, κ(uF, z) satisﬁes a version of (12) w.r.t. variations of u in U (see [17, Lem. 16]). That is, we have dP(ζi, ζi+1) ≲∆t κ(uiF, ζi). (27) (27) For the other ingredient, we deﬁne, for f ∈Hd and z ∈Pn, γFrob(f, z) := ( supk≥2 1 k!∥Dzf∥−1∥Dk zf∥Frob 1 k−1 if Dzf is nonzero ∞ otherwise and we let and we let ¯γ2 Frob(F, z) := γ2 Frob(f1, z) + · · · + γ2 Frob(fn, z). In contrast with κ(F, z), ¯γFrob(F, z) does not depend on how the hypersurfaces Z(fi) intersect at z but only on the geometry of each individual hypersurface at z. The split Frobenius γ number ˆγFrob(F, z) := κ(F, z) ¯γFrob(F, z) (28) captures both aspects. Moreover, it bounds γ(F, z) as we have γ(F, z) ≤ˆγFrob(F, z) (29) ˆγFrob(F, z) := κ(F, z) ¯γFrob(F, z) (28) Moreover, it bounds γ(F, z) as we have rob(F, z) := κ(F, z) ¯γFrob(F, z) (28) eover, it bounds γ(F, z) as we have γ(F, z) ≤ˆγFrob(F, z) (29) ˆγFrob(F, z) := κ(F, z) ¯γFrob(F, z) (28) (28) captures both aspects. Moreover, it bounds γ(F, z) as we have captures both aspects. length of the form ∆t = c κ(uiF, zi) ˆγFrob(uiF, zi) ∆t = c κ(uiF, zi) ˆγFrob(uiF, zi) ∆t = κ(uiF, zi) ˆγFrob(uiF, zi) ( ) ( ) for some constant c. This, in turn, leads to a bound for the number of steps in the homotopy of the form (compare with Proposition 3.1) ( ) ( ) for some constant c. This, in turn, leads to a bound for the number of steps in the homotopy of the form (compare with Proposition 3.1) K = KF (u, ζ0) ≤C Z T 0 κ(utF, ζt) ˆγFrob(utF, ζt)dt (30) (30) for some constant C. Furthermore, Lairez [17, Prop. 17] shows that, for a given F ∈Hd with square-free components, E (u,ζ)∼ρU κ2(uF, ζ) ≤6n2 (31) (31) and that [17, Lem 38], for a Gaussian f ∈Hd, E f∼N(0,I) E ζ∼Z(f) γ2 Frob(f, ζ) ≤1 4d3(d + n). We can use this inequality to bound, for a Gaussian F ∈Hd, the value of EF ∼N(0,I) E(u,ζ)∼ρU ¯γ2 Frob(uF, ζ). Indeed, if (u, ζ) ∈VU is ρU-distributed then u1ζ, . . . , unζ are zeros of f1, . . . , fn, uni- formly distributed in Z(f1), . . . , Z(fn), respectively, and independent [17, Thm. 8]. Also, if F is Gaussian, so are its components fi. It follows that E F ∼N(0,I) E (u,ζ)∼ρU ¯γ2 Frob(uF, ζ) = E (u,ζ)∼ρU n X i=1 E fi∼N(0,I) γ2 Frob(uifi, ζ) = E (u,ζ)∼ρU n X i=1 E fi∼N(0,I) γ2 Frob(fi, uiζ) = n X i=1 E fi∼N(0,I) E ζi∼Z(fi) γ2 Frob(fi, ζi) ≤ 1 4nD3(D + n). = n X i=1 E fi∼N(0,I) E ζi∼Z(fi) γ2 Frob(fi, ζi) ≤ 1 4nD3(D + n). (32) (32) At this stage, the reasoning continues as in §3.2. One ﬁrstly shows that when F is Gaussian (or, equivalently, when F ∼S(Hd)) and (u, ζ) ∼ρU, the pair (ut, ζt) in (30) is also ρU-distributed in VU. Hence At this stage, the reasoning continues as in §3.2. One ﬁrstly shows that when F is Gaussian (or, equivalently, when F ∼S(Hd)) and (u, ζ) ∼ρU, the pair (ut, ζt) in (30) is also ρU-distributed in VU. 4. Subsequent Progress Moreover, it bounds γ(F, z) as we have γ(F z) ≤ˆγF b(F z) (29) γ(F, z) ≤ˆγFrob(F, z) (29) (29) thus providing us with a replacement for (14) which, we will see, turns out to be better for our purposes. With (27) and (29) respectively replacing (12) and (14) we can reason as in §3.1 and use now Lipschitz bounds for κ and γFrob to derive an expression for the step SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 249 length of the form length of the form length of the form Hence E F ∼N(0,I) E (u,ζ)∼ρU K ≤ C E F ∼N(0,I) Z T 0 E (ut,ζt)∼ρU κ(utF, ζt) ˆγFrob(utF, ζt)dt ≤ 4Cn E F ∼N(0,I) E (u,ζ)∼ρU κ(uF, ζ) ˆγFrob(uF, ζ) = (28) 4Cn E F ∼N(0,I) E (u,ζ)∼ρU κ2(uF, ζ) ¯γFrob(uF, ζ) = 4Cn E F ∼N(0,I) E (u,ζ)∼ρU κ2(uF, ζ) E F ∼N(0,I) E (u,ζ)∼ρU ¯γFrob(uF, ζ) ≤ (31−32) 4Cn · 6n2 · r 1 4nD3(D + n) (33) = O(n4D2). (33) 250 FELIPE CUCKER For the second equality we used that κ and ¯γFrob are independent. For the last in- equality we used that F has square-free components almost surely (along with (31)) and Jensen’s inequality (along with (32)). For the second equality we used that κ and ¯γFrob are independent. For the last in- equality we used that F has square-free components almost surely (along with (31)) and Jensen’s inequality (along with (32)). This is the bound we wished for. It shows that the average number of steps in the rigid homotopy is polynomial in n and D. It only remains to be seen that the cost of each such step is still linear in N. Lairez [17, Cor. 34] shows that this is the case: the cost of each step is O(n2D2N). It may be worth to describe, nonetheless, how γFrob(fi, z) is computed for a given fi ∈Hdi and a point z ∈Cn+1 as this computation is not straightforward. The crucial observation is the following. Let g(X) ∈Hdi be the shifted poly- nomial fi(z + X). This is no longer a homogeneous polynomial. For k = 0, . . . , di let g[k] denote the homogeneous component of degree k of g. Then Dk zf = Dk 0g and [17, Lem. 30] 1 k!Dk zf Frob = 1 k!Dk 0g Frob = g[k] (34) (34) where, we emphasize, the norm of the last term is the Weyl norm. This equality provides a way to compute the left-hand side: compute g, then its component g[k], and ﬁnally the Weyl norm of the latter. The cost of this preocedure is dominated by the cost of the computation of g. Let Ni = dimC Hdi = n+di n . A naive approach to compute g leads to an Ω(N 2 i ) which is too large. But Lairez devised a procedure to compute g whose cost is O(nd2 i Ni) [17, Prop. 32]. length of the form Since the computation of ∥Dzfi∥−1 has cost O(Ni) it follows that we can compute γFrob(fi, z) with cost O(nd2 i Ni). Furthermore, if ui ∈U(n + 1) we have γFrob(uifi, z) = γFrob(fi, uiz) so that we can compute γFrob(uifi, z) with the same cost. We conclude that, given F ∈Hd, u ∈U, and z ∈Cn+1, we can compute ¯γFrob(uF, z) with cost O(nD2N). The other ingredients in the computation of the step-length being simpler, one concludes the analysis with a total cost for this computation of O(n2D2N). In conjunction with the bound for the average number of steps in (33), and the O(n4+ nD4) cost of computing the initial pair, we end up with a O(n6D4N) for the average total cost of computing one zero of F. The exponent 1 on N is optimal. 4.3. Structured systems. Consider the polynomial a0X3 0X2 1X3 2 + a1X5 1X3 2 + a2X8 0 + a3X0X2 1X5 2. (35) We can describe it via the list {(a0, (3, 2, 3)), (a1, (0, 5, 3)), (a2, (8, 0, 0)), (a3, (1, 2, 5))} (36) 4.3. Structured systems. Consider the polynomial a0X3 0X2 1X3 2 + a1X5 1X3 2 + a2X8 0 + a3X0X2 1X5 2. (35) We can describe it via the list a0X3 0X2 1X3 2 + a1X5 1X3 2 + a2X8 0 + a3X0X2 1X5 2. (35) (35) We can describe it via the list We can describe it via the list {(a0, (3, 2, 3)), (a1, (0, 5, 3)), (a2, (8, 0, 0)), (a3, (1, 2, 5))} (36) (36) {(a0, (3, 2, 3)), (a1, (0, 5, 3)), (a2, (8, 0, 0)), (a3, (1, 2, 5))} which, even with the integers 3, 2, 3, . . . written in binary, is substantially shorter than the list of all the 8+3 3 = 165 coeﬃcients of a generic polynomial in H8 (in X0, X1, X2). The list in (36) is a sparse encoding of the polynomial above; the list of all coeﬃcients (in some preestablished order) is known as dense encoding. It is the one we have been considering in all the previous sections and has a size of Θ(dimC Hd). It is easy to see that the sparse encoding of a polynomial cannot be much bigger than its dense encoding but can be way smaller. A price for this suc- cinctness is fragility: a number of operations with polynomials destroy sparseness. Think for instance in quotient and remainder applied to the polynomials Xn 0 −Xn 1 and X0 −X1. They are both sparse (in the sense of having only two monomials) which, even with the integers 3, 2, 3, . . . written in binary, is substantially shorter than the list of all the 8+3 3 = 165 coeﬃcients of a generic polynomial in H8 (in X0, X1, X2). The list in (36) is a sparse encoding of the polynomial above; the list of all coeﬃcients (in some preestablished order) is known as dense encoding. It is the one we have been considering in all the previous sections and has a size of Θ(dimC Hd). It is easy to see that the sparse encoding of a polynomial cannot be much bigger than its dense encoding but can be way smaller. A price for this suc- cinctness is fragility: a number of operations with polynomials destroy sparseness. Think for instance in quotient and remainder applied to the polynomials Xn 0 −Xn 1 and X0 −X1. 4.3. Structured systems. Consider the polynomial They are both sparse (in the sense of having only two monomials) SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 251 but their quotient is Xn−1 0 + Xn−2 0 X1 + · · · + Xn−1 1 which has n monomials. Only writing this quotient has a high cost in the sparse size of the data, in contrast with its cost as a function on the dense size, which is small. Another way to describe a polynomial, which can be even more succint than a sparse encoding, is as a straight-line program (SLP for short). The sequence of operations (starting with the variables X0 and X1) X0 + X1 → (X0 + X1)2 →(X0 + X1)4 →(X0 + X1)8 →(X0 + X1)16 → (X0 + X1)2n = 2n X j=0 2n j X2n−j 0 Xj 1 performs n + 1 operations and ends up in a polynomial f of degree 2n for which both the dense and sparse encodings have size Ω(2n). We can encode f via the sequence of operations above and this encoding has size O(n). Not unexpectedly, the catalog of operations which are expensive in terms of the SLP encoding is bigger. The only operation that is clearly eﬃciently performed in terms of the SLP size, besides arithmetic operations with polynomials, is evaluation: given an SLP in n variables X1, . . . , Xn encoding a polynomial f and a point z ∈Cn, the computation of f(z) ∈C takes time linear in the SLP size. A fundamental result of Baur and Strassen [5] shows that we can compute all of f(z), ∂X1f(z), . . . , ∂Xnf(z) with essentially the same cost. The question which is naturally posed is the following: (S17-Sparse) What is the cost of computing a zero (S17-Sparse) of a system in terms of its sparse (or SLP) size? What is the cost of computing a zero (S17-Sparse) of a system in terms of its sparse (or SLP) size? Before attempting any answer to this question it is worth to look at it with more detail. Consider the sparse size, to ﬁx ideas. A ﬁrst remark is that the set of polynomials in Hd having some coeﬃcient equal to 0 has measure zero in Hd. 4.3. Structured systems. Consider the polynomial For systems in such an L any linear homotopy will loop forever and the average complexity of the algorithm will be inﬁnity. because all the zeros of F on the hypersurface {X0 = 0} are double. For systems in such an L any linear homotopy will loop forever and the average complexity of the algorithm will be inﬁnity. Secondly, L is not unitary invariant. In general, given F ∈L and u ∈U, we don’t have uF ∈L. This deprives us from a very powerful technical tool. Nonetheless research in homotopy methods for sparse systems has been carried out for decades. And even though there are no conclusive results, advances have been made. The state of the art in this quest can be found in the papers [18, 19] and in the references therein. 4.4. Low-complexity systems. The framework of rigid homotopies introduced by Lairez naturally considers (possibly small) subsets of Hd. Indeed, for a given input F ∈Hd the space where the homotopy path leading to F lives in is not the whole of Hd (as in Sections 2 and 3) but its subset UF := {uF \| u ∈U}. The initial system G is not arbitrary in Hd but of the form vF for some arbitrary v ∈U. In addition, if L(F) denotes the evluation complexity of F (that is, the length of the shortest SLP computing F) then L(uF) ≤L(F) + (n + 1)4. In particular, if F has a low evaluation complexity then all systems utF in the homotopy path have low evaluation complexity as well. These considerations are the motivation behind [11], where the goal is to devise an eﬃcient rigid homotopy for low-complexity systems. On a ﬁrst approach, a system F ∈Hd is ﬁxed and random inputs are considered with the form uF where u is drawn from the uniform distribution in U. This mimics the framework in §3.2 replacing random F, G ∈S(Hd) by random u, v ∈U. At a ﬁrst glance, it would seem that the algorithm in [17] can be applied without modiﬁcations. Unfortunately, a careful look at the details shows that there is a diﬃculty. For f ∈Hd, the computation of γFrob(f, z) described at the end of §4.2 relies on computing Weyl norms ∥gk∥for the homogeneous components gk of the polynomial g := f(z + X). Theorem 4.1. There is a randomized algorithm which, given f ∈C[X0, . . . , Xn] as a black-box function, an upper bound d on its degree, a point z ∈Cn+1, and 4.3. Structured systems. Consider the polynomial Consequently, the average cost over Hd in terms of the sparse size of systems in Hd will be the same as that in terms of its dense size. One may reﬁne the question above and consider only polynomials with a given monomial structure. For instance, one of the polynomials may be imposed to have the form in (35). The average (regarding this polynomial) would then be taken, not over the whole of H8, but over the tuples (a0, a1, a2, a3) of coeﬃcients, say from a Gaussian distribution on C4. Other components of the input system F ∈Hd may also have a (possibly diﬀerent) ﬁxed monomial structure. Before attempting any answer to this question it is worth to look at it with more detail. Consider the sparse size, to ﬁx ideas. A ﬁrst remark is that the set of polynomials in Hd having some coeﬃcient equal to 0 has measure zero in Hd. Consequently, the average cost over Hd in terms of the sparse size of systems in Hd will be the same as that in terms of its dense size. One may reﬁne the question above and consider only polynomials with a given monomial structure. For instance, one of the polynomials may be imposed to have the form in (35). The average (regarding this polynomial) would then be taken, not over the whole of H8, but over the tuples (a0, a1, a2, a3) of coeﬃcients, say from a Gaussian distribution on C4. Other components of the input system F ∈Hd may also have a (possibly diﬀerent) ﬁxed monomial structure. This determines a space of inputs which is a linear subspace L of Hd over which a Gaussian measure is naturally deﬁned (or a uniform measure on its associated unit sphere). And suggests that an approach as described in Sections 2 and 3 could be possible. Unfortunately though, such an approach is plagued with diﬃculties. Firstly, one observes that it may happen that all systems in L are singular. Indeed, this is the case, for instance, if a component of F has the form a0X3 0X2 1X3 2 + a1X2 0X3 1X3 2 + a2X8 0 + a3X2 0X1X5 2 = X2 0(a0X0X2 1X3 2 + a1X4 1X2 2 + a2X6 0 + a3X1X5 2) 252 FELIPE CUCKER because all the zeros of F on the hypersurface {X0 = 0} are double. some ε > 0, returns a number G ≥0 satisfying γFrob(f, z) ≤G ≤192n2dγFrob(f, z) γFrob(f, z) ≤G ≤192n2dγFrob(f, z) with probability at least 1−ε. The computation takes O d log( d ε)(L(f)+n+log d) operations. 2 2 The algorithms in §3.2, §3.3, and §4.2 are all of Las Vegas type. This means that they are randomized algorithms (they draw random numbers during their execution) whose output is guaranteed to be correct: only their running time is random (for each input data). The algorithm in Theorem 4.1 is also a randomized one but of Monte Carlo type. Its running time is deterministic but its output is correct only with a given probability. One can replace in the rigid homotopy the computation of γFrob(f, z) described at the end of §4.2 by the randomized algorithm in Theorem 4.1. In doing so, however, the rigid homotopy may be aﬀected in two ways. If the γFrobs computed along the homotopy are overestimated (the upper bound in Theorem 4.1 does not hold) then the corresponding step-lengths are too short and the complexity bounds no longer hold. If, instead, some γFrob is underestimated (the lower bound in Theorem 4.1 does not hold) then the correctness of the rigid continuation is compromised. The algorithm devised in [11], called there BoostBlackBoxSolve, deals with both problems. The next result (Theorem 1.1 in [11]) states its properties regarding termination and correctness. Theorem 4.2. Let F = (f1, . . . , fn) be a homogeneous polynomial system with degrees at most D in n + 1 variables having only regular zeros. On input F, given as a black-box evaluation program, and ε > 0, Algorithm BoostBlackBoxSolve terminates almost surely and computes a point z ∈Pn, which is an approximate zero of F with probability at least 1 −ε. 2 Because of its reliance on the Monte-Carlo computation of the γFrobs, Algo- rithm BoostBlackBoxSolve is itself of Monte Carlo type: the output is only correct with given probability. Nonetheless, this probability is bounded below inde- pendently of F. The algorithm succeeds with probability at least 1−ε for any input F with regular zeros (of course, it may not terminate if this regularity hypothesis is not satisﬁed as the homotopy path may lead to a singular zero). We are next interested on the average cost of BoostBlackBoxSolve over random data of the form uF for a ﬁxed F and random u ∈U. 4.3. Structured systems. Consider the polynomial If f is given by a short SLP then g is given by an equally short SLP. This is clear. Lemma 3.4 in [11] shows that we can actually do more: for any w ∈Cn+1 we can compute the values g0(w), . . . , gd(w) with cost O(dL(f)+n+log d). So, the evaluation complexity of the gks is also small. We are left with the problem of computing their Weyl norms (obviously, without expanding the gks to obtain their coeﬃcients; this would be too expensive). The way out lies on (yet another) useful property of the Weyl norm, namely, that for g ∈Hd, ∥g∥2 = n + d d E w∼S(Cn+1) \|g(w)\|2. (37) (37) This allows for a randomized algorithm to approximate ∥g∥: sample s points w1, . . . , ws from S(Cn+1), compute the empirical average M := 1 s P \|g(wi)\|2 and This allows for a randomized algorithm to approximate ∥g∥: sample s points w1, . . . , ws from S(Cn+1), compute the empirical average M := 1 s P \|g(wi)\|2 and return qn+d d M. The cost of this algorithm is low (it relies on evaluating g) but one pays for it both in terms of precision —we only obtain an approximation of ∥g∥— and of certainty —we only have a probabilistic guarantee of correctness. More precisely, the following is Theorem 3.3 in [11]. return qn+d d M. The cost of this algorithm is low (it relies on evaluating g) but one pays for it both in terms of precision —we only obtain an approximation of ∥g∥— and of certainty —we only have a probabilistic guarantee of correctness. More precisely, the following is Theorem 3.3 in [11]. Theorem 4.1. There is a randomized algorithm which, given f ∈C[X0, . . . , Xn] as a black-box function, an upper bound d on its degree, a point z ∈Cn+1, and SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 253 some ε > 0, returns a number G ≥0 satisfying As F itself is ﬁxed, we should expect this cost to depend with the geometry of F. We can express this dependence in terms of γFrob. We deﬁne, for f ∈Hd, Γ(f)2 := E z∈Z(f) γFrob(f, z)2 where the distribution on Z(f) ⊂P(Cn+1) is the uniform, and, for F ∈Hd, Γ(F) := Γ(f1)2 + · · · + Γ(fn)21/2 . This quantity is a measure of regularity (or conditioning) of the set of hypersurfaces {Z(f1), . . . , Z(fn)}. If all these hypersurfaces have only algebraically regular points then Γ(F) < ∞. But the converse is not true. A necessary and suﬃcient condition for the ﬁniteness of Γ(F) is yet to be found. 254 FELIPE CUCKER The complexity of BoostBlackBoxSolve, is bounded in the next result [11, Thm. 1.2]. Theorem 4.3. Let F = (f1, . . . , fn) be a homogeneous polynomial system with degrees at most D in n + 1 variables. Assume that, for all i ≤n, fi is square- free. Let u ∈U be uniformly distributed. Then, on input uF, given as a black-box evaluation program, and ε ∈[0, 1 4], Algorithm BoostBlackBoxSolve terminates after (n, D)O(1) · L(F) · Γ(F) log Γ(F) + log log ε−1 operations on average. “On average” refers to expectation over both the random draws made by the algorithm and the random variable u, but F is ﬁxed. 2 operations on average. “On average” refers to expectation over both the random draws made by the algorithm and the random variable u, but F is ﬁxed. 2 The hypothesis on F (the fact that all the Z(fi) are regular) implies that each fi is square-free. Hence, by Theorem 4.2, ensures that the algorithm terminates almost surely. It further implies that the quantity Γ(F) is ﬁnite and, therefore, so is the bound in Theorem 4.3. We note that this bound is polynomial in n and D, linear in L(F), almost linear in Γ(F), and linear in log log ε−1. The latter means that we can take ε = 2−2100 without afterthoughts as to the eﬀect on the running’s cost. For all practical purposes the algorithm behaves as one with certiﬁed outputs. 4.5. Algebraic branching programs. Theorem 4.3 can be extended to subsets M ⊂Hd other than UF (for a given F ∈Hd). some ε > 0, returns a number G ≥0 satisfying The crucial requirement on M is that it is unitarily invariant in the following sense: M is endowed with a probability distribution such that for all F ∈M and all u ∈U, uF ∈M , and F and uF are identically distributed. Trivially, the set UF is unitarily invariant. Final average complexity bounds will now depend not on Γ(F) for a particular F but on the quantity Ef∈M Γ(F)2. The second main result in [11] estimates this quantity for a family of well-known subsets M given by SLPs known as Algebraic Branching Programs (ABP) introduced by Nissan [20]. ( ) [ ] To understand ABPs let us consider a simple example. Consider the matrices A1 = 6X0 + X1 −3X0 + 2X1 5X0 4X0 −3X1 2X0 + 7X1 6X0 −5X1 and and and A2 =   3X0 + 2X1 −X0 + 4X1 X0 −X1 2X0 −5X1 2X0 −X1 −3X0 −X1   whose entries are linear forms in (X0, X1). We can take the product whose entries are linear forms in (X0, X1). We can take the product A1 · A2 = 25X2 0 + 15X0X1 −27X2 0 + 37X0X1 −6X2 1 26X2 0 −12X0X1 −8X2 1 −18X2 0 + 32X0X1 −42X2 1 as well as its trace as well as its trace fA(X) = 7X2 0 + 47X0X1 −42X2 1. The coeﬃcients of this polynomial in H2 are simple functions of the entries in A1 and A2. In this example, the former is an element in C3 and the latter an element in C24. Clearly, the map (A1, A2) 7→fA is surjective. But this needs not to be so. ( ) Let r = (r1, . . . , rd) ∈Nd, r0 = rd, and Mr(n + 1) be the space of d-tuples of matrices A = (A1(X), . . . , Ad(X)) where Aj(X) is a rj−1 ×rj matrix whose entries SMALE’S 17TH PROBLEM: ADVANCES AND OPEN DIRECTIONS 255 are linear forms on (X0, . . . , Xn). This is a complex linear space of dimension (n + 1) Pd j=1 rj−1rj. F A ∈M ( + 1) id th h l i l f d d ) P j 1 j j For A ∈Mr(n + 1) we consider the homogeneous polynomial of degree d fA(X) := tr(A1(X) · . . . · Ad(X)). For large enough r1, . . . some ε > 0, returns a number G ≥0 satisfying , rd the map A 7→fA is a surjection Mr(n + 1) →Hd, but for smaller values dimC Mr(n + 1) < dimC Hd. Tuples of polynomials described by ABPs give us a natural example of an M ⊂Hd for which we can actually eﬃciently run the rigid homotopy. To show this, we endow Mr(n + 1) with the Hermitian norm ∥A∥2 := d X j=1 n X i=0 ∥Aj(ei)∥2 Frob ∥A∥2 := d X j=1 n X i=0 ∥Aj(ei)∥2 Frob where ei is the ith unit vector in Cn+1. With this norm at hand we can deﬁne the standard Gaussian distribution on Mr(n + 1), via the density πdimC Mr(n+1)e−∥A∥2. We say that an ABP is irreducible when r1, . . . , rd−1 ≥2. The second main result in [11], Theorem 1.5, is then the bound for irreducible random ABPs where ei is the ith unit vector in Cn+1. With this norm at hand we can deﬁne the standard Gaussian distribution on Mr(n + 1), via the density πdimC Mr(n+1)e−∥A∥2. We say that an ABP is irreducible when r1, . . . , rd−1 ≥2. The second main result in [11], Theorem 1.5, is then the bound for irreducible random ABPs E A∼Mr(n+1) Γ(fA)2 ≤3 4d3(d + n) log d. (38) (38) Because the distribution of a Gaussian ABP is unitarily invariant one can derive from Theorems 4.2 and 4.3 the following [11, Corollary 1.6]. Because the distribution of a Gaussian ABP is unitarily invariant one can derive from Theorems 4.2 and 4.3 the following [11, Corollary 1.6]. Corollary 4.4. Let f1, . . . , fn be given by independent irreducible Gaussian ABPs of degree at most D and evaluation complexity at most L, and ε ∈[0, 1 4]. Then Boost- BlackBoxSolve returns an approximate zero of F = (f1, . . . , fn) with probability at least 1 −ε in at least 1 −ε in (n, D)O(1)L log log ε−1 (n, D)O(1)L log log ε−1 2 operation on average. operation on average. It is remarkable that the values rj do not occur on the right-hand side of (38). They do occur, however in the bound in Corollary 4.4 as, it is easy to see using iterated matrix multiplication, L = O(nDr3) where r = max rj. References [1] N.H. Abel. M´emoire sur les ´equations alg´ebriques, ou l’on d´emontre l’impossibilit´e de la r´esolution de l’´equation g´en´erale du cinqui`eme degr´e. In L. Sylow and S. Lie, editors, Œuvres Compl`etes de Niels Henrik Abel, volume I, pages 28–33. Grøndahl & Søn, 1881. [2] D. Armentano, C. Beltr´an, P. B¨urgisser, F. Cucker, and M. Shub. Condition length and complexity for the solution of polynomial systems. Found. Comput. Math., 16:1401–1422, 2016. [3] D. Armentano, C. Beltr´an, P. B¨urgisser, F. Cucker, and M. Shub. 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https://openalex.org/W4386647322	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0291493&type=printable	English	null	Hypnotic suggestion versus sensory modulation of bodily awareness	PloS one	2,023	cc-by	13,862	PLOS ONE RESEARCH ARTICLE Hypnotic suggestion versus sensory modulation of bodily awareness C. ApelianID1,2, D. B. Terhune3,4, F. De Vignemont1 1 De´partement d’e´tudes Cognitives, Institut Jean Nicod, ENS, EHESS, CNRS, PSL University, Paris, France, 2 ARCHE, Formation, Paris, France, 3 Department of Psychology, Goldsmiths, University of London, London, United Kingdom, 4 Department of Psychology, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, United Kingdom 1 De´partement d’e´tudes Cognitives, Institut Jean Nicod, ENS, EHESS, CNRS, PSL University, Paris, France, 2 ARCHE, Formation, Paris, France, 3 Department of Psychology, Goldsmiths, University of London, London, United Kingdom, 4 Department of Psychology, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, United Kingdom 1 De´partement d’e´tudes Cognitives, Institut Jean Nicod, ENS, EHESS, CNRS, PSL University, Paris, France, 2 ARCHE, Formation, Paris, France, 3 Department of Psychology, Goldsmiths, University of London, London, United Kingdom, 4 Department of Psychology, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, United Kingdom clement.apelian@arche-hypnose.com * clement.apelian@arche-hypnose.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Apelian C, Terhune DB, De Vignemont F (2023) Hypnotic suggestion versus sensory modulation of bodily awareness. PLoS ONE 18(9): e0291493. https://doi.org/10.1371/journal. pone.0291493 Editor: Jane Elizabeth Aspell, Anglia Ruskin University, UNITED KINGDOM Received: December 2, 2022 Accepted: August 30, 2023 Published: September 12, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0291493 Copyright: © 2023 Apelian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Bodily awareness arises from somatosensory, vestibular, and visual inputs but cannot be reduced to these incoming sensory signals. Cognitive factors are known to also impact bodily awareness, though their specific influence is poorly understood. Here we systemati- cally compared the effects of sensory (bottom-up) and cognitive (top-down) manipulations on the estimated size of body parts. Toward this end, in a repeated-measures design, we sought to induce the illusion that the right index finger was elongating by vibrating the biceps tendon of the left arm whilst participants grasped the tip of their right index finger (Lackner illusion; bottom-up) and separately by hypnotic suggestion (top-down), with a sham version of the Lackner illusion as an active control condition. The effects of these manipulations were assessed with perceptual and motor tasks to capture different components of the representation of body size. We found that hypnotic suggestion significantly induced the illu- sion in both tasks relative to the sham condition. The magnitudes of these effects were stronger than those in the Lackner illusion condition, which only produced a significantly stronger illusion than the sham condition in the perceptual task. We further observed that illusion magnitude significantly correlated across tasks and conditions, suggesting partly shared mechanisms. These results are in line with theories of separate but interacting repre- sentational processes for perception and action and highlight the influence of cognitive fac- tors on low-level body representations. * clement.apelian@arche-hypnose.com PLOS ONE PLOS ONE Introduction have declared that no competing interests exist. It is generally assumed that bodily illusions depend on misleading sensory signals [18, 19]. For example, in the Lackner illusion, perceived limb extension is caused by vibrating a tendon, which alters proprioceptive signals. By contrast, the extent to which these illusions are attribut- able to, or influenced by, top-down factors, has received only scant attention. In contrast to sensory-driven methods, a top-down approach aims to modify bodily awareness by inducing changes in the subject’s beliefs, motivations, and expectations, with outcomes shaped by per- sonal learning history, current social environment, and germane psychological traits [20]. Most experiments adopting this top-down approach target high-level representations of the body. For example, participants are exposed to photographs of thin fashion models, which have been shown to modify bodily satisfaction, as indexed by psychometric measures [21]. A possibly more promising, albeit understudied, route for investigating the role of top-down mechanisms in the modulation of bodily awareness is provided by hypnotic suggestion [22, 23]. It involves direct verbal suggestions following hypnotic induction, which consists of a prior extended suggestion to enter a “state of hypnosis” aimed at raising suggestibility [24–27]. A wealth of data has demonstrated that hypnotic suggestion can be used to impact processes long thought to be beyond social influence. It has thus been shown that hypnotic suggestion can reliably induce analgesia [28], subjective blindness [29], and visual hallucinations [30–32], and that it can reduce response conflict in cognitive control tasks [33]. Hypnotic suggestion has also been used to influence various dimensions of self-awareness, including the sense of agency [34–36], mirror self-recognition [37], sex change delusion [38], and the sense of body ownership [39]. Some of these studies specifically target bodily awareness [40–42] or have used expectation manipulation to modulate body perception [43] but to our knowledge, hyp- notic suggestion has not yet been systematically used to modify the relatively low-level prop- erty of body metrics. Bottom-up modulation through distorted sensory signals and top-down modulation through hypnotic suggestion may be conceived as distinct modes by which bodily awareness can be manipulated but it remains an open question to what extent they are fully independent. For example, several studies have reported positive correlations between the magnitude of the rubber hand illusion (RHI) [8], as well as other germane bodily illusions, and (hypnotic) sug- gestibility [44–48]. Introduction The experience of the body may seem self-evident, coherent, and stable, to the point of being almost invisible. However, in some circumstances bodily awareness can be dramatically altered. Not only can one experience phantom sensations in long-lost amputated limbs [1], but one can also fail to locate one’s body parts (autotopagnosia) [2], or experience them as being elongated (macrosomatognosia) [3], alien (somatoparaphrenia) [4], or as having a will of their own (anarchic hand syndrome) [5]. Critically, these distortions of bodily awareness are not restricted to psychiatric or neurological disorders and can be experimentally induced in neuro- typical individuals through manipulation of sensory inputs [6–8]. Data Availability Statement: Data are publicly available on the Open Science Framework (https:// osf.io/jxek3/). Funding: This research was supported by ARCHE formation (scholarship of A.C. and experimental 1 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness The present study focused on body metrics, namely the representation of the length of body parts. Body metrics comprise relatively basic properties, whose accurate representation is essential for action planning [9]. It is also relatively central to the way we perceive ourselves, as distortions of these metrics are known to contribute to disorders such as anorexia nervosa [10, 11]. Yet, body metrics can be manipulated relatively easily. For instance, the vibration of the insertion site of the biceps brachii can elicit the experience of illusory arm extension. If, at the same time, one holds a part of one’s body with the arm that one feels moving away, one can report feeling that the grasped body part is elongating in the direction of the illusory motion (the Lackner illusion) [6, 12–17]. One can induce similar body part elongation by altering visual inputs, for example through virtual reality [7]. expenses, no grant number, https://www.arche- hypnose.com/), FrontCog (ANR-17-EURE-0017 FrontCog, https://www.psl.eu/en/front-cog) and PSL (ANR-10-IDEX-0001-02 PSL, https://psl.eu/ en). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. expenses, no grant number, https://www.arche- hypnose.com/), FrontCog (ANR-17-EURE-0017 FrontCog, https://www.psl.eu/en/front-cog) and PSL (ANR-10-IDEX-0001-02 PSL, https://psl.eu/ en). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: A.C have read the journal’s policy and the authors of this manuscript have the following competing interests: scholarship from ARCHE formation. T.D. and V.F. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 Introduction However, it should be noted that a correlation between hypnotic suggest- ibility and a specific bodily illusion does not necessarily entail that the illusion is driven by suggestion and expectations. It may merely indicate that highly suggestible individuals are more responsive to the illusion induction method via bottom-up mechanisms. Furthermore, it has been recently debated to what extent the RHI could be explained by demand characteris- tics [45, 49, 50], that is, by participants’ explicit compliance, or unconscious changes in behav- iour and experience in accordance with implicit or explicit experimental demands [51]. These debates highlight the need for a better understanding of top-down influences on bodily aware- ness. In turn, this will allow us to critically examine current models of body representation and pave the way forward to better understand the diverse factors that shape bodily awareness. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 2 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness A now classic taxonomy distinguishes between visuospatial aspects of body representation, labelled body image, and sensorimotor aspects of body representation, often referred to under the label of body schema. This distinction raises a number of methodological and theoretical challenges but it can be summarized as follows: body image is the body you perceive, whereas body schema is the body you act with [52]. Here we define body image as the perceptual report of a body part size. Body schema on the other hand is defined here as the perceived distance between the position of a body part and a target one aims for when only proprioception is available to reach that target. Accordingly, bodily awareness can be selectively impaired specif- ically at one level, and not at the other [53]. In Alice in Wonderland syndrome, for instance, some patients report feeling very tall, as if they were walking on stilts, yet they walk normally [54]. In this case, only body image is disturbed, and body schema remains intact. This dissocia- tion can also be experimentally induced in healthy participants. In several instances, illusions impact perceptual responses considerably more than motor responses [14, 55, 56]. Illusory arm movement due to tendon vibration, for instance, does not significantly affect motor plan- ning [14]. Introduction Nonetheless, it is believed that body image and body schema interact in most instances [54, 57, 58], in part because the brain tends to avoid conflict whenever possible and favours unity and consistency. Consequently, in everyday life the body we perceive generally coincides with the body that we act with. The central aim of this study was to induce the experience of finger elongation and to con- trast bottom-up (Lackner illusion) and top-down (hypnotic suggestion) manipulations of both body image and body schema. To measure the effects on these bodily representations, we used two tasks that are considered as reliable behavioural measures of body image and body schema. We assessed body image by using a continuous version of body size estimation [16, 59, 60], in which a participant adjusted a visual representation of their hand where the index finger size is changed using a slider (finger length perception task) and body schema by a line reaching task wherein participants matched the position of different lines with their fingertip hidden inside of a box [14, 61]. First, we sought to determine whether hypnotic suggestion can effectively modulate body image and/or body schema. Secondly, we compared the magnitude of such effects against corresponding bottom-up influences on these representations (Lackner illusion) and an active sham condition. Since models of body image and body schema are mostly informed by bottom-up manipulations, our third aim was to assess the robustness of these models when cognitive factors were manipulated. More precisely, we wanted to clarify the interaction of body image and body schema, as reflected in correlations of their respective measures under different sets of constraints, either sensory (Lackner illusion) or cognitive (hypnotic suggestion). Insofar as responsiveness to direct verbal suggestions varies consider- ably in the general population [20], we measured hypnotic suggestibility and expected that it would predict the magnitude of the hypnotic suggestion effect. Material and methods We report how we determined our sample size, all data exclusions, all inclusion/exclusion cri- teria, whether inclusion/exclusion criteria were established prior to data analysis, all manipula- tions, and all measures in the study. Participants The sample size, exclusion criteria, manipulations and measures were established prior to data acquisition. We specified the a priori criterion that a mean effect compared to the sham condi- tion below the test-retest reliability of our measurements would not be considered meaningful. A pilot study led us to estimate this minimal effect at 10 mm, corresponding to an effect size of PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 3 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness (Hedges’s) g = .53. Using a statistical power estimate (1-β) of 0.95 and an α-level of .05 with a two-tailed paired-samples t-test, a power analysis run using GPower 3.1 [62] yielded a mini- mum sample size of 49 participants. To account for potential outliers and/or attrition, we pre- specified a sample size of 52, allowing for around 5% of data loss. 51 participants (31 women, 20 men) completed the experiment (Mage = 38.7; SD = 13.2; range: 20–65 years). All but two were right-handed according to the Edinburgh Handedness Inventory [63]. None reported psychiatric or neurologic disorders nor use of psychoactive drugs (medical or recreational) in the past six months. All participants had two valid, functional arms and were fluent French speakers. 27 participants had never experienced hypnosis, 11 reported having experienced hypnosis in the past at least once, and 13 were current hypnosis users (self-hypnosis, counsel- ling, therapy). Participants were recruited via two communication networks. One was the RISC (Relais d’Information sur les Sciences de la Cognition; RISC-UAR 3332 CNRS), a service advertising experiment opportunities in cognitive science to potential participants. The second one was the first author’s personal communication network. The sample was diverse in age, gender, ethnicity and prior knowledge of hypnosis. The whole experiment was approved by the ethics committee of Paris Descartes under IRB n˚00012019–19 in agreement with the Dec- laration of Helsinki (2008). Informed written consent was obtained from each participant before the beginning of the study. Materials The “target” window (Fig 1A) separating the index finger from the rest of the hand PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 4 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness was positioned and sized by the experimenter at the beginning of the experiment just after the Fig 1. Experimental apparatuses in the finger length perception task (a) and line reaching task (b). (a) Participants adjusted the slider so that the graphical representation matched their perceived finger length. As the slider is moved, the target window is resized to change the displayed length of the finger. The window is set to enclose only the index finger and no other element that could give away the true finger size. (b) Participants hovered above the estimated location of one of three parallel lines with their right index fingertip whilst their hand was hidden from view in a box. The position of the right hand in (b) corresponds to the starting position in each trial; the left hand simply rests on the table during measurements. https://doi.org/10.1371/journal.pone.0291493.g001 Fig 1. Experimental apparatuses in the finger length perception task (a) and line reaching task (b). (a) Participants adjusted the slider so that the graphical representation matched their perceived finger length. As the slider is moved, the target window is resized to change the displayed length of the finger. The window is set to enclose only the index finger and no other element that could give away the true finger size. (b) Participants hovered above the estimated location of one of three parallel lines with their right index fingertip whilst their hand was hidden from view in a box. The position of the right hand in (b) corresponds to the starting position in each trial; the left hand simply rests on the table during measurements. https://doi org/10 1371/journal pone 0291493 g001 Fig 1. Experimental apparatuses in the finger length perception task (a) and line reaching task (b). (a) Participants adjusted the slider so that the graphical representation matched their perceived finger length. As the slider is moved, the target window is resized to change the displayed length of the finger. The window is set to enclose only the index finger and no other element that could give away the true finger size. Materials Social desirability was indexed using the Balanced Inventory of Desirable Responding short form (BIDR-16) [64]. The BIDR-16 consists of sixteen items in which participants rate the extent to which different statements apply to them using a 7-point Likert scale. This scale includes two subscales: self-deceptive enhancement, corresponding to the tendency to give honest but positively-biased reports, and image management, corresponding to conscious dis- simulation of responses in order to give a socially-favourable image of oneself. These subscales have good test-retest reliability and correlate modestly. In our sample, the self-deceptive enhancement subscale displayed acceptable internal consistency (Cronbach’s α = 0.74) whereas the image management subscale did not (α = 0.52). Hypnotic suggestibility was measured using the French version of the Waterloo-Stanford Group Scale of Hypnotic Susceptibility: Form C (WSGC) [65, 66]. This scale consists of a relaxa- tion-based hypnotic induction followed by twelve suggestions for alterations in motor control, cognition, and perception. Following a de-induction, participants dichotomously self-rate their responsiveness to each of the suggestions. Hypnotic suggestibility is subsequently quanti- fied as the total score (range: 0 to 12). The WSGC is a widely-used measure of hypnotic sug- gestibility that has previously been found to exhibit acceptable internal consistency [66, 67] although it was borderline in the present sample (α = 0.61), according to commonly used con- ventions [68]. In addition to hypnotic suggestibility, we asked participants to report their familiarity with hypnosis with the question “Report your familiarity with hypnosis (being hyp- notized) or auto-hypnosis” using a Likert 5-point scale (1: “None” to 5: “Daily”). A finger length perception task, a continuous version of body size estimation [16, 59, 60], was used to measure body image. The task was performed using a graphical user interface dis- playing a picture of the participant’s right hand and a slider (Fig 1). This GUI was presented on a laptop (Lenovo ideapad 100S; screen size: 14”). A picture of the participant’s left hand was taken at the start of the experiment against a white backdrop with ambient light from above (no directional light to prevent hard shadows) using a web camera (F/#2.0; f: 4.8mm - 1). The GUI was developed by the first author using python and the OpenCV library (available upon request). Materials (b) Participants hovered above the estimated location of one of three parallel lines with their right index fingertip whilst their hand was hidden from view in a box. The position of the right hand in (b) corresponds to the starting position in each trial; the left hand simply rests on the table during measurements. https://doi.org/10.1371/journal.pone.0291493.g001 was positioned and sized by the experimenter at the beginning of the experiment, just after the picture of the hand was taken. The participant was instructed to adjust the slider until the pic- ture of their index finger matched their perceived length of their finger. Adjusting the slider resized the “target” window laterally, thus shrinking or expanding the displayed length of the finger. In each condition, perceived finger length was measured as the mean of two trials, one with the starting position of the slider at the maximum distortion (three times the normal size) PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 5 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness and one starting at the minimum distortion (one third of the normal size). This was done to prevent anchoring effects [69]. By comparing the reported finger size at baseline to the actual finger size, we can derive an estimate of the bias in the measurement. In this study, a mean bias of 9.6mm (SD = 7.4) was found, indicating that participants overestimated the size of their index finger. This motivated us to use the mean bias as the dependent measure for this task, which was operationalized as the difference between the perceived finger length in each experi- mental condition relative to baseline, with negative and positive values denoting perceptual contraction and elongation, respectively. We refer to this bias measure as the perceptual effect. A line reaching task was used to measure body schema, similar to classic reaching or match- ing tasks [14, 61]. In this task, the participant’s right arm was hidden in a box on a table before removing their blindfold. Three parallel lines {1,2,3} with an inter-line distance of 40mm were displayed in the field of view of the participant next to the box, with a number displayed next to each line (Fig 1). In each condition, participants completed seven trials in which they were asked to match the position of one of the three parallel lines with their right index fingertip {1,2,3,2,1,3,1}. Materials The lines were only visible to the participants outside the box, so they could not see the position of their right hand. Position recording was performed by placing a sheet of paper at a standard position at the bottom of the box and marking down the position of the fingertip with a marker for each trial. Positions were extracted by measuring the distance between the marks and the edge of the sheet. When comparing the difference of finger posi- tions at baseline relative to the target lines positions, the mean variation was 6mm (SD = 18.9mm), again indicating participants’ tendency to overestimate their fingertip distance (resulting in undershooting). Hence, as in the finger perception task, we used the mean bias in our analyses, computed as the mean finger position in each experimental condition relative to the mean finger position at baseline with positive values corresponding to undershooting (pointing closer to the body relative to the baseline trial) and negative values corresponding to overshooting (pointing further away from the body relative to the baseline trial). We refer to this bias measure as the pointing effect. Given the lack of an a priori expectation regarding the direction of the effect on this task and the difficulty participants reported to consciously locate their fingertip, this task played the role of an implicit measure of body schema. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 https://doi.org/10.1371/journal.pone.0291493.g002 Procedure Lackner illusion. In the Lackner illusion condition we vibrated (80Hz ± 10Hz) the tendon at the insertion site of the left biceps brachii for 30s (Fig 2) whilst the participant, blindfolded, held their right-hand index finger with their left hand in a pincer grip. This frequency and duration of stimulation has previously been shown to be optimal in the induction of the Fig 2. Depiction of the stimulation point for the (a) Lackner illusion procedure and (b) sham. (a) The stimulation was applied to the insertion site of the biceps brachii, while (b) stimulation on the brachio-radialis. In this figure the arm is open for clearly displaying the stimulation landmarks, however in the experiment, the participant’s left arm was set at a 90˚ angle. Fig 2. Depiction of the stimulation point for the (a) Lackner illusion procedure and (b) sham. (a) The stimulation was applied to the insertion site of the biceps brachii, while (b) stimulation on the brachio-radialis. In this figure the arm is open for clearly displaying the stimulation landmarks, however in the experiment, the participant’s left arm was set at a 90˚ angle. https://doi.org/10.1371/journal.pone.0291493.g002 6 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness illusion [12]. The vibrating device used in this study was a handheld wand massager (CE; Palo- queth). The participant’s left arm was set at a 90˚ angle and remained stationary for the whole duration of the condition. Participants were informed that they might feel their right index fin- ger changing in size or not, but no reference was made regarding whether it might contract or elongate. Insofar as the Lackner illusion persists after vibration has stopped, the illusion was tested in the same way as other conditions, without vibration or index finger being held by the contralateral hand. Sham. In the sham condition, all instructions were the same as the Lackner illusion proce- dure, except the vibration was applied to the skin next to the tendon (~4cm distance) on the brachio-radialis where no physiological effect should arise [6, 16]. To ensure that expectations were similar between the sham manipulation and the Lackner illusion manipulation, we sur- veyed a group of 72 participants (online) that did not know of the Lackner illusion. Procedure Partici- pants were described the manipulations and measurements with text and images, but the expected effect of the manipulation was not mentioned similarly to the actual study. Manipula- tions were labelled as “illusion 1” (sham) and “illusion 2” (Lackner illusion) for participants. Participants were then asked to estimate if the manipulation, compared to baseline, would make a person receiving the illusion “overshoot, pointing past the lines”, “correctly point the lines”, or “undershoot, being short of reaching the lines”. This was done for both manipula- tions in the same order as the experiment, with the sham manipulation first then the Lackner illusion manipulation. Similar questions were asked about the effects in the finger length per- ception task with options: “perceive the index finger shorter”, “perceive the index finger as normal”, “perceive the index finger longer”. Analyses reveal that for the line reaching task expectancies are balanced between manipulations as proportion P = .465 was not significantly different from .5 with p = .635, B = 0.13 (See Table 1). However, this was not the case for the perceptual task where the sham manipulation was expected to lead to perceptual elongation more than the Lackner illusion, proportion P = .333 was significantly different from .5 with p = .007, B = 4.45 (See Table 1). Overall, we are confident that the sham manipulation played its role as a control condition. Sham. In the sham condition, all instructions were the same as the Lackner illusion proce- dure, except the vibration was applied to the skin next to the tendon (~4cm distance) on the brachio-radialis where no physiological effect should arise [6, 16]. To ensure that expectations were similar between the sham manipulation and the Lackner illusion manipulation, we sur- veyed a group of 72 participants (online) that did not know of the Lackner illusion. Partici- pants were described the manipulations and measurements with text and images, but the expected effect of the manipulation was not mentioned similarly to the actual study. Manipula- tions were labelled as “illusion 1” (sham) and “illusion 2” (Lackner illusion) for participants. Hypnotic suggestion. The hypnotic suggestion condition was conducted following a script developed for this experiment and included an induction followed by a suggestion to experience the right index finger elongating. Procedure The first session involved the administration of the French version of the WSGC in small groups (3–8) in a small, quiet classroom without windows or distractors. In the second session, participants completed the finger length perception task and the line reaching task four times: at baseline without manipulation (baseline); after vibrating an irrele- vant location near the participant’s elbow (sham); after vibrating the insertion site of the biceps brachii (Lackner illusion); and after a hypnotic induction and a verbal suggestion for finger elongation (hypnotic suggestion). In all four conditions–except baseline–the participants held their right index finger with their left hand in a pincer grip whilst blindfolded. Baseline mea- surements were performed first, allowing the participant to develop familiarity with the tasks. The sham condition always preceded the Lackner illusion procedure so that participants had no reference level concerning the real illusion. The order of the sham and Lackner illusion conditions, and hypnotic suggestion condition were counterbalanced. Participants were told that the illusions were independent so that they might experience the first one only, the second one only, neither, or both. In each condition, the line reaching task always preceded the finger length perception task. At the end of the experiment, participants were debriefed regarding the sham condition and explained the motivation for this deception. No participant reported being aware that one of the conditions was a sham. A question-and-answer time was offered to dissipate any uneasy feelings that might arise due to the unusual experience of hypnosis. Par- ticipants were compensated 16€ for their time. Analyses. In each task, we quantified finger elongation by subtracting the baseline score from the mean score in the respective condition. A single multivariate outlier (>M+3 SDs in 3 conditions) was identified and removed from the dataset. We tested the effect of each condi- tion on each task relative to baseline with a repeated measures ANOVA followed by post hoc analyses, employing two-tailed paired-samples t-tests (both frequentist and Bayesian). In post hoc tests, the Holm-Bonferroni correction was applied to p values. We also computed and compared the correlations (Pearson’s r) between perceptual and pointing effects in the differ- ent conditions using the cocor library [71]. In Bayesian post hoc tests, we used default priors corresponding to a Cauchy distribution centred on zero with a width of .707 (i.e., 1/p2) [72]. Procedure The induction consisted of focusing attention on the bodily sensations felt in the hands, reducing agency for hand motion (similar to the item “moving hands together” in the WSGC [67]), and in using imagery to change the sensation experienced in the hands, thus fostering a positive set of attitudes toward hypnosis and a pas- sive response set. The hypnotic suggestion was phrased as “the finger is being pulled and Table 1. Number of participants expecting each combination of manipulation effect direction in an online survey (N = 72). Line reaching task Finger length perception task Lackner illusion leads to undershooting Lackner illusion has no effect Lackner illusion leads to overshooting Lackner illusion leads to shorter perceived index Lackner illusion has no effect Lackner illusion leads to longer perceived index Sham leads to undershooting 2 4 13 Sham leads to shorter perceived index 3 1 8 Sham has no effect 2 19 3 Sham has no effect 0 19 4 Sham leads to overshooting 17 4 8 Sham leads to longer perceived index 19 7 11 https://doi.org/10.1371/journal.pone.0291493.t001 Number of participants expecting each combination of manipulation effect direction in an online survey (N = 72). PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 7 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness elongated”. This suggestion was repeated three times and participant was given 60s to experi- ence the suggestion without further suggestions or cues. This was done to allow for optimal responding, as the perceived effect of the suggestion can take several seconds and often a cou- ple of minutes to peak (see e.g., ref. [70]). Afterwards, and before testing, participants were told that they would go through the measurement tasks, eyes open, while remaining hypno- tized with their index finger keeping the elongated size for the whole duration of the measure- ments. When testing was complete, a standard de-induction was performed (a short procedure aimed at reorienting participants to normal functioning by signing the end of the hypnotic procedure, similar to ref. [67]) with an emphasis on the index finger returning to its normal size. Participants where then instructed to rub their hands and take a short break. General procedure. Participants’ involvement in this study consisted of two sessions sep- arated by at least one day. Condition differences on perceived finger elongation Our first analysis contrasted the magnitude of the condition manipulations on responses in the two tasks. To that end, we performed a repeated measures one-way ANOVA for testing the influence of condition (baseline, sham, Lackner illusion, hypnotic suggestion) on the outcome measure for each task separately. We found significant main effects of condition in both the perceptual task, F(3,49) = 28.9, p < .001, η 2 = 0.37, B10 = 3.2 x 1012, RR = [<0.001, >2], and the line reaching task, F(3,49) = 19.6, p < .001, η2 = 0.29, B10 = 1.2 x 108, RR = [<0.001, >2]. Mauchly’s test of sphericity indicated that the assumption of sphericity was violated (p < .05). Nonetheless, applying either the Greenhouse-Geisser or Huynh-Feldt correction did not change the reported results. Hence, the results demonstrate that responses on both tasks varied across conditions. As can be seen in Fig 3, Tables 2 and 3, post hoc t-tests revealed that the sham condition did not have a significant effect compared to baseline in either task. However, corresponding Bayesian analyses provided evidence in favour of the null hypothesis (no condition difference) only for the line reaching task with only ambiguous evidence for the length perception task. The Lackner illusion procedure had a moderate significant effect (with corresponding Bayes- ian evidence) on responses in the perceptual task, but no significant effect could be detected on the line reaching task, with corresponding insensitive Bayesian evidence. Similarly, relative to the sham condition, the Lackner illusion only produced a significant effect on responses in the length perception task, although the Bayesian evidence for this difference was insensitive. Responses in the line reaching task also did not significantly differ between the sham and Lack- ner illusion conditions and the Bayesian evidence was insensitive as to a difference between these conditions. By comparison, relative to both baseline and the sham condition, the hyp- notic suggestion condition yielded reliably strong, significant effects (with corresponding Bayesian evidence) on responses in both tasks. Finally, the hypnotic suggestion condition had a strong significant effect on both tasks compared to the Lackner illusion procedure, with cor- responding Bayesian evidence in favour of a condition difference. Overall, 42 out of 50 (84%) participants had a stronger effect in the hypnotic suggestion condition compared to the Lack- ner illusion procedure in the perceptual task, and 40 (80%) in the line reaching task. Sample description The distribution of hypnotic suggestibility in our sample, as indexed by the WSGC, M = 5.65; SD = 2.4; range: 0–11, was commensurate with the general Francophone population (M = 4.84; SD = 2.15; range: 1–10 [66]). Our sample included 8 low (score 3) and 8 high (score 9) suggestible participants. Body mass index (kg.m-2), MBMI = 22; SD = 3.3; range: [17.4–36.6], was comparable to the general French population, MBMI = 24.8; SD = 5.4 [75]. Social desirability, MSDE = 4.32; SD = 0.97, MIM = 4.63; SD = 0.78, was also commensurate with previously reported samples [64]. Procedure For correlations, we also used default priors, corresponding to a stretched beta distribution with width 1.0 [72]. These analyses were performed using JASP [73]. We set alpha error proba- bility thresholds at .05, with p-values below this threshold interpreted as statistically significant. Bayes Factors (B10) below .33 were interpreted to reflect moderate evidence in favour of the null hypothesis, those greater than 3.0 were taken to provide moderate evidence for the alter- native hypothesis and in-between values were interpreted as being ambiguous [72]. When pos- sible, we report robustness regions (RR) alongside Bayesian analyses by indicating the range of prior widths (either the scale of the Cauchy distribution for t-tests or the stretched Beta distri- bution width for correlations) consistent with the conclusion derived from the Bayes factor (eg. for a Bayes factor B = 10, we report the smallest and largest prior widths for which B>3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 8 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness Hence, no RR are reported for B between 1/3 and 3. Prior width were varied from 0 to 2, which covers the reasonable range of prior distributions. We use p-values, effect sizes, and Bs for interpreting significant effects but only effect sizes and Bs for interpreting non-significant effects. Figures were generated using Python 3, and the matplotlib and seaborn libraries [74]. Data are publicly available on the Open Science Framework (https://osf.io/jxek3/). No part of the study procedures or analyses was pre-registered prior to the research being conducted and thus the analyses can be considered exploratory. Condition differences on perceived finger elongation These results demonstrate that the Lackner illusion manipulation produced a moderate-sized illusion PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 9 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness Fig 3. Raincloud plots of perceptual and pointing effects (mm) as a function of condition (N = 50). Distributions reflect kernel density estimation plots and markers reflect individual measurements. Boxplot whisker limits are at 1.5 interquartile range from the hinges, diamonds represent individual values outside the whiskers. (a) raw data, (b) baseline corrected data. https://doi.org/10.1371/journal.pone.0291493.g003 Fig 3. Raincloud plots of perceptual and pointing effects (mm) as a function of condition (N = 50). Distributions reflect kernel density estimation plots and markers reflect individual measurements. Boxplot whisker limits are at 1.5 interquartile range from the hinges, diamonds represent individual values outside the whiskers. (a) raw data, (b) baseline corrected data. https://doi.org/10.1371/journal.pone.0291493.g003 Fig 3. Raincloud plots of perceptual and pointing effects (mm) as a function of condition (N = 50). Distributions reflect kernel density estimation plots and markers reflect individual measurements. Boxplot whisker limits are at 1.5 interquartile range from the hinges, diamonds represent individual values outside the whiskers. (a) raw data, (b) baseline corrected data. https://doi.org/10.1371/journal.pone.0291493.g003 Table 2. Post hoc comparison of the effect of conditions on responses in the finger length perception task (N = 50). Finger length perception task Mean difference a SE Cohen’s d p b B10 RR Baseline Sham 3.37 4.21 0.11 .425 0.60 [1.407, >2] Lackner illusion 13.95 4.21 0.47 .003 9.48 [0.027, >2] Hypnotic suggestion 35.49 4.21 1.19 < .001 6.73 x 106 [<0.001, >2] Sham Lackner illusion 10.58 4.21 0.36 .026 2.68 [0.105, 0.578] Hypnotic suggestion 32.12 4.21 1.08 < .001 1.55 x 106 [<0.001, >2] Lackner illusion Hypnotic suggestion 21.54 4.21 0.72 < .001 1426 [<0.001, >2] Notes a = means in mm b = Holm-Bonferroni corrected Significant effects are in bold. RR = robustness region. Prior size for reported Bs: p2/2 https://doi.org/10.1371/journal.pone.0291493.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 10 / 22 Table 2. Post hoc comparison of the effect of conditions on responses in the finger length perception task (N = 50). stness region. Prior size for reported Bs: p2/2 PLOS ONE Table 3. Post-hoc comparison of the effect of conditions on response in the line reaching task (N = 50). Line reaching task Mean difference a SE Cohen’s d p b B10 RR Baseline Sham 2.34 4.72 0.07 .68 0.26 [0.52, >2] Lackner illusion 6.86 4.72 0.21 .45 1.94 - Hypnotic suggestion 32.08 4.72 0.96 < .001 3715 [<0.001, >2] Sham Lackner illusion 4.52 4.72 0.14 .68 0.88 - Hypnotic suggestion 29.74 4.72 0.89 < .001 790 [<0.001, >2] Lackner illusion Hypnotic suggestion 25.23 4.72 0.76 < .001 266 [0.002, >2] Notes a = means in mm b = Holm-Bonferroni corrected. Significant effects are in bold. RR = robustness region. Prior size for reported Bs: p2/2 https://doi org/10 1371/journal pone 0291493 t003 le 3. Post-hoc comparison of the effect of conditions on response in the line reaching task (N = 50). = means in mm b = Holm-Bonferroni corrected. Significant effects are in bold. RR = robustness region. Prior size for reported Bs: p2/2 of finger elongation relative to the sham procedure, when indexed with the perceptual effect, but this procedure did not produce robust responses relative to the sham procedure nor reli- able effects in the line reaching task. Hypnotic suggestion, on the other hand yielded reliably strong effects on both tasks relative to baseline, sham, and the Lackner illusion procedure. Our results with the Lackner illusion procedure are in the same range as those of previous experiments [16], or on a different body part such as the arm [15] and nose [13]. For example, in ref. [16] the typical elongation for the subgroup (10 of 30 participants) feeling their finger elongated was about one phalanx (about 2 cm), ref. [15] reported a mean elongation (for the responsive subgroup) of 51.5 mm and 33.3 mm in two experiments, and in ref. [13] the mean elongation for participants reporting an elongation was 44 mm. For the 13 participants clearly responding to the illusion (perceptual elongation greater than 20 mm) the mean elongation was 55 mm. Hence, it appears that the Lackner illusion manipulation produced results similar to previous studies. Nevertheless, we did not observe robust differences relative to the sham condition in the line reaching task. We next sought to assess the potential role of control variables: age, gender, condition order, BMI, hypnotic suggestibility (WSGC), social desirability (self-deceptive enhancement and image management), and familiarity with hypnosis. Notes. Covariates were introduced separately in each model; a = comparison of models including the condition and the covariate to the model only including the condition. Significant effects are in bold. Condition differences on perceived finger elongation Finger length perception task Mean difference a SE Cohen’s d p b B10 RR Baseline Sham 3.37 4.21 0.11 .425 0.60 [1.407, >2] Lackner illusion 13.95 4.21 0.47 .003 9.48 [0.027, >2] Hypnotic suggestion 35.49 4.21 1.19 < .001 6.73 x 106 [<0.001, >2] Sham Lackner illusion 10.58 4.21 0.36 .026 2.68 [0.105, 0.578] Hypnotic suggestion 32.12 4.21 1.08 < .001 1.55 x 106 [<0.001, >2] Lackner illusion Hypnotic suggestion 21.54 4.21 0.72 < .001 1426 [<0.001, >2] on of the effect of conditions on responses in the finger length perception task (N = 50). Table 2. Post hoc comparison of the effect of conditions on responses in the finger length perception task (N = 50). Table 2. Post hoc comparison of the effect of conditions on responses in the finger length perception task (N = 50). PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 10 / 22 Hypnotic suggestion versus sensory modulation of bodily awareness PLOS ONE https://doi.org/10.1371/journal.pone.0291493.g004 Nonetheless, including this covariate in the model did not notably change the foregoing condi- tion effects. All of the covariates yielded Bayesian evidence in favour of the null except age (fin- ger length perception task), gender, familiarity with hypnosis, image management and condition order (line reaching task), for which the evidence was insensitive in discriminating between the null and alternative hypotheses. PLOS ONE We did so by including each variable in turn in the repeated measure ANOVA model as a covariate. As presented in Table 4, only hypnotic suggestibility had a significant effect, and only on responses in the perceptual task. Table 4. Covariate effects in repeated-measures ANOVAs examining condition effects on responses in the finger length perception task and line reaching task (N = 50). Finger length perception task Line reaching task Covariate F p η2 B10 a F p η2 B10 a Age 3.53 .066 .069 0.88 0.02 .896 < .01 0.27 Gender 0.74 .394 .015 0.30 1.09 .301 .022 0.44 BMI 0.56 .457 .012 0.29 0.43 .514 < .01 0.32 Hypnotic suggestibility 8.91 .004 .157 7.35 0.25 .622 < .01 0.31 Familiarity with hypnosis 0.48 .491 < .01 0.27 0.99 .325 .020 0.41 Self-deceptive enhancement 0.84 .365 .017 0.30 0.03 .855 < .01 0.28 Image management 0.08 .784 < .01 0.24 1.70 .198 .034 0.55 Condition order 0.14 .708 < .01 0.24 3.91 .054 .075 1.31 ANOVAs examining condition effects on responses in the finger length perception task and line reaching task Table 4. Covariate effects in repeated-measures ANOVAs examining condition effects on responses in the finger length perception task and line reaching task (N = 50). variate effects in repeated-measures ANOVAs examining condition effects on responses in the finger length perception t Table 4. Covariate effects in repeated-measures ANOVAs examining condition effects on responses in the finger length (N = 50). Table 4. Covariate effects in repeated-measures ANOVAs examining condition effects on responses in the fi (N = 50). PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 11 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness Fig 4. Scatterplots depicting the association between pointing effects and perceptual effects in the sham, Lackner illusion, hypnotic suggestion conditions and at baseline. Lines denote least squares regression slopes. (a) raw data, (b) baseline corrected data. B10 is the Bayes factor for the alternate hypothesis (correlation between variables). p < .01, * p < .001. Fig 4. Scatterplots depicting the association between pointing effects and perceptual effects in the sham, Lackner illusion, hypnotic suggestion conditions and at baseline. Lines denote least squares regression slopes. (a) raw data, (b) baseline corrected data. B10 is the Bayes factor for the alternate hypothesis (correlation between variables). p < .01, * p < .001. Correlations In our next series of analyses, we assessed the correlations between responses in the two tasks in the different conditions. As can be seen in Fig 4, the perceptual and pointing effects were positively and significantly correlated in all conditions. This association was strong in the hyp- notic suggestion condition but moderate in the Lackner illusion and sham conditions (with corresponding Bayesian evidence in favour of a strong positive association in all conditions). These results suggest a moderate to strong association between effects in the two tasks. Finally, we examined whether the observed effects correlated with two measures of social desirability (SDE and IM) (Table 5). These effects were uniformly weak (rs < .19) and in yielded Bayesian evidence in favour of the null hypothesis in all cases except for the effect of hypnotic suggestion on pointing effects for SDE, which was insensitive. Cumulatively, these data strongly suggest the observed effects are unrelated to social desirability. PLOS ONE PLOS ONE Table 5. Pearson correlation coefficients (with p-values and Bayes factors) between social desirability and responses in both tasks for each condition relative to baseline (N = 50). Sham perceptual effect Sham pointing effect Lackner illusion perceptual effect Lackner illusion pointing effect Hypnotic suggestion perceptual effect Hypnotic suggestion pointing effect Self-deceptive enhancement (SDE) r -.03 .11 .14 .12 -.01 .18 95% CI [-.29,.23] [-.17,.37] [-.14,.37] [-.16,.37] [-.27,.25] [-.10,.43] p .83 .46 .34 .42 .96 .20 B10 0.18 0.23 0.27 0.24 0.18 0.39 RR [0.316, >2] [0.506, >2] [0.694, >2] [0.552, >2] [0.304, >2] [1.315, >2] Image management (IM) r .00 .08 .04 .01 -.04 .11 95% CI [-.26,.26] [-.20,.35] [-.23,.29] [-.27,.28] [-.29,.23] [-.17,.37] p .98 .57 .80 .97 .80 .46 B10 0.18 0.21 0.18 0.18 0.18 0.23 RR [0.304, >2] [0.413, >2] [0.321, >2] [0.303, >2] [0.321, >2] [0.506, >2] RR = robustness region. Bs and RR are derived from a stretched beta distribution prior with width 1.0 https://doi org/10 1371/journal pone 0291493 t005 RR = robustness region. Bs and RR are derived from a stretched beta distribution prior with width 1.0 but also that these effects were significantly stronger than the corresponding magnitude of the sensory-driven Lackner illusion [13, 15, 16]. Furthermore, we report a pervasive correlation between perceptual and motor responses across all conditions, suggesting that these measures of body image and body schema are moderately to strongly interrelated. These results demon- strate that hypnotic suggestion is an effective technique for transient modulations of body image and body schema. Discussion This study compared the effects of hypnotic suggestion and the Lackner illusion manipulation on the perceived size of participants’ index finger (body image) and on the way they act with it (body schema). Body metrics comprise relatively low-level properties, which one might have expected to be immune to cognitive penetration. However, we found that not only did hyp- notic suggestion induce illusory finger elongation at both visuospatial and sensorimotor levels, PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 12 / 22 Hypnotic suggestion versus sensory modulation of bodily awareness PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 Theoretical accounts of the effect of hypnotic suggestion The strong effect of the hypnotic suggestion on body schema compared to the Lackner illusion, can be interpreted at least in two different ways. Since the hypnotic suggestion explicitly revealed the intended purpose of the manipulation, it is possible that participants enacted what they interpreted to be the “correct” hypnotic behaviour. This interpretation aligns with some sociocognitive accounts of hypnosis [26, 80] that conceptualize hypnotic behaviour and experi- ence as a culturally devised role and an active process. According to this account, the hypnotic suggestion did not directly alter body representations; rather, participants responded according to their expectations. This interpretation nicely explains the strong correlation between effects in the finger length perception and in the line reaching tasks. Indeed, if participants retained a normal body schema, they might index their pointing on their perceptual effect–i.e., the bias in the line reaching task was intentionally planned and enacted by the participant. According to this interpretation, participants imagined having a longer finger without critical appraisal, reported it in the perceptual task, and then intentionally undershot the targets in the line reach- ing task (with unbiased body schema). In other words, the perceptual effect could be interpreted as reporting imagery, and the pointing effect as adjusting the pointing motion to match the make-believe finger. In support of this interpretation, some highly suggestible individuals use active goal-directed strategies to bring about the suggested effect [81]. According to the predictive processing framework, representations are updated on the basis of their prior probability distribution and new available evidence [82–85]. In our case, the size of the index finger is usually inferred from prior knowledge stored in memory, and (new) sensory evidence. In the Lackner illusion condition, body image is computed based on incorrectly integrating information from illusory proprioceptive signals (arm extension caused by vibrations) and touch. These sensory signals are inconsistent with a normal finger length prior, or in other terms, the probability of having a normal sized finger given the sensory evi- dence is low. Hence, the corresponding finger representation shifts for some participants to an elongated finger on the basis of (misleading) sensory evidence. In contrast to the Lackner illu- sion manipulation, in the case of the hypnotic suggestion, no relevant sensory evidence is pres- ent; rather, the normal finger prior representation retrieved from memory and the elongated finger expectation derived from the suggestion compete based on their relative probabilities. Effects on body image and body schema Although the mechanisms underlying the effects of hypnotic suggestion are still debated, it is widely accepted that they involve top-down regulation [22]. This implies that when relevant sensory evidence was minimized (participants were blindfolded and had no tactile inputs), body image was primarily shaped by high-level information originating from the verbal sug- gestion even though it was incongruent with prior knowledge about the finger size. By con- trast, in the Lackner illusion condition there was no systematic expectation and misleading sensory signals induced by vibrations were present. In this condition, the contribution of prior knowledge was plausibly more predominant for most participants, as reflected in a weaker effect of this procedure relative to the hypnotic suggestion. It can indeed be safer for a cogni- tive system to rely more on the learning history of the organism than on noisy, and sometimes contradictory, sensory inputs. This finding is in line with other studies showing that partici- pants’ perception is shaped more strongly by prior information in contexts of high uncertainty [76]. Accordingly, we interpret the condition difference as a tendency to favour data derived from high level representations (hypnotic suggestion) compared to sensory data (Lackner illu- sion) for updating body metrics. The hypnotic suggestion also had a significant effect on body schema. This effect seemed tightly linked to the effect on body image as the two measurements were strongly correlated. Although the Lackner illusion modified body image, we did not find that it significantly modi- fied body schema. It could be that we failed to observe a significant Lackner illusion effect on body schema because the effect was too noisy, too weak, or it could be that this illusion only PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 13 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness influenced body image. The Bayesian evidence in favour of this effect was insensitive and thus we are unable to determine which of these outcomes led to the current results. Regardless, pre- vious research has observed a dissociation showing that action can be immune to bodily illu- sions in some circumstances [14, 55, 56, 77] (but not in others e.g., [78]). Dissociations like this motivate the conceptual distinction between the two bodily representations [52]. Dissociations, however, are rare. Effects on body image and body schema Their scarcity, even in the clinical literature, can be explained by the fact that these representations normally interact and shape each other [54, 57, 58]. Such interaction can account for the correlation between perceptual and pointing effects in all conditions. In other words, regardless of the condition, variations in body image tend to be followed by corresponding changes in body schema. Our data further revealed a positive correlation between perceptual effects in the hypnotic suggestion and Lackner illusion conditions, as we observed in another experiment [79]. This might reflect a sensitivity to alterations of bodily experiences towards bottom-up and top- down influences. Yet, detailed theoretical accounts integrating these influences on body repre- sentations are lacking. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 Theoretical accounts of the effect of hypnotic suggestion This process may explain why participants responded on a continuum rather than according to a bimodal distribution. In turn, body image is shaped by the weighted average of the two PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 14 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness priors with the weights corresponding to the precision of each (precision-weighting). When the suggested expectancies are unlikely for a participant, as might be the case for a difficult sug- gestion such as visual hallucination, the memory prior exerts greater influence. This yields a small effect and vice versa, if the more precise suggestion prior is heavily weighted. More broadly, beyond the setting of this experiment, the suggested prior could be supported by imagery and selective attention to seemingly confirming evidence while downplaying discon- firming evidence, though the relationship of these variable with response to suggestion is prob- ably complex and controversial (on imagery e.g., ref. [79, 86]; on attention e.g., ref. [87–90]). Further research is needed to stringently test the predictions of this account. Nonetheless, dif- ferential predictions can be derived from sociocognitive and predictive processing accounts in the context of the modulation of body representation through hypnotic suggestion. In particular, in cases wherein the hypnotic suggestion is incongruent with the close coupling of body image and body schema, sociocognitive accounts [26, 80] would predict that suggestion effects would dominate, whereas the predictive processing account would predict that the influence of sugges- tion would be weak. In particular, according to a sociocognitive account, behaviour can be adapted according to the suggestion irrespective of body image-schema coupling. By contrast, according to a predictive processing account, suggestions would have to compete with prior knowledge and thus suggestion effects would plausibly be diminished. For example, if partici- pants were led to believe that the effect of experiencing one’s finger as longer was to point past the lines in the line reaching task (i.e., overshooting rather than undershooting), then sociocognitive accounts would predict a quasi-reversal of the association between perceptual and pointing effects in individuals who form this expectation. By contrast, a predictive processing account only pre- dicts a small reduction of the effect of the hypnotic suggestion due to marginal compliance effects, but overall the same positive association between effects (see ref. [91] for a test). Theoretical accounts of the effect of hypnotic suggestion Lastly, these two views discussed here do not overlap with awareness of intentions, in par- ticular the sociocognitive view does not envision participants as lying or being deceitful. Meta- cognitive theories of hypnosis [92, 93] propose that intentional alteration of responses can occur outside of awareness. One version of this theory [92] maintains that intentions are pre- served during hypnosis whereas high-order thoughts representing these first order intentions are inaccessible, thus resulting in a lack of awareness of one’s own intention (for supporting evidence, see [94, 95]). Regardless of awareness of intentions, which we did not measure in this study, our data suggest that the effect of hypnotic suggestion is not driven by compliance. If this effect were driven by compliance in response to the perceived requirements of the situa- tion, social desirability would be expected to correlate with responses, at least in the hypnotic condition. Insofar as we observed a null correlation for this association, this suggests that our observed effects are unlikely to be driven by compliance, but we cannot rule out the possibility of broader effects related to demand characteristics. Limitations of the study Despite the advances afforded by the present work, we acknowledge some limitations of this study. Firstly, we did not perform a non-hypnotic suggestion condition, nor an induction without suggestion. These would serve as potentially valuable controls for the hypnotic sugges- tion manipulation as they would have allowed us to better dissociate effects attributable to the induction and the suggestion. These conditions were not included because these questions were outside the scope of the present work and because we did not expect an induction to pro- duce spontaneous finger elongation. Insofar as non-hypnotic direct verbal suggestibility is a reliable correlate of hypnotic suggestibility [96, 97], we expect that the present results would generalize to applications involving direct verbal suggestions without a formal induction PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 15 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness procedure (as suggested by ref. [79]). Secondly, we did not measure participants’ expectancies for finger elongation. This would have allowed us to clarify to what extent the observed effects were mediated by this variable. We did not index response expectancies because doing so may provide participants with explicit indicators of how they are expected to respond. Relatedly, indexing response expectancies may introduce a confound wherein participants feel compelled to respond congruently with their expectations (consistency motivation) [98], thereby artifi- cially biasing response patterns towards expectations. It should also be noted that different expectancies in different conditions does not necessarily mean that an effect (condition differ- ence) is driven by expectancies. Nonetheless, future work would benefit from investigating the role of expectancies such as by formally manipulating them in different ways. Thirdly, we included all participants regardless of their responsiveness to the Lackner illusion procedure. This was intentional, as we wanted to assess the general effect of this illusion compared to the impact of hypnotic suggestion on perceived finger elongation. The effect of the illusion was significant on body image. Nonetheless, we observed relatively few participants who unequivo- cally responded to this illusion and as a result we could not perform analyses on this subgroup alone. A comparison of hypnotic suggestion and Lackner illusion manipulations in a homoge- neous group displaying both effects would prove more informative regarding the interplay of bottom-up and top-down effects on body metrics, but the recruitment of such a population would be resource intensive. Limitations of the study Fourth, the hypnotic suggestibility scale we used [66] displayed internal consistency that is marginally acceptable and it suffers from many problems that plague classical hypnotic suggestibility scales [99]. However, this scale is one of the most widely used scales in contemporary experimental hypnosis research [65, 100] and was deemed to the most optimal among French-adapted scales. Fifth, it would have been valuable to conduct sys- tematic phenomenologically-oriented interviews with participants to elucidate the diversity of the experiences in the various conditions [81]. This research approach was not the primary tar- get of our study, but it would be worthwhile to pursue in future research on this topic. Future work should similarly aim to compare and contrast the manipulations used here against other bottom-up manipulations, such as virtual reality (e.g., [7]). Sixth, in the present experiment the Lackner illusion was always preceded by the sham illusion condition. Although this can be seen as a limitation because of the lack of counterbalancing, a fixed order was implemented to avoid the possibility of participants detecting the sham illusion based on their previous experi- ence of the Lackner illusion condition. Importantly, including the sham condition first is likely to provide a more conservative estimate of the experimental manipulations because it should not produce any perceptual distortion of the body (body image). Finally, our conclusion that body image and body schema were altered by the Lackner illu- sion and hypnotic suggestion are based on inverse inference. Caution is advised in interpreting results of behavioural tests as indicators of latent unobservable variables [101]. Nonetheless, our claim that these behavioural tests are credible measures of body image and body schema rests on a wealth of prior data [14, 16, 59–61]. Furthermore, the link between the two body rep- resentations and behavioural measures is definitional. This link would be compromised in cases where participants misunderstood the task (e.g., reporting the preferred size of the finger instead of the perceived size) but no evidence points towards such a conclusion. We are confi- dent that the association between our behavioural indicators and body metrics is sound but provisional, and it will depend on future development of the field. Conclusion Our objective was to compare sensory and cognitive contributions to bodily awareness. We showed a strong effect of hypnotic suggestion on body metrics at both sensorimotor and visuo- spatial levels. This effect was larger than the Lackner illusion manipulation on both levels. Bot- tom-up (Lackner illusion) and top-down (hypnotic suggestion) effects were correlated, suggesting that they both partly index a latent sensitivity of body metrics to modulation. We compared and contrasted two alternative accounts of these effects and outline ways by which they can be discriminated in future research. Clinical significance The results presented here pave the way to future clinical research involving the use of hyp- notic suggestion to modulate body representation. Bodily awareness is believed to be disturbed PLOS ONE \| https://doi.org/10.1371/journal.pone.0291493 September 12, 2023 16 / 22 PLOS ONE Hypnotic suggestion versus sensory modulation of bodily awareness in eating disorders, such as anorexia nervosa [10, 11, 59, 60], and body image flexibility seems to be a valuable predictor of several psychopathological conditions [102, 103]. Hypnosis has demonstrated efficacy as an adjunct to traditional treatments [104–107], in particular in the treatment of psychosomatic disorders [108–111]. Hypnotic suggestibility is also known to be elevated in conditions involving aberrant body representation, such as functional paralysis [112, 113]. However, greater understanding of hypnotic suggestion pathways to modulate bodily awareness could delineate more clearly the areas where hypnosis could be clinically effi- cacious and where its use should be avoided. In particular, future research is necessary to eluci- date the roles of hypnotic suggestibility and body representation flexibility as possible risk or protective factors in psychopathologies involving body awareness. Author Contributions Conceptualization: C. Apelian, F. De Vignemont. Conceptualization: C. Apelian, F. De Vignemont. Conceptualization: C. Apelian, F. De Vignemont. Formal analysis: C. Apelian. Investigation: C. Apelian. Methodology: C. Apelian, D. B. Terhune, F. De Vignemont. Writing – original draft: C. Apelian. Writing – review & editing: C. Apelian, D. B. Terhune, F. De Vignemont. Formal analysis: C. Apelian. Methodology: C. Apelian, D. B. Terhune, F. De Vignemont. Writing – original draft: C. Apelian. Writing – review & editing: C. Apelian, D. B. Terhune, F. De Vignemont. Writing – review & editing: C. Apelian, D. B. Terhune, F. De Vignemont. Writing – review & editing: C. Apelian, D. B. Terhune, F. De Vignemont. 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https://openalex.org/W3011126334	https://europepmc.org/articles/pmc7153263?pdf=render	English	null	The Rational Design and Biological Mechanisms of Nanoradiosensitizers	Nanomaterials	2,020	cc-by	20,595	Received: 13 February 2020; Accepted: 7 March 2020; Published: 11 March 2020 Abstract: Radiotherapy (RT) has been widely used for cancer treatment. However, the intrinsic drawbacks of RT, such as radiotoxicity in normal tissues and tumor radioresistance, promoted the development of radiosensitizers. To date, various kinds of nanoparticles have been found to act as radiosensitizers in cancer radiotherapy. This review focuses on the current state of nanoradiosensitizers, especially the related biological mechanisms, and the key design strategies for generating nanoradiosensitizers. The regulation of oxidative stress, DNA damage, the cell cycle, autophagy and apoptosis by nanoradiosensitizers in vitro and in vivo is highlighted, which may guide the rational design of therapeutics for tumor radiosensitization. Keywords: nanoradiosensitizers; radiosensitization; radiotherapy; biological mechanisms; physicochemical properties gn and Biological Mechanisms tizers and Shumei Zhai 1,* nterface Chemistry of the Ministry of Education, School of Chemistry and g University, Jinan 250100, Shandong, China; wangxiaoling@mail.sdu.edu.cn (X.W.) Light Industry, Zibo 255300, Shandong, China du.cn; Tel.: +86-531-8836-4464 d: 7 March 2020; Published: 11 March 2020 s been widely used for cancer treatment. However, the intrinsic toxicity in normal tissues and tumor radioresistance, promoted zers. To date, various kinds of nanoparticles have been found ncer radiotherapy. This review focuses on the current state of the related biological mechanisms, and the key design strategies ers. The regulation of oxidative stress, DNA damage, the cell cycle, noradiosensitizers in vitro and in vivo is highlighted, which may peutics for tumor radiosensitization. rs; radiosensitization; radiotherapy; biological mechanisms; threats to human health [1]. Despite the great advances in cancer recent years, the survival rate of cancer patients has only slightly arious methods, such as surgery, chemotherapy, and radiotherapy, tment [3,4]. Among these methods, radiotherapy is based on destroy cancer cells and can be used alone or combined with other backs of radiotherapy, such as toxic side eﬀects to the human body cells [7–9], promote the investigation of radiosensitizers, which can t eﬀect of radiotherapy. ysicochemical properties, nanoparticles (NPs) are widely used in mer product, energy, and biomedical ﬁelds [10–13]. Various kinds of ancer cells to radiotherapy due to their intrinsic radiosensitive action nd siRNAs [14]. Moreover, in vitro and in vivo experiments have osensitization eﬀects by regulating multiple biological mechanisms, mage, cell cycle arrest, apoptosis, autophagy, and hypoxia-related ze the recent progress made in NPs-induced radiosensitization gn and Biological Mechanisms tizers and Shumei Zhai 1,* nterface Chemistry of the Ministry of Education, School of Chemistry and g University, Jinan 250100, Shandong, China; wangxiaoling@mail.sdu.edu.cn (X.W.) Light Industry, Zibo 255300, Shandong, China du.cn; Tel.: +86-531-8836-4464 d: 7 March 2020; Published: 11 March 2020 s been widely used for cancer treatment. However, the intrinsic toxicity in normal tissues and tumor radioresistance, promoted zers. To date, various kinds of nanoparticles have been found ncer radiotherapy. This review focuses on the current state of the related biological mechanisms, and the key design strategies ers. The regulation of oxidative stress, DNA damage, the cell cycle, noradiosensitizers in vitro and in vivo is highlighted, which may peutics for tumor radiosensitization. rs; radiosensitization; radiotherapy; biological mechanisms; threats to human health [1]. Despite the great advances in cancer recent years, the survival rate of cancer patients has only slightly arious methods, such as surgery, chemotherapy, and radiotherapy, tment [3,4]. Among these methods, radiotherapy is based on destroy cancer cells and can be used alone or combined with other backs of radiotherapy, such as toxic side eﬀects to the human body cells [7–9], promote the investigation of radiosensitizers, which can t eﬀect of radiotherapy. ysicochemical properties, nanoparticles (NPs) are widely used in mer product, energy, and biomedical ﬁelds [10–13]. Various kinds of ancer cells to radiotherapy due to their intrinsic radiosensitive action nd siRNAs [14]. Moreover, in vitro and in vivo experiments have osensitization eﬀects by regulating multiple biological mechanisms, mage, cell cycle arrest, apoptosis, autophagy, and hypoxia-related ze the recent progress made in NPs-induced radiosensitization nanomaterials nanomaterials Hainan Sun 1,2, Xiaoling Wang 1 and Shumei Zhai 1,* 1 Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education, Scho Chemical Engineering, Shandong University, Jinan 250100, Shandong, China; sunhainan1986@126.com (H.S.); wangxiaoling@mail.sdu.edu.cn (X.W.) 2 Shandong Vocational College of Light Industry, Zibo 255300, Shandong, China * Correspondence: smzhai@sdu.edu.cn; Tel.: +86-531-8836-4464 1 Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education, Sc Chemical Engineering, Shandong University, Jinan 250100, Shandong, China; sunhainan1986@126.com (H.S.); wangxiaoling@mail.sdu.edu.cn (X.W.) 2 Shandong Vocational College of Light Industry, Zibo 255300, Shandong, China * Correspondence: smzhai@sdu.edu.cn; Tel.: +86-531-8836-4464 Nanomaterials 2020, 10, 504; doi:10.3390/nano10030504 The Rational Design and Biological Mechanisms of Nanoradiosensitizers Hainan Sun 1,2, Xiaoling Wang 1 and Shumei Zhai 1,* Hainan Sun 1,2, Xiaoling Wang 1 and Shumei Zhai 1,* 1. Introduction Cancer is one of the leading threats to human health [1]. Despite the great advances in cancer biology and clinical treatment in recent years, the survival rate of cancer patients has only slightly improved in recent decades [2]. Various methods, such as surgery, chemotherapy, and radiotherapy, are used clinically in cancer treatment [3,4]. Among these methods, radiotherapy is based on high-energy ionizing radiation to destroy cancer cells and can be used alone or combined with other therapies [5,6]. The intrinsic drawbacks of radiotherapy, such as toxic side eﬀects to the human body and radiation resistance in cancer cells [7–9], promote the investigation of radiosensitizers, which can signiﬁcantly enhance the treatment eﬀect of radiotherapy. Due to their outstanding physicochemical properties, nanoparticles (NPs) are widely used in various ﬁelds, including the consumer product, energy, and biomedical ﬁelds [10–13]. Various kinds of NPs have been found to sensitize cancer cells to radiotherapy due to their intrinsic radiosensitive action and loading capacity for drugs and siRNAs [14]. Moreover, in vitro and in vivo experiments have demonstrated that NPs exhibit radiosensitization eﬀects by regulating multiple biological mechanisms, such as oxidative stress, DNA damage, cell cycle arrest, apoptosis, autophagy, and hypoxia-related mechanisms [15–17]. In this review, we summarize the recent progress made in NPs-induced radiosensitization by modulating multiple biological mechanisms. Hypoxia-related radiosensitive eﬀects have been systematically reviewed by Li and Zhang et al. [18,19] and, therefore, are not addressed in this review. The regulation of crucial cellular processes by the physicochemical properties of NPs in cancer cells is reviewed, which may guide the design of nanoradiosensitizers in the future. Nanomaterials 2020, 10, 504; doi:10.3390/nano10030504 www.mdpi.com/journal/nanomaterials www.mdpi.com/journal/nanomaterials Nanomaterials 2020, 10, 504 2 of 30 2. Oxidative Stress Reactive oxygen species (ROS) are generated from oxygen in physiological environments mainly in mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In the mitochondrial respiratory chain, leaked electrons are captured by oxygen, resulting in ROS generation [20]. The activation of NADPH oxidase in the cell membrane also leads to the catalyzed transformation of oxygen into ROS [21]. In addition to these two intrinsic pathways, high-energy ionizing radiation can also elicit ROS generation by directly reacting with water and/or oxygen molecules [22]. The accumulation of ROS in cells leads to oxidative stress [23]. In this section, we summarize the radiosensitization eﬀect of NPs based on this oxidative stress mechanism (Table 1) and the regulation of oxidative stress in cancer cells by the physicochemical properties of NPs. Table 1. Summary of nanoradiosensitizers based on oxidative stress mechanism. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Gold 20 Polyethylene glycol (PEG) MDA-MB-231 2, 4, 6, 8, 10 Gy; 320 kVp Increased cytotoxicity [24] Gold 10 Amino-PEG A549 25 keV/µm protons or 225 kV X-rays 1.14 (protons), 1.22 (X-rays) [25] Gold 2 Levonorgestrel EC1 cells/EC1 tumor-bearing nude mice 4 Gy Increased cytotoxicity [26] Gold 3 Histidine U14 6 Gy 1.54 [27] Gold 14 Thio-glucose SK-OV-3 90 kVp, 6 MV Increased cytotoxicity [28] Gold 6 Octaarginine (R8) LS180 6 MV X-rays, 0–10 Gy 1.59 [29] Gold 3 Mitochondria-targeting peptide (CCYKFR) MCF-7 4 Gy 1.31 [30] Gold-TiO2 18 Triphenylphosphine (TPP) MCF-7/4T1 tumor-bearing Balb/c mouse 4, 6 Gy Tumor volume inhibition [31] Gold 54 HS-PEG-CH3, HS-PEG-NH2, HS-PEG-folate (FA), cell-penetrating peptide TAT KB/U14 tumor-bearing mice 4 Gy TAT > FA > NH2 > CH3 [32] Gold 2 Glutathione (GSH) MCF-7 2.25 Gy Reduced survival rates [33] Gold GNPs (53), gold nanospikes (GNSs, 54), gold nanorods (GNRs, 50 × 12) PEG KB 4 Gy 1.62 (GNPs), 1.37 (GNSs), 1.21 (GNRs) [34] Fe3O4-Gold 12.5 1-methyl-3-(dodecylphosphonic acid) imidazolium bromide MCF-7, A549 1, 2, 3 Gy Reduced survival rates [35] α-Fe2O3@Au 49 Dopamine 4T1 6 Gy Suppressed tumor growth [36] Au@Se 120 Positively-charged chitosan A375 4 Gy Suppressed tumor growth [37] TiO2-gold 70.1 PEG SUM159 5, 10 Gy Suppressed tumor growth [27] Silver N.A. N.A. HepG2 6 Gy 1.98 [38] Table 1. Summary of nanoradiosensitizers based on oxidative stress mechanism. 3 of 30 Nanomaterials 2020, 10, 504 Table 1. Cont. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. 2. Oxidative Stress Silver 15 Polyvinylpyrrolidone U251 4 Gy Increased cell death [39] Silver 20–30 Polyvinylpyrrolidone MDA-MB-231 0–4 Gy Increased cell death [40] Iron oxide 10 Cetuximab U87MGEGFRvIII 10 Gy Increased cell death [41] Ferrocene 75 PEG 4T1 4 Gy Increased cell death [42] Selenium 27.5 GSH MCF-7 0–8 Gy Increased cell death [29] Selenium 200 Chitosan, transferrin C6, A375 2 Gy Increased cell death [43] Selenium <40 PEG HeLa 8 Gy Increased cell death [44] Gadolinium oxide 3 N.A. A549, NH1299, NH1650 2 Gy Increased cell death [45] Gd2O3, CeO2-Gd <100 N.A. U-87MG 3 Gy Increased cell death [46] Gd-doped titania 20 4-carboxybutyl triphenylphosphonium bromide MCF-7 4 Gy 1.7 [47] Cerium oxide N.A. N.A. L3.6pl, Panc1 5 Gy Increased cell death [48] CuO 5.4 N.A. MCF-7 cells, U14 tumor-bearing nude mice 0–8 Gy Increased cell death [49] Silicon <5 2-methyl 2-propenoic acid methyl ester rat glioma C6 cells 0–3 Gy Promoted ROS production [50] Silicon N.A. NH2 MCF-7 3 Gy Promoted ROS production [51] C60 90–100 N.A. B16, SMMU-7721 0–10 Gy Increased cell death [52] Hydrogenated nanodiamonds 16 N.A. Caki-1, ZR75.1S, ZR75.1R 4 Gy Increased cell death [53] MWCNTs loaded with ruthenium polypridyl complex Diameter: 225 NH2 R-HepG2 8 Gy Increased cell death [54] Abbreviations: DEF, dose enhancement factor; SER, sensitization enhancement ratio; N.A., not available; Ref., references. 2.1. Nanoradiosensitizers Based on Oxidative Stress Gold-based nanomaterials have been extensively investigated as a new type of NP-based radiosensitizer involved in cancer radiotherapy. Gold nanoparticles (GNPs) with various decorations can sensitize cancer cells to radiation through the induction of oxidative stress. For example, both polyethylene glycol (PEG)-functionalized GNPs (20 nm) and amino-PEG-functionalized GNPs (10 nm) radiosensitized cancer cells through the induction of oxidative stress [24,25]. Under X-ray irradiation, gold-levonorgestrel nanoclusters (2 nm) consisting of Au8(C21H27O2)8 (Au8NC) with bright luminescence (58.7% quantum yield) and satisfactory biocompatibility promoted the production of ROS and induced cytotoxicity in human esophageal squamous cancer cells (EC1 )cells and EC1 tumor-bearing nude mice [26]. The overexpression of glutathione (GSH) in tumors impaired these radiotherapy eﬀects. Histidine-capped gold nanoclusters (Au NCs@His) (3 nm) improved the radiotherapy eﬀects by depleting intracellular GSH in U14 cells [27]. 4 of 30 Nanomaterials 2020, 10, 504 Enhancement of cellular uptake is beneﬁcial to the radiosensitization eﬀects induced by GNPs. For example, thioglucose decoration on GNPs (14 nm) increased cellular uptake by approximately 31% in human ovarian cancer cells (SK-OV-3) compared with the uptake of naked GNPs, with the former inducing elevated levels of ROS production and enhanced radiotherapy [28]. The modiﬁcation of GNP-PEG with positively charged cell-penetrating peptides signiﬁcantly increased GNP internalization and ROS production in the LS180 colorectal cancer cell line, leading to enhanced radiosensitivity [29]. Mitochondria-targeting NPs exhibited excellent radiosensitization eﬀects. Peptide (CCYKFR)-templated Au nanoclusters (AuNCs, 3 nm) eﬃciently targeted mitochondria in human breast cancer MCF-7 cells. CCYKFR–AuNCs irradiated by 4 Gy of X-rays elicited a burst of mitochondrial ROS, resulting in cancer cell death (Figure 1) [30]. In another study, a mitochondria-targeted nanoradiosensitizer was constructed by covalently attaching mitochondria targeting triphenylphosphine (TPP) groups to TiO2-Au NPs. When irradiated with X-rays, TiO2-Au-TPP NPs (18 nm) triggered ROS production in mitochondria, caused a decrease in mitochondrial membrane potential, and induced the death of MCF-7 cells. In a MCF-7 and 4T1 tumor-bearing Balb/c mouse model, TiO2-Au-TPP dramatically improved the therapeutic eﬃcacy of radiotherapy [31]. Nanomaterials 2020, 10, 504 5 of 33 Figure 1. Enhanced radiosensitivity of cancer cells by gold nanoclusters (AuNCs) with mitochondria targeting. Peptide-templated AuNCs were synthesized through a green synthetic route, featured with highly efficient co-localization onto mitochondria after endocytosis. Under 4 Gy X-ray irradiation, peptide (CCYKFR)–AuNCs can introduce the burst of mitoROS and cell death. Reproduced with permission [30]. Copyright The Royal Society of Chemistry, 2017. Figure 1. 2.1. Nanoradiosensitizers Based on Oxidative Stress Enhanced radiosensitivity of cancer cells by gold nanoclusters (AuNCs) with mitochondria targeting. Peptide-templated AuNCs were synthesized through a green synthetic route, featured with highly eﬃcient co-localization onto mitochondria after endocytosis. Under 4 Gy X-ray irradiation, peptide (CCYKFR)–AuNCs can introduce the burst of mitoROS and cell death. Reproduced with permission [30]. Copyright The Royal Society of Chemistry, 2017. Figure 1. Enhanced radiosensitivity of cancer cells by gold nanoclusters (AuNCs) with mitochondria targeting. Peptide-templated AuNCs were synthesized through a green synthetic route, featured with highly efficient co-localization onto mitochondria after endocytosis. Under 4 Gy X-ray irradiation, peptide (CCYKFR)–AuNCs can introduce the burst of mitoROS and cell death. Reproduced with permission [30]. Copyright The Royal Society of Chemistry, 2017. Figure 1. Enhanced radiosensitivity of cancer cells by gold nanoclusters (AuNCs) with mitochondria targeting. Peptide-templated AuNCs were synthesized through a green synthetic route, featured with highly eﬃcient co-localization onto mitochondria after endocytosis. Under 4 Gy X-ray irradiation, peptide (CCYKFR)–AuNCs can introduce the burst of mitoROS and cell death. Reproduced with permission [30]. Copyright The Royal Society of Chemistry, 2017. Figure 1. Enhanced radiosensitivity of cancer cells by gold nanoclusters (AuNCs) with mitochondria targeting. Peptide-templated AuNCs were synthesized through a green synthetic route, featured with highly efficient co-localization onto mitochondria after endocytosis. Under 4 Gy X-ray irradiation, peptide (CCYKFR)–AuNCs can introduce the burst of mitoROS and cell death. Reproduced with permission [30]. Copyright The Royal Society of Chemistry, 2017. Figure 1. Enhanced radiosensitivity of cancer cells by gold nanoclusters (AuNCs) with mitochondria targeting. Peptide-templated AuNCs were synthesized through a green synthetic route, featured with highly eﬃcient co-localization onto mitochondria after endocytosis. Under 4 Gy X-ray irradiation, peptide (CCYKFR)–AuNCs can introduce the burst of mitoROS and cell death. Reproduced with permission [30]. Copyright The Royal Society of Chemistry, 2017. The surface physicochemical properties of GNPs can modulate this radiosensitization effect. Gold nanospikes (GNSs, 54 nm) were decorated with different ligands, including HS-PEG-CH3 (GNSs), HS-PEG-NH2 (NH2-GNSs), HS-PEG-folate (FA-GNSs), and cell-penetrating peptide TAT (TAT-GNSs). These GNSs exhibited the following radiosensitization effects in human oral squamous KB cells in descending order of magnitude: TAT-GNSs > FA-GNSs > NH2-GNSs > GNSs; these effects are correlated dosely with their levels of cellular uptake. Among these GNSs, the sensitization enhancement ratio (SER) for the TAT-GNSs reached 2.30. The radiosensitization mechanisms of these GNSs involved increased ROS generation and mitochondrial depolarization. 2.1. Nanoradiosensitizers Based on Oxidative Stress Radiotherapy of U14 tumor-bearing mice in vivo demonstrated that TAT-GNSs exhibited the best radiosensitization effect [32]. Zwitterionic glutathione-coated AuNPs (GS-AuNPs) sensitized MCF-7 cells to radiation delivered with a clinically appropriate megavoltage photon beam at a low dose of approximately 2.25 The surface physicochemical properties of GNPs can modulate this radiosensitization eﬀect. Gold nanospikes (GNSs, 54 nm) were decorated with diﬀerent ligands, including HS-PEG-CH3 (GNSs), HS-PEG-NH2 (NH2-GNSs), HS-PEG-folate (FA-GNSs), and cell-penetrating peptide TAT (TAT-GNSs). These GNSs exhibited the following radiosensitization eﬀects in human oral squamous KB cells in descending order of magnitude: TAT-GNSs > FA-GNSs > NH2-GNSs > GNSs; these eﬀects are correlated dosely with their levels of cellular uptake. Among these GNSs, the sensitization enhancement ratio (SER) for the TAT-GNSs reached 2.30. The radiosensitization mechanisms of these GNSs involved increased ROS generation and mitochondrial depolarization. Radiotherapy of U14 tumor-bearing mice in vivo demonstrated that TAT-GNSs exhibited the best radiosensitization eﬀect [32]. Zwitterionic glutathione-coated AuNPs (GS-AuNPs) sensitized MCF-7 cells to radiation delivered with a clinically appropriate megavoltage photon beam at a low dose of approximately 2.25 Gy, while PEG decoration 5 of 30 Nanomaterials 2020, 10, 504 exhibited protective eﬀects against radiation. The ligand eﬀect on radiosensitization is thought to be independent of cellular uptake but may be related to ROS regulation [33]. The shape of gold (Au) nanomaterials can also regulate the eﬃciency of radiosensitization. GNPs (53 nm), GNSs (54 nm), and gold nanorods (GNRs, 50 nm × 12 nm) with the same PEG coatings exhibiting diﬀerent radiosensitization eﬀects in KB cells, the eﬀects of which followed the same order as cellular uptake level (GNPs > GNSs > GNRs). The radiosensitization mechanisms involve ROS production, with GNPs inducing the highest ROS and cytotoxicity levels under irradiation [34]. p g g y y The hybridization of GNPs with other NPs also exhibited excellent radiosensitization eﬀects. Fe3O4-Au nanoparticles (Fe3O4-Au pNPs, 12.5 nm) signiﬁcantly enhanced ROS levels in MCF-7 and A549 cells and sensitized both kinds of cells to X-rays. However, in MCF-10A noncancer cells, Fe3O4-Au pNPs only slightly promoted the production of ROS and ultimately failed to exhibit a radiosensitization eﬀect [35]. α-Fe2O3 coated with ultrasmall gold nanoseeds (α-Fe2O3@Au, 49 nm) sensitized 4T1 cells to radiotherapy through ROS generation. Near-infrared (NIR) treatment induced ablation of α-Fe2O3 and the aggregation of GNPs of approximately 13 nm, which was the optimal size for ROS production and radiotherapy [36]. 2.1. Nanoradiosensitizers Based on Oxidative Stress GNRs (61 nm × 17 nm) encapsulated with Se shells (Au@Se-R/A NCs, R/A: arginine-glycine-aspartic acid (RGD)/activatable cell-penetrating peptide (ACPP), 120 nm) were used as sensitizers in radiochemotherapy by synergizing the radiosentization eﬀects of GNRs and the anticancer eﬀects of the Se shells. The radiosentization mechanism of the Au@Se-R/A NCs involved ROS overproduction [37]. Dumbbell-like Au-TiO2 NPs (DATs, 70.1 ± 4.9 nm) showed a synergistic therapeutic eﬀect on radiation therapy, mainly because of strong asymmetric electric coupling of the metals with high atomic number, and the dielectric oxides at their interfaces. DATs signiﬁcantly sensitized SUM159 triple-negative breast cancer (TNBC) cells to X-rays through oxidative stress and reduced tumor volume in SUM159-tumor-bearing mice, increasing their median survival [27]. In addition to gold-based NPs, other kinds of NPs can enhance radiation sensitivity. AgNPs induced radiation sensitivity by decreasing the levels of catalase (CAT), superoxide dismutase (SOD), and total GSH in human hepatocellular carcinoma HepG2 cells [38]. The induction of ROS played an essential role in the radiosensitization of AgNPs (15 nm) in human U251 glioma cells [39]. TNBC cells are more vulnerable to reagents that elicit oxidative stress than are non-TNBC cells. AgNPs (20–30 nm) were found to induce higher levels of oxidative damage in TNBC cells than they did in non-TNBC cells, leading to the reduction of TNBC growth and improvement of radiation therapy [40]. Iron-based NPs can also sensitize cancer cells to radiation through an oxidative stress mechanism. For example, magnetic iron-oxide NPs (IONPs, 10 nm) decorated with the epidermal growth factor receptor (EGFR) antibody cetuximab were targeted EGFRvIII-overexpressing glioblastoma (GBM) cells. A signiﬁcant antitumor eﬀect was found in vitro after treatment with cetuximab-IONPs and ionizing radiation. Moreover, in vivo experiments indicated that the overall survival of nude mice was signiﬁcantly increased after the combination treatment described above was administered to them. These radiosensitization mechanisms involve ROS production [41]. The spherical aggregates of PEGylated ferrocene (Fc-PEG, 75 nm) sensitized 4T1 cells to X-rays through the enhanced generation of ROS [42]. SeNPs (27.5 nm) induced both endogenous and radiation-induced ROS formation in MCF-7 cells under irradiation [29]. The surface decoration regulated the radiosensitization eﬀects of SeNPs. Speciﬁcally, SeNPs (200 nm) decorated with chitosan and transferrin signiﬁcantly enhanced the radiotherapy eﬀects of 125I seeds through the activation of ROS production and p53-mediated apoptotic pathways in C6 and A375 cells [43]. 2.1. Nanoradiosensitizers Based on Oxidative Stress In another study, PEG-SeNPs sensitized HeLa cells to radiotherapy through an oxidative stress mechanism. However, polyvinylpyrrolidone (PVP)-SeNPs did not exhibit radiosensitization eﬀects [44]. A series of metal oxide NPs exhibited radiosensitization eﬀects based on an oxidative stress mechanism. For example, gadolinium oxide NPs (3 nm) induced hydroxyl radical production and oxidative stress in a dose- and concentration-dependent manner under X-ray irradiation in non-small-cell lung cancer (NSCLC) cells [45]. Gd2O3 and CeO2-Gd NPs (<100 nm) sensitized U-87 6 of 30 Nanomaterials 2020, 10, 504 MG cells to radiation through induced oxidative stress [46]. Gd-doped titania NPs ((TiO2(Gd) NPs), 20 nm) decorated with 4-carboxybutyl triphenylphosphonium bromide (TPP) targeted mitochondria in MCF-7 cells. TiO2(Gd) NPs boosted ROS production in mitochondria under X-ray irradiation and greatly ampliﬁed the antitumor eﬃcacy of radiotherapy [47]. Cerium oxide NPs increased ROS production in pancreatic cancer cells, resulting in the activation of thioredoxin 1 (TRX1)-apoptosis signaling kinase 1 (ASK1)-c-Jun terminal kinase (JNK) (TRX1-ASK1-JNK) redox-sensing pathway and apoptosis [48]. In contrast, another work showed that cerium nanoparticles (CNPs) exhibited higher SOD-mimetic activity, which could be modulated by the change in the anion of the precursor salt [55]. CuO NPs (5.4 nm) signiﬁcantly increased oxidative stress levels, resulting in radiosensitization eﬀects in MCF-7 cells and U14 tumor-bearing nude mice [49]. SiNPs (<5 nm) signiﬁcantly enhanced ROS production in rat glioma C6 cells under X-ray irradiation. In the absence of SiNPs, ROS levels were not enhanced upon X-ray irradiation [50]. Positively charged NH2-SiNPs penetrated the mitochondrial membrane and signiﬁcantly increased intracellular ROS levels in MCF-7 cells under X-ray irradiation; however, uncapped SiNPs did not increase intracellular ROS levels [51]. Carbon-based NPs can also induce radiosensitization eﬀects based on an oxidative stress mechanism. For example, the combination of nano-C60 (90–100 nm) administration and 60Co γ irradiation induced higher ROS levels and enhanced cytotoxicity in B16 and human hepatocellular carcinoma SMMU-7721 cells than did 60Co treatment alone [52]. Hydrogenated nanodiamonds (16 nm) with a positive charge enhanced the radiotherapy eﬀects in three radioresistant cancer cell lines (Caki-1, ZR75.1S, ZR75.1R). These radiosensitization mechanisms involved oxidative stress [53]. NH2-decorated multiwalled carbon nanotubes (NH2-MWCNTs) were loaded with ruthenium polypridyl complex (RuPOP@MWCNTs) via π-π and hydrogen bond interactions. The positive charge on the MWCNTs promoted NP cellular uptake into cancer cells. RuPOP@MWCNTs signiﬁcantly enhanced the radiation eﬀects of clinically appropriate X-ray irradiation of drug-resistant R-HepG2 cells through an oxidative stress mechanism [54]. 2.2. The Impact of the Nanoparticles’ (NP) Physicochemical Properties on Oxidative Stress Generally, the size of NPs is negatively correlated with the oxidative stress level induced in cancer cells. Triphenylphosphine monosulfonate (TPPMS)-GNPs (1.4 nm) induced higher ROS levels than their 15 nm-sized counterparts in HeLa cells [56]. GNPs of diﬀerent sizes (30, 50, 90 nm) regulated oxidative stress levels in HL-60 and HepG2 cells, with GNPs of 30 nm treatment resulting in the lowest GSH level, followed by that induced by GNPs of 50 nm and 90 nm [57]. GNPs of 5 nm elicited the highest ROS level in HepG2 and L02 cells, followed by 20 nm- and 50 nm-sized GNPs [58]. The intracellular ROS level induced by PEG-GNPs (6.2–61.2 nm) was also negatively correlated with NP size in HepG2 and HeLa cells [59]. In addition to GNPs, NPs of other sizes were also negatively correlated with oxidative stress levels in cancer cells, such as silica NPs [60,61], AgNPs [62], PVP-AgNPs [63] and Cu2-xSe NPs [64]. The shape of NPs can regulate oxidative stress levels in cancer cells. Rod-like NPs induce higher levels of ROS in cells than do spherical NPs [65,66], while octahedral-shaped Cu2O NPs induce higher ROS levels and cytotoxicity than hexagonal or cubic Cu2O NPs [67]. The surface charge of NPs can regulate the oxidative stress level in cancer cells. We and other researchers have found that the intracellular ROS level is positively correlated with positively charged NPs, such as GNPs [68–70], polyethylenimine (PEI)-decorated GNRs [71], single-walled carbon nanotubes (SWCNTs) [72], ZnO NPs (98 nm, 11.1 mV) [73], upconversion (UCNP)@SiO2 NPs with NH2 decoration (37 nm, 26.2 mV) [74], and glucose-decorated iron oxide NPs (70 nm) [75]. In addition, we found that, in A549 cells, hydrophobic GNPs are more likely to induce oxidative stress than are hydrophilic NPs [68]. Furthermore, the length of the hydrophobic moieties in positively charged ligands was found to be positively correlated with intracellular ROS levels induced in HeLa cells treated with GNPs (2 nm) [76]. 7 of 30 7 of 30 Nanomaterials 2020, 10, 504 3. DNA Damage DNA in cells continually incurs various types of damage, and cells have devised ingenious mechanisms to repair DNA damage [77]. DNA damage leads to many kinds of diseases, including cancer [78]. However, DNA damage induced by radiation also plays key roles in cancer treatment [79]. In this section, we summarize the radiosensitization eﬀect of NPs on DNA damage mechanisms (Table 2). The regulation of DNA damage in cancer cells by physicochemical properties of NPs is also summarized because it is beneﬁcial to the design of nanoradiosensitizers. Table 2. Summary of nanoradiosensitizers based on DNA damage mechanism. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Gold 2 N.A. MDA-MB-231 2 Gy Enhanced DNA damage [80] Gold 12 PEG U251 4 Gy (in vitro), 20 Gy (in vivo) 1.3 [81] Gold and superparamagnetic iron oxide nanoparticles (SPION)-loaded micelles 100 Dodecanethiol, oleic acid U251, U373 4 Gy Enhanced DNA damage [82] Au@Se 120 Positively-charged chitosan A375 4 Gy Suppressedtumor growth [37] Nanogel containing GNPs N.A. PEG SCCVII 15 Gy Increased cell death [83] Gold 4.5 DNA U251MG-P1 1–5 Gy Increased cell death [84] Gold 50 Cisplatin GBM cell lines (S2) 10 Gy Inhibited cell proliferation [85] Gold 30 Herceptin SK-BR-3 0–1 Gy Enhanced DNA damage [86] Gold 30 Herceptin MDA-MB-361 0–7 Gy 1.6 [87] Gold 5 Doxorubicin HeLa 3 Gy Increased cell death [88] Gold 18 Chitosan, Doxorubicin MCF-7 0.5, 1, 3 Gy Increased cell death [89] Gold 8 Goserelin, PEG PC3 4 Gy Increased cell death [90] Gold 14 Polyethylenimine A712 80 mGy/min Increased cell death [91] Gold <2 GSH, BSA HeLa cells and U14 tumor-bearing nude mice 0–8 Gy 1.3 (GSH), 1.21 (BSA) [92] Silver 20–30 Polyvinylpyrrolidone MDA-MB-231 0–4 Gy Increased cell death [40] Silver 20 Epidermal growth factor receptor-speciﬁc antibody nasopharyngeal carcinoma epithelial (CNE) cells 0, 2, 4, 6, 8 Gy 1.4 [93] Iridium <5 RGD, transactivator of transcription (TAT) 4T1 4, 6, 8 Gy Increased cell death [94] Table 2. Summary of nanoradiosensitizers based on DNA damage mechanism. 8 of 30 Nanomaterials 2020, 10, 504 Table 2. Cont. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Gadolinium-doped ZnO 9 N.A. SKLC-6 0–8 Gy 1.47 (10 µg/mL), 1.61 (20 µg/mL) [95] Iron-oxide 10 Oleic acid WEHI-164 2 Gy Inhibited cell proliferation [96] SPION 4–6 Chitosan, PEG, PEI, siRNA UW228, Res196 2 Gy Increased cell death [97] Thulium oxide 40–45 N.A. 3. DNA Damage 9L gliosarcoma 0–8 Gy 1.32 [98] Tantalum oxide 80 Doxorubicin 4T1 0–6 Gy Increased cell death [99] ZnO 7 N.A. SKLC-6 2 Gy 1.23 (10 µg/mL), 1.31 (20 µg/mL) [100] Polymeric NPs containing camptothecin 20–30 N.A. HT-29 0–6 Gy 2.2 [101] Polymer NPs loaded with JNK inhibitor <100 N.A. Lewis lung carcinoma (LLC) cells 0–10 Gy Suppressedtumor growth [102] PLGA NPs containing DNA double-strand repair inhibitors 87 PEG H460 0–8 Gy Increased cell death [74] PEI carrying DSB bait 140 Folate PC-3, 22Rv1 0–8 Gy Increased cell death [103] BSA NPs loaded organic selenocompound 255 Folate HeLa 8 Gy Increased cell death [104] Abbreviations: DEF, dose enhancement factor; SER, sensitization enhancement ratio; N.A., not available; Ref., references. 3.1. Nanoradiosensitizers Based on DNA Damage The interaction between GNPs and DNA regulates radiosensitization eﬀects. GNPs (<5 nm) can bind to plasmid DNA through electrostatic interactions, resulting in DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) under irradiation [105]. The distance between GNPs and DNA can aﬀect the radiosensitization eﬀect because GNPs with the shortest possible linker induce the greatest radiosensitization eﬀect [106]. GNP-induced DNA damage contributes to the radiosensitization eﬀect on cells. GNPs (2 nm) sensitized MDA-MB-231 breast cancer cells to radiation through the induction of DNA damage [80]. PEG-decorated GNPs (12 nm) increased cellular DNA damage under irradiation in human GBM-derived cell lines and enhanced the survival of orthotopic GBM tumor-bearing mice [81]. Gold and superparamagnetic iron oxide nanoparticles (SPION)-loaded micelles (GSMs, 100 nm) in combination with radiotherapy led to an ~2-fold increase in DSBs in GBM cells [82]. The combination of Au@Se NPs (120 nm) treatment with irradiation induced DNA damage by enhancing ROS generation and the phosphorylation levels of related proteins, including Ataxia-telangiectasia mutated (ATM), Checkpoint kinase 2 (Chk2), Breast cancer susceptibility protein 1 (BRCA1), p53 and histones [37]. The inhibition of DNA repair induced by GNPs also contributes to the radiosensitization eﬀect. For example, homologous recombination (HR) and non-homologous end joining (NHEJ) are intrinsic DSB repair pathways. PEGylated nanogels containing GNPs inhibited the expression of HR and NHEJ-related proteins, including Rad51 and Ku70, resulting in the suppression of the repair of radiation-induced DSBs in murine squamous carcinoma SCCVII cells [83]. DNA-GNPs (4.5 nm) eﬀectively abrogated the repair of radiation-induced DNA DSBs and sensitized U251MG-P1 cancer stem cell-like cells to radiotherapy through the induction of mitotic catastrophe [84]. 9 of 30 5 nm) Nanomaterials 2020, 10, 504 radiation-induced DSB ff i l b d GNPs conjugated with anticancer drugs exhibited a synergistically eﬀective cancer treatment by eﬀectively delivering drugs to tumor cells and enhancing cell radiosensitization activity, such as through DNA damage induction. For example, the combined treatment of cisplatin-tethered GNPs (50 nm) and irradiation signiﬁcantly enhanced DNA DSBs, as evidenced by the enhanced density of γ-H2AX foci, resulting in the apoptosis of patient-derived treatment-resistant glioblastoma multiforme (GBM) cells (Figure 2) [85]. HER-2-targeted Herceptin-GNPs (30 nm) in combination with X-rays induced higher levels of DNA DSBs in HER2-positive human breast cancer cells than X-ray irradiation alone [86,87]. Furthermore, the combined treatment of Herceptin-GNPs and X-ray irradiation signiﬁcantly inhibited tumor growth compared to X-ray irradiation alone [87]. 3.1. Nanoradiosensitizers Based on DNA Damage Doxorubicin (DOX)-loaded GNPs (5 nm) with dual-targeting decoration were more likely to be internalized by HeLa cells than were monotargeting or nontargeting decorations, leading to higher levels of DNA DSBs under irradiation [88]. Chitosan-capped GNPs (18 nm) loaded with doxorubicin (CS-GNPs-DOX) enhanced the chemoradiotherapeutic eﬀect by signiﬁcantly decreasing cancer cells viability by increasing DNA DSBs in MCF-7 breast cancer cells [89]. Goserelin-decorated pegylated GNRs (gGNRs, 8 nm) were preferentially internalized by PC3 cells through a gonadotropin-releasing hormone receptor-mediated mechanism. The combined treatment of gGNR and irradiation induced higher levels of γ-H2AX foci than the combination of pegylated GNR and irradiation [90]. stem cell-like cells to radiotherapy through the induction of mitotic catastrophe [84]. GNPs conjugated with anticancer drugs exhibited a synergistically effective cancer treatment by effectively delivering drugs to tumor cells and enhancing cell radiosensitization activity, such as through DNA damage induction. For example, the combined treatment of cisplatin-tethered GNPs (50 nm) and irradiation significantly enhanced DNA DSBs, as evidenced by the enhanced density of γ-H2AX foci, resulting in the apoptosis of patient-derived treatment-resistant glioblastoma multiforme (GBM) cells (Figure 2) [85]. HER-2-targeted Herceptin-GNPs (30 nm) in combination with X-rays induced higher levels of DNA DSBs in HER2-positive human breast cancer cells than X-ray irradiation alone [86,87]. Furthermore, the combined treatment of Herceptin-GNPs and X-ray irradiation significantly inhibited tumor growth compared to X-ray irradiation alone [87]. Doxorubicin (DOX)-loaded GNPs (5 nm) with dual-targeting decoration were more likely to be internalized by HeLa cells than were monotargeting or nontargeting decorations, leading to higher levels of DNA DSBs under irradiation [88]. Chitosan-capped GNPs (18 nm) loaded with doxorubicin (CS-GNPs-DOX) enhanced the chemoradiotherapeutic effect by significantly decreasing cancer cells viability by increasing DNA DSBs in MCF-7 breast cancer cells [89]. Goserelin-decorated pegylated GNRs (gGNRs, 8 nm) were preferentially internalized by PC3 cells through a gonadotropin-releasing hormone receptor-mediated mechanism. The combined treatment of gGNR and irradiation induced higher levels of γ-H2AX foci than the combination of pegylated GNR and irradiation [90]. Figure 2. Multifunctional nanosphere for combined chemo-radiotherapy. Principle of nanosphere action based on gold and platinum mediated radiosensitization and cisplatin induced genotoxic damage. Reproduced with permission [85]. Copyright The Royal Society of Chemistry, 2014. Figure 2. Multifunctional nanosphere for combined chemo-radiotherapy. Principle of nanosphere action based on gold and platinum mediated radiosensitization and cisplatin induced genotoxic damage. Reproduced with permission [85]. Copyright The Royal Society of Chemistry, 2014. Figure 2. Multifunctional nanosphere for combined chemo-radiotherapy. Principle of nanosphere action based on gold and platinum mediated radiosensitization and cisplatin induced genotoxic damage. Reproduced with permission [85]. Copyright The Royal Society of Chemistry, 2014. Figure 2. Multifunctional nanosphere for combined chemo-radiotherapy. Principle of nanosphere action based on gold and platinum mediated radiosensitization and cisplatin induced genotoxic damage. Reproduced with permission [85]. Copyright The Royal Society of Chemistry, 2014. Figure 2. Multifunctional nanosphere for combined chemo-radiotherapy. Principle of nanosphere action based on gold and platinum mediated radiosensitization and cisplatin induced genotoxic damage. Reproduced with permission [85]. Copyright The Royal Society of Chemistry, 2014. Figure 2. Multifunctional nanosphere for combined chemo-radiotherapy. Principle of nanosphere action based on gold and platinum mediated radiosensitization and cisplatin induced genotoxic damage. Reproduced with permission [85]. Copyright The Royal Society of Chemistry, 2014. The size and surface chemistry of GNPs can regulate the radiosensitization eﬀect through the generation of DNA damage. Citrate-coated GNPs (6, 10, 25 nm) induced diﬀerent levels of plasmid DNA damage under irradiation. The number of SSB increased as the size of the GNP was decreased [107]. The uptake level of positively charged GNPs (14 nm) was higher than that of negatively charged GNPs by A712 human glioblastoma cells. Upon irradiation, positively charged GNPs induced higher levels of DNA damage and apoptosis than did their negatively charged counterparts [91]. The surface chemistry of the GNPs (32 nm) regulated the DNA damage-mediated radiosensitization eﬀects. Citrate decoration on GNPs induced the highest level of plasmidic DNA damage under irradiation, followed by PEG1000, human serum albumin (HSA), and PEG4000 decoration, for which the level of DNA damage was correlated with hydroxyl radical (HO·) production [108]. Both GSH- and bovine serum albumin 10 of 30 Nanomaterials 2020, 10, 504 (BSA)-decorated Au25NCs (<2 nm) with biocompatible coating surfaces preferentially accumulated in tumors via an improved enhanced permeability and retention (EPR) eﬀect, which led to a greater enhancement of cancer radiotherapy than was induced by the much larger Au NPs. Under irradiation, GSH-coated Au25NCs induced more signiﬁcant DNA damage, decreased tumor weight and showed more eﬃcient renal clearance than did BSA-GNPs [92]. In addition to gold-based NPs, other metal NPs can induce DNA damage-related radiosensitization eﬀects. For example, AgNPs (20–30 nm) induced more DNA damage with concurrent radiation treatment in triple-negative MDA-MB-231 cells than they did in non-triple-negative MCF-7 and MCF-10A cells, with the eﬀect based on an oxidative stress mechanism and resulting in the inhibition of tumor growth [40]. In another study, AgNPs (20 nm) inhibited the expression of DNA damage/repair proteins, including Rad51, Ku-70, and Ku-80, in nasopharyngeal carcinoma epithelial (CNE) cells under irradiation. The targeted epidermal growth factor receptor-speciﬁc antibody decoration on AgNPs can signiﬁcantly enhance radiation-induced DNA damage, possibly by elevated cellular uptake [93]. Arg-Gly-Asp (RGD) andtransactivator of transcription (TAT) decoration on iridium (Ir) NPs (<5 nm) accumulated in cancer cells and showed cell-nucleus targeting. RGD-Ir-TAT NPs elicited ROS overproduction and DNA lesions in 4T1 cells under X-ray irradiation and exhibited satisfactory destruction of tumor tissues [94]. Metal oxide NPs exhibited radiosensitization eﬀects through DNA damage or the inhibition of DNA repair. Thulium oxide NPs (40–45 nm) enhanced DSBs in radioresistant 9L brain gliosarcoma cells under irradiation [98]. The combined chemoradiotherapy of DOX-loaded mesoporous tantalum oxide (mTa2O5, 80 nm) NPs led to a strong synergistic therapeutic eﬀect in a mouse tumor model. The interaction of Ta with X-rays induced signiﬁcant DNA damage during radiotherapy [99]. ZnO NPs (7 nm) sensitized SKLC-6 lung cancer cells to radiation through the induction of DNA damage; however, ZnO NPs exhibited no radiosensitization eﬀect on MRC-5 normal lung cells [100]. Gadolinium-doped ZnO NPs (9 nm) impaired DNA repair by downregulating the mRNA levels of XRCC2 and XRCC4 genes under irradiation and induced apoptosis in SKLC-6 lung carcinoma cells [95]. Oleic acid decorated iron-oxide NPs (MN-OA, 10 nm) downregulated proteins involved in DNA double-strand break repair, such as RAD51 and BRCA1, resulting in DNA damage in mouse ﬁbrosarcoma WEHI-164 cells under irradiation [96]. Apurinic endonuclease 1 (Ape1) is an enzyme involved in base excision repair. The SPION (4–6 nm)-based siRNA delivery system knocked down the expression of Ape1 and sensitized brain tumor cells to radiotherapy [97,109]. A series of polymer NPs can be used as drug carriers to enhance the radiosensitization eﬀect by promoting DNA damage. Polymeric NPs containing camptothecin (CRLX101, 20–30 nm) promoted the formation and persistence of radiation-induced DSBs and inhibited radiation-induced HIF1α activation, which resulted in enhanced radiosensitization of HT-29 cells and xenograft models [101]. Irradiation can induce site-speciﬁc expression of receptors in tumor cells, such as tax-interaction protein 1 (TIP-1). TIP-1-targeted polymer NPs (<100 nm) loaded with JNK inhibitor molecules signiﬁcantly inhibited DNA repair in Lewis lung carcinoma (LLC) cells under irradiation and induced greater apoptosis and inhibition of tumor growth compared to irradiation alone [102]. The application of DNA double-strand repair inhibitors (DSBRIs) is a promising strategy to improve radiotherapy. KU55933, a DSBRI, was loaded into PLGA NPs (87 nm). The resulting NP KU55933 improved the radiosensitization of H460 cells and tumor tissues through the downregulation of ATM and AKT phosphorylation [74]. EGF-decorated PLGA NPs (130–140 nm) incorporating a ruthenium-based radiosensitizer preferentially bound to EGFR-overexpressing oesophageal cancer cells and exhibited radiosensitization eﬀects through the induction of DNA damage [110]. Folate-decorated PEI NPs were used to construct a new class of DNA damage repair inhibitors, nanoparticle Dbait (NP Dbait, 140 nm), which were internalized by prostate cancer cells overexpressing folate receptors. Dbait in the nucleus inhibited DNA damage repair signaling pathways by mimicking DNA DSBs, resulting in the activation of DNA-PK and H2AX phosphorylation. DNA damage repair factors were assembled at the end of Dbait and sequestered away from the real DSB sites, resulting in defects in DSB repair in 11 of 30 Nanomaterials 2020, 10, 504 cells under irradiation [103]. X-ray repair cross-complementing protein 1 (XRCC-1) is overexpressed in X-ray-resistant HeLa cells and is critical for the inhibition of DNA repair. Folate decorated-BSA NP (255 nm) loaded with organic selenocompounds increased ROS overproduction and inhibited XRCC-1 expression in HeLa cells under irradiation [104]. 3.2. The Impact of NP Physicochemical Properties on DNA Damage Generally, the size of NPs is negatively correlated with DNA damage level. Small GNPs (5 nm) induced DNA damage in HepG2 cells and clastogenic damage in vivo, while larger GNPs (20 nm, 50 nm) did not induce these eﬀects [111]. AgNPs (4.7 nm) induced higher genotoxicity in HepG2 and HL-60 cells than did AgNPs (42 nm), as evidenced by DNA strand breaks and oxidative DNA damage [112]. Small silica NPs (19 nm) induced higher DNA damage levels in HepG2 cells than did larger NPs (43 nm, 68 nm) [61]. The shape of NPs can regulate DNA damage. MWCNTs (10–30 µm/8–15 nm, 0.5–2 µm/8–15 nm) induced single-strand DNA damage and elevated DNA repair gene levels in HepG2 cells, while MWCNTs (10–30 µm/20–30 nm) caused no damage to DNA [113]. Another study reported that the length and diameter of the MWCNTs were positively correlated with the DNA damage level in A549 cells [114]. Needle-shaped PLGA-PEG NPs (30 × 540 nm) caused DNA fragmentation and cytotoxicity in HepG2 cells, while no DNA damage was found in HepG2 cells treated with spherical NPs (90 nm) [115]. In addition to the size and shape of NPs, the NP surface charge can also regulate DNA damage. GNPs (3.1 nm, 24.5 mV) with a positive charge induced the highest DNA damage level in A549 cells, followed by negatively charged GNPs [116]. 4. Cell-Cycle Arrest The cell cycle contains four phases: The G1, S, G2 and M phase. The G2/M phase is the most sensitive phase to radiation. Therefore, NPs that can induce cell-cycle arrest at the G2/M phase enhance the radiotherapeutic eﬀect on cancer cells. In recent years, various kinds of NPs were found to exhibit radiosensitization eﬀects through the induction of cell-cycle arrest. They are summarized in this section (Table 3). Table 3. Summary of nanoradiosensitizers based on cell-cycle arrest mechanism. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Gold 5 N.A. Huh7, HepG2 5 Gy of γ, 5 GyE of neutron radiation 1.16–1.80 [117] Gold 50 N.A. HTB-72 0–4 Gy Increased cell death [118] Gold 11 Glucose DU-145 2 Gy Growth inhibition [119] Gold 44 × 15 Arg-Gly-Asp peptides (RGD) A375 0–8 Gy 1.35 [120] Gold 55 Epidermal growth factor receptor (EGFR) antibody HeLa 5, 10 Gy Increased cell death [121] Gold 16, 49 Glucose MDA-MB-231 0–10 Gy 1.86 (49 nm), 1.49 (16 nm) [54] Gold-silver 150 l-ascorbic acid HepG2 0–10 Gy 1.8 [122] Graphene Lateral size: 18 N.A. SW620, HCT116 c 3, 6 Gy Increased cell death [123] Bi2O3 45 Hyaluronic acid SMMC-7721 0–9 Gy Increased cell death [124] Iron-oxide 10 Oleic acid WEHI-164 2 Gy Inhibited cell proliferation [96] Gadolinium oxide 2–5 N.A. A549, NH1299, NH1650 0–4 Gy 1.10 (A549), 1.11 (NH1299), 1.20 (NH1650) [125] Table 3. Summary of nanoradiosensitizers based on cell-cycle arrest mechanism. 12 of 30 Nanomaterials 2020, 10, 504 Table 3. Cont. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Titanate nanotubes 10 N.A. SNB-19, U87MG 0.5, 1, 2, 5, 10 Gy Increased cell death [126] TiO2 45 SN-38, nucleus-targeting moieties 4T1-Luc 4, 6 Gy Inhibited cell proliferation [127] Hydroxycamptothecin- loaded micelles 132 Folate HeLa Total dose of 20 Gy Inhibited tumor growth [128] Ceria 3–5 Neogambogic acid MCF-7 0–8 Gy Inhibited cell proliferation [129] Micelles 85 Paclitaxel Lewis lung carcinoma cells Total dose of 12 Gy Inhibited tumor growth [130] PLGA 200–500 Paclitaxel HepG2, HeLa 0–10 Gy Increased cell death [131] PLGA 500 Paclitaxel MCF-7 0–10 Gy Increased cell death [132] PLGA 130–150 Docetaxel A549, CNE-1 0–8 Gy 1.68 (A549), 1.61 (CNE-1) [133] Fe3O4@ZnO <200 Doxorubicin, transferrin receptor antibody SMMC-7721 3 Gy Inhibited tumor growth [134] Abbreviations: DEF, dose enhancement factor; SER, sensitization enhancement ratio; N.A., not available; Ref., references. 4.1. Nanoradiosensitizers Based on Cell-Cycle Arrest GNPs can sensitize cancer cells to radiation through the induction of cell-cycle arrest. For example, the combined treatment of GNPs (5 nm) and neutron/γ irradiation induced cell-cycle arrest in the G2/M phase, resulting in the inhibition of migration and invasion of Huh7 and HepG2 cells [117]. The combined treatment of irradiation and GNPs (50 nm) signiﬁcantly increased the proportion of melanoma cells in the G2/M phase, which enhanced the next radiation treatment [118]. Glucose-GNPs (Glu-GNPs, 11 nm) induced the G2/M arrest of radiation-resistant DU-145 human prostate cancer cells through activation of checkpoint kinases CDK1 and CDK2, resulting in these cells being sensitized to ionizing radiation [119]. The decoration of GNPs with targeting moieties can enhance the radiosensitization eﬀect. Arg-Gly-Asp peptide (RGD)-GNRs sensitized melanoma A375 cells exposed to radiation through the downregulation of radiation-induced integrin αvβ3 and induction of cell-cycle arrest in the G2/M phase [120]. EGFR antibody-decorated hollow gold nanospheres (anti-EGFR/HGNs, 55 nm) were more eﬃciently internalized by HeLa cells than were naked HGNs. More cells were arrested in the G2/M phase after induction by anti-EGFR/HGNs than they were after induction by naked HGNs. The combination of anti-EGFR/HGNs and megavoltage irradiation signiﬁcantly enhanced the number of apoptotic cells than did irradiation alone [121]. GNP size aﬀected the radiosensitization eﬀects of cell-cycle arrest. For example, the size of Glu-GNPs regulated the radiosensitization eﬀects in MDA-MB-231 cells. Glu-GNPs (49 nm) were internalized more eﬃciently than their counterparts (16 nm) and induced higher levels of G2/M cell-cycle arrest under irradiation [54]. Cho et al. reported that the surface structure of NPs played key roles in enhancing these radiosensitizing eﬀects. They found that day-ﬂower-like nanoparticle (D-NP), which has a large surface area, induced more ROS production in and G2/M stage arrest of HepG2 cells and exhibited signiﬁcant radiosensitization eﬀects. However, spherical night-ﬂower-like nanoparticle (N-NP), which has a small surface area, did not aﬀect the cell-cycle distribution and exhibited no radiosensitization eﬀects [122]. In addition to GNPs, other NPs also exhibit radiosensitization effects through the induction of G2/M arrest. Graphene quantum dots (GQDs) sensitize colorectal carcinoma cells to ionizing radiation through the induction of ROS generation, G2/M stage arrest, and apoptosis [123]. Hyaluronic acid-functionalized bismuth 13 of 30 13 of 30 Nanomaterials 2020, 10, 504 oxide nanoparticles (HA-Bi2O3 NPs, 45 nm) exhibited excellent biocompatibility and radiosensitization effects in SMMC-7721 cells and tumor-bearing mice. 4.1. Nanoradiosensitizers Based on Cell-Cycle Arrest The combined treatment of HA-Bi2O3 NPs and irradiation drove a higher proportion of cells into the G2/M phase than did irradiation alone [124]. HSP90 was found to play key roles in the radiosensitization effects of oleic acid-decorated iron-oxide NPs (MN-OA, 10 nm) on WEHI-164 cells and tumor tissues. The interaction between MN-OA and HSP90 led to the downregulation of proteins involved in cell cycle progression, such as cyclin B1 and CDC2, ultimately resulting in G2/M stage arrest [96]. Gadolinium oxide NPs (2–5 nm) sensitized three NSCLC cell lines (A549, NH1299, and NH1650) to carbon ion radiation through the induction of cell cycle arrest in the G2/M phase and cytotoxicity [125]. Titanate nanotubes (TiONts, 10 nm in diameter) were internalized by human glioblastoma cells (SNB-19 and U87 MG cell lines) through endocytosis and diffusion mechanisms. Upon irradiation, TiONts enhanced G2/M cell cycle arrest [126]. Nanomaterials 2020, 10, 504 15 of 33 involved in cell cycle progression, such as cyclin B1 and CDC2, ultimately resulting in G2/M stage arrest [96] Gadolinium oxide NPs (2 5 nm) sensitized three NSCLC cell lines (A549 NH1299 and y NPs loaded with cell-cycle blockers and anticancer drugs can enhance the radiosensitization effects on cells. 7-Ethyl-10-hydroxy-camptothecin (SN-38) induced G2/M cell-cycle arrest. Mesoporous TiO2 NPs (45 nm) carrying SN-38 and nucleus-targeting moieties (MTiO2(SN-38)-TAT-RGD) accumulated in the nucleus of 4T1-Luc cells and induced G2/M arrest. The combination of MTiO2(SN-38)-TAT-RGD and X-ray irradiation inhibited cell proliferation in vitro and decreased tumor volume in vivo (Figure 3) [127]. Folate-decoratedhydroxycamptothecin(HCPT)-loadedmicelles(HFOL,132nm)wereeffectivelyinternalized by HeLa cells. In vivo experiments indicated that HFOL induced the G2/M phase arrest of tumor tissue cells and inhibited tumor growth upon irradiation [128]. Ceria NPs loaded with the anticancer drug neogambogic acid (NGA-CNPs, 3–5 nm) induced the G2/M phase cell cycle arrest of MCF-7 cells and significant radiosensitization effects [129]. Paclitaxel (PTX)-loaded micelle NPs (NK105, 85 nm) induced more severe G2/M stage arrest and higher radiosensitization of Lewis lung carcinoma cells than did PTX alone [130]. In another study, PTX-loaded poly(D,L-lactide-co-glycolide) (PLGA, 200–500 nm) NPs sensitized HepG2 and HeLa cells to radiotherapy through the induction of G2/M stage arrest [131]. Coculture of MCF-7 cells with paclitaxel-loaded PLGA NPs (500 nm) demonstrated that released paclitaxel blocked cells in the G2/M phase and sensitized MCF-7 cells to radiation [132]. Docetaxel-loaded PLGA NPs sensitized A549 and CNE-1 cells to radiation through enhanced G2/M arrest and apoptosis [133]. 4.1. Nanoradiosensitizers Based on Cell-Cycle Arrest The Fe3O4@ZnO nanocomposites functionalized with transferrin receptor antibody (TfR Ab) delivered DOX into hepatocellular carcinoma SMMC-7721 cells, resulting in G2/M cell cycle arrest in combination with irradiation. In vivo studies demonstrated that tumor growth was significantly inhibited after radiotherapy mediated by Fe3O4@ZnO/DOX/TfR Ab [134]. arrest [96]. Gadolinium oxide NPs (2–5 nm) sensitized three NSCLC cell lines (A549, NH1299, and NH1650) to carbon ion radiation through the induction of cell cycle arrest in the G2/M phase and cytotoxicity [125]. Titanate nanotubes (TiONts, 10 nm in diameter) were internalized by human glioblastoma cells (SNB-19 and U87 MG cell lines) through endocytosis and diffusion mechanisms. Upon irradiation, TiONts enhanced G2/M cell cycle arrest [126]. NPs loaded with cell-cycle blockers and anticancer drugs can enhance the radiosensitization effects on cells. 7-Ethyl-10-hydroxy-camptothecin (SN-38) induced G2/M cell-cycle arrest. Mesoporous TiO2 NPs (45 nm) carrying SN-38 and nucleus-targeting moieties (MTiO2(SN-38)-TAT- RGD) accumulated in the nucleus of 4T1-Luc cells and induced G2/M arrest. The combination of MTiO2(SN-38)-TAT-RGD and X-ray irradiation inhibited cell proliferation in vitro and decreased tumor volume in vivo (Figure 3) [127]. Folate-decorated hydroxycamptothecin (HCPT)-loaded micelles (HFOL, 132 nm) were effectively internalized by HeLa cells. In vivo experiments indicated that HFOL induced the G2/M phase arrest of tumor tissue cells and inhibited tumor growth upon irradiation [128]. Ceria NPs loaded with the anticancer drug neogambogic acid (NGA-CNPs, 3–5 nm) induced the G2/M phase cell cycle arrest of MCF-7 cells and significant radiosensitization effects [129]. Paclitaxel (PTX)-loaded micelle NPs (NK105, 85 nm) induced more severe G2/M stage arrest and higher radiosensitization of Lewis lung carcinoma cells than did PTX alone [130]. In another study, PTX-loaded poly(D,L-lactide-co-glycolide) (PLGA, 200–500 nm) NPs sensitized HepG2 and HeLa cells to radiotherapy through the induction of G2/M stage arrest [131]. Coculture of MCF-7 cells with paclitaxel-loaded PLGA NPs (500 nm) demonstrated that released paclitaxel blocked cells in the G2/M phase and sensitized MCF-7 cells to radiation [132]. Docetaxel-loaded PLGA NPs sensitized A549 and CNE-1 cells to radiation through enhanced G2/M arrest and apoptosis [133]. The Fe3O4@ZnO nanocomposites functionalized with transferrin receptor antibody (TfR Ab) delivered DOX into hepatocellular carcinoma SMMC-7721 cells, resulting in G2/M cell cycle arrest in combination with irradiation. In vivo studies demonstrated that tumor growth was signiﬁcantly inhibited after radiotherapy mediated by Fe3O4@ZnO/DOX/TfR Ab [134] Figure 3. Preparation of MTiO2(SN-38)- transactivator of transcription (TAT)-Arg-Gly-Asp (RGD) nanoparticles (NPs) and its application for enhanced radiotherapy. Reproduced with permission [127]. 4.2. The Impact of NP Size on Cell-Cycle Arrest Citrate-GNPs of 56, 33, and 15 nm regulated the proportion of HepG2 cells in the G2/M phase, with 15 nm citrate-GNPs inducing the highest level of G2/M cell-cycle arrest [135]. ZnO NPs of 20 nm enhanced the proportion of HeLa cells in the G2/M phase, while ZnO NPs of 40 nm and 80 nm did not induce G2/M arrest [136]. Therefore, it seems that smaller NPs are more likely to induce G2/M arrest. The impact of NP shape and surface chemistry on G2/M arrest has rarely been reported. 4.1. Nanoradiosensitizers Based on Cell-Cycle Arrest Copyright The Royal Society of Chemistry, 2019. Figure 3. Preparation of MTiO2(SN-38)- transactivator of transcription (TAT)-Arg-Gly-Asp (RGD) nanoparticles (NPs) and its application for enhanced radiotherapy. Reproduced with permission [127]. Copyright The Royal Society of Chemistry, 2019. Figure 3. Preparation of MTiO2(SN-38)- transactivator of transcription (TAT)-Arg-Gly-Asp (RGD) nanoparticles (NPs) and its application for enhanced radiotherapy. Reproduced with permission [127]. Copyright The Royal Society of Chemistry, 2019. Figure 3. Preparation of MTiO2(SN-38)- transactivator of transcription (TAT)-Arg-Gly-Asp (RGD) nanoparticles (NPs) and its application for enhanced radiotherapy. Reproduced with permission [127]. Copyright The Royal Society of Chemistry, 2019. 14 of 30 Nanomaterials 2020, 10, 504 14 of 30 5. Apoptosis There are two types of apoptosis: extrinsic and intrinsic apoptosis. Extrinsic apoptosis is triggered by the activation of death receptor superfamily proteins on the cell membrane. Intrinsic apoptosis is triggered through the endoplasmic reticulum- or mitochondria-related mechanisms [137,138]. The radiosensitization eﬀects of the NPs based on the apoptosis mechanism are summarized in this section (Table 4). Moreover, the regulation of cancer cell apoptosis by NP physicochemical properties is also summarized. Table 4. Summary of nanoradiosensitizers based on apoptosis mechanism. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Gold 5 N.A. HSC-3 2, 4, 8 Gy Increased cell death [139] Gold 18 BSA U87 0–8 Gy 1.37 [140] Au@Fe2O3 44 Folate KB 2, 4 Gy Increased cell death [141] Gold 55 EGFR antibody HeLa 5, 10 Gy Increased cell death [121] GNRs@mSiO2 76 × 33 RGD MDA-MB-231 0–10 Gy 1.52 [142] Gold 20 Cyclic RGD NCI-H446 tumor-bearing mice 5 Gy Inhibited tumor growth [143] Gold 8, 50, 187 BSA H22 hepatoma-bearingmice 5 Gy 1.93 (8 nm), 2.02 (50 nm) [144] Gold 5, 12, 27, 49 PEG HeLa 0–8 Gy 1.41 (5 nm), 1.65 (12 nm), 1.58 (27 nm), 1.42 (49 nm) [145] GNPs+17-AAG N.A. Folate HCT-116 2 Gy Increased cell death [146] Gold 78 Cisplatin B16 0–8 Gy 1.29 [147] Gold 30 Cetuximab A431 tumor xenograft 25 Gy Inhibited tumor growth [148] Gold 56 Anti-c-Met antibodies CaSki 0–10 Gy Increased cell death [149] Gold 50–70 × 35 siRNA HNSCC 2.5 Gy Increased cell death [150] Silver 15 Citrate U251 4 Gy 1.64 [151] Gadolinium based NPs 3 N.A. SQ20B 0–4 Gy Increased cell death [152] Selenium 80 PEG A549 N.A. Increased cell death [153] QDs 48 Amine H460 6 Gy Increased cell death [154] Cu2(OH)PO4 5 Poly(acrylic acid) sodium HeLa N.A. Inhibited tumor growth [155] nMOFs <100 N.A. MC38 0–16 Gy 2.68 [156] Mesoporous silica <200 Valproic acid C6, U87 0–8 Gy 1.71 [157] Silica 40 Hyperbranched polyamidoamine SK-BR3 0–8 Gy Inhibited tumor growth [158] Black phosphorus QDs 3 PLGA A375 0–4 Gy Inhibited tumor growth [159] Table 4. Summary of nanoradiosensitizers based on apoptosis mechanism. 15 of 30 Nanomaterials 2020, 10, 504 Table 4. Cont. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Black phosphorus nanosheets 300 × 25 N.A. A375 4 Gy Inhibited tumor growth [160] Dendrimer 20 N.A. 5.1. Nanoradiosensitizers Based on Apoptosis GNPs exhibit radiosensitization eﬀects based on apoptosis. First, GNPs alone in combination with irradiation induce higher apoptosis rates of cancer cells [139,140]. GNPs conjugated with targeting moieties also exhibit excellent radiosensitization eﬀects that culminate in apoptosis induction. For example, folate-conjugated Au@Fe2O3 NPs (44 nm) exhibited radiosensitization eﬀects in KB cells by elevating the apoptosis rate [141]. EGFR antibody-decorated hollow gold nanospheres (anti-EGFR/HGNs, 55 nm) induced the downregulation of Bcl-2 and the upregulation of caspase 3, Bax, and Bad in HeLa cells under irradiation [121]. RGD decoration signiﬁcantly increased the cellular uptake of GNRs@mSiO2 through a receptor-mediated mechanism in integrin αVβ3-overexpressing breast cancer cells, resulting in an increased apoptosis rate and inhibition of tumor growth under irradiation compared to the apoptosis and growth rates induced by non-decorated GNRs@mSiO2 [142]. Cyclic RGD (cRGD)-conjugated GNPs (20 nm) induced higher apoptosis rates in small cell lung cancer NCI-H446 cell tumor-bearing mice than did irradiation treatment alone. The long-term exposure of cRGD-GNPs in combination with radiotherapy signiﬁcantly inhibited the growth of tumor tissues [143]. The size of the GNP can regulate the radiosensitization eﬀect. Without radiation exposure, BSA-GNPs (8, 50, 187 nm) exhibited no induced cytotoxicity in hepatocellular carcinoma. Under irradiation, small BSA-GNPs induced higher levels of caspase-3 and Bax expression and lower levels of Bcl-2 expression in mouse tumor tissues than larger GNP counterparts [144]. The size of the PEG-GNPs can also regulate the radiosensitization eﬀect. PEG-GNPs with diameters of 12 nm and 27 nm had stronger sensitization eﬀects than did PEG-GNPs with diameters of 5 nm and 49 nm, as indicated by the induction of apoptosis and necrosis, resulting in the inhibition of tumor growth [145]. GNPs loaded with multiple drugs, antibodies, and siRNA exhibit radiosensitization eﬀects. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is an inhibitor of HSP90 that induces the apoptosis of cancer cells. The combined treatment of 17-AAG, GNPs and irradiation induced the highest expression of caspase-3 in HCT-116 cells compared with single treatments or combinations of two treatments [146]. Cisplatin-loaded gold nanoparticles (Au@PAH-Pt/DMMA, 78 nm) aggregated rapidly through electrostatic interactions in the acidic tumor microenvironment. Under irradiation, the aggregated GNPs induced higher apoptosis rates and inhibited tumor growth than were induced by irradiation alone [147]. Cetuximab-GNPs (30 nm) increased the apoptosis rates in A431 tumor xenografts when administered in combination with irradiation [148]. 5. Apoptosis OCM-1 2 Gy Increased cell death [161] PEG-Pep-PCL 85 Docetaxel BGC823, SGC7901, MKN45, GES-1 0–8 Gy 1.24 [162] Cationic copolymer N.A. Plasmid encoding HGFK1 gene U87, U251 0–10 Gy 1.38 [163] HSA 180–220 Antibody, miRNA U87MG, LN229 0–10 Gy 1.64 (U87MG), 1.25 (LN229) [164] Abbreviations: DEF, dose enhancement factor; SER, sensitization enhancement ratio; N.A., not available; Ref., references. 5.1. Nanoradiosensitizers Based on Apoptosis 5.1. Nanoradiosensitizers Based on Apoptosis The MET proto-oncogene receptor tyrosine kinase (c-Met) is overexpressed in multiple malignancies, such as the cervical cancer cell line CaSki, and is related to the metastasis, proliferation, and invasion of cancer. The combined treatment of anti-c-Met antibody-loaded hollow GNPs (56 nm) induced the overexpression of caspase-3 and Bax, resulting in a higher apoptosis rate than did irradiation alone [149]. Head and neck squamous cell carcinoma exhibits an inherent anti-apoptotic mechanism through the upregulation of the sphingosine kinase (SphK1) gene. GNRs (50–70 nm × 35 nm) loaded with siRNA of the SphK1 16 of 30 gh the siRNA Nanomaterials 2020, 10, 504 neck squamous cell upregulation of the sph gene signiﬁcantly enhanced the expression of caspase 3 under irradiation in human squamous cell carcinoma xenografts [150]. squamous cell carcinoma xenografts [150]. In addition to GNPs, other metal-based NPs can also exhibit radiosensitization effects through i h i Ci A NP (15 ) hibi d f l di i i i bili b In addition to GNPs, other metal-based NPs can also exhibit radiosensitization eﬀects through apoptosis mechanisms. Citrate-AgNPs (15 nm) exhibited powerful radiosensitizing ability by eliciting high apoptosis rates of U251 cells under radiation [151]. The combination of gadolinium-based NPs (3 nm) with irradiation signiﬁcantly inhibited tumor growth accompanied by an enhanced number of cells in late apoptosis [152]. PEGylated selenium NPs (80 nm) combined with X-ray irradiation exhibited higher caspase-3 activity and apoptosis in A549 cells than did irradiation or PEGylated selenium NPs alone [153]. The combination of QDs (48 nm) and irradiation induced higher levels of cleaved caspase 3 and apoptosis in H460 cells than did irradiation alone [154]. Cu2(OH)PO4 NPs (5 nm) generated CuI sites under irradiation, which catalyzed the decomposition of H2O2 into hydroxyl radicals in the tumor microenvironment, resulting in the apoptosis and necrosis of HeLa cells [155]. Cationic nanoscale metal–organic frameworks (nMOFs, <100 nm) containing Hf and Ru elements were targeted to mitochondria, resulting in signiﬁcant depolarization of the mitochondrial membrane, an increased apoptosis rate, and inhibition of tumor growth in mouse models under irradiation (Figure 4). apoptosis mechanisms. Citrate-AgNPs (15 nm) exhibited powerful radiosensitizing ability by eliciting high apoptosis rates of U251 cells under radiation [151]. The combination of gadolinium- based NPs (3 nm) with irradiation significantly inhibited tumor growth accompanied by an enhanced number of cells in late apoptosis [152]. 5.1. Nanoradiosensitizers Based on Apoptosis PEGylated selenium NPs (80 nm) combined with X-ray irradiation exhibited higher caspase-3 activity and apoptosis in A549 cells than did irradiation or PEGylated selenium NPs alone [153]. The combination of QDs (48 nm) and irradiation induced higher levels of cleaved caspase 3 and apoptosis in H460 cells than did irradiation alone [154]. Cu2(OH)PO4 NPs (5 nm) generated CuI sites under irradiation, which catalyzed the decomposition of H2O2 into hydroxyl radicals in the tumor microenvironment, resulting in the apoptosis and necrosis of HeLa cells [155]. Cationic nanoscale metal–organic frameworks (nMOFs, <100 nm) containing Hf and Ru elements were targeted to mitochondria, resulting in significant depolarization of the mitochondrial membrane, an increased apoptosis rate, and inhibition of tumor growth in mouse models under irradiation (Figure 4). Figure 4. Mitochondria-targeted radiotherapy (RT)-radiodynamic therapy (RDT) mediated by Hf- DBB-Ru. Hf-DBB-Ru was internalized by tumor cells efﬁciently and enriched in mitochondria due to Figure 4. Mitochondria-targeted radiotherapy (RT)-radiodynamic therapy (RDT) mediated by Hf-DBB-Ru. Hf-DBB-Ru was internalized by tumor cells eﬃciently and enriched in mitochondria due to dispersed cationic charges in the nMOF framework. Hf6 SBUs preferentially absorb X-rays over tissues to enhance RT by sensitizing hydroxyl radical generation and enable RDT by transferring energy to Ru(bpy)32+-based bridging ligands to generate singlet oxygen. The RT-RDT process trigger mitochondrial membrane potential depolarization, membrane integrity loss, respiratory chain inactivation, and cytochrome c release to initiate apoptosis of cancer cells. Reproduced with permission. Copyright Springer Nature, 2018. Figure 4. Mitochondria-targeted radiotherapy (RT)-radiodynamic therapy (RDT) mediated by Hf- DBB-Ru. Hf-DBB-Ru was internalized by tumor cells efﬁciently and enriched in mitochondria due to Figure 4. Mitochondria-targeted radiotherapy (RT)-radiodynamic therapy (RDT) mediated by Hf-DBB-Ru. Hf-DBB-Ru was internalized by tumor cells eﬃciently and enriched in mitochondria due to dispersed cationic charges in the nMOF framework. Hf6 SBUs preferentially absorb X-rays over tissues to enhance RT by sensitizing hydroxyl radical generation and enable RDT by transferring energy to Ru(bpy)32+-based bridging ligands to generate singlet oxygen. The RT-RDT process trigger mitochondrial membrane potential depolarization, membrane integrity loss, respiratory chain inactivation, and cytochrome c release to initiate apoptosis of cancer cells. Reproduced with permission. Copyright Springer Nature, 2018. Non-metal NPs also exhibit radiosensitization eﬀects. Valproic acid (VPA) sensitized multiple cancer cell types to radiation through the inhibition of histone deacetylase (HDAC). 5.2. The Impact of NP Physicochemical Properties on Apoptosis The translocation of Bax from the cytoplasm to mitochondria is a crucial step in apoptosis. Smaller nano-C60 were more likely to induce the translocation of Bax than were larger NPs in MCF-7 cells [168]. The shape of NPs can also regulate the apoptosis rate of cancer cells. ZnO nanorods (65 × 150 nm) induced higher rates of HeLa and SiHa cell apoptosis than did ZnO nanosphere (60 nm), as evidenced by the reduction of phospho-Bad and PARP cleavage [169]. The aspect ratio of silica NPs can determine the apoptosis rate. Silica NPs (aspect ratio = 4) induced the highest apoptosis rates of A375 cells, followed by silica NPs with aspect ratio = 2 and aspect ratio = 1 [170]. Polystyrene nanospheres (20.6 nm) induced a higher rate of HeLa cell apoptosis than did nanodisks (19.7 nm) [171]. Needle-shaped PLGA-PEG NPs (30 × 540 nm) induced a higher expression of caspase 3 in HepG2 cells than did spherical NPs (90 nm) [115]. In addition, the hydrophobicity of GNPs (20–25 nm) regulated the rate of A549 cell apoptosis. Hydrophobic GNPs induced higher apoptosis rates than did hydrophilic GNPs [172]. 60 y g [ ] The shape of NPs can also regulate the apoptosis rate of cancer cells. ZnO nanorods (65 × 150 nm) induced higher rates of HeLa and SiHa cell apoptosis than did ZnO nanosphere (60 nm), as evidenced by the reduction of phospho-Bad and PARP cleavage [169]. The aspect ratio of silica NPs can determine the apoptosis rate. Silica NPs (aspect ratio = 4) induced the highest apoptosis rates of A375 cells, followed by silica NPs with aspect ratio = 2 and aspect ratio = 1 [170]. Polystyrene nanospheres (20.6 nm) induced a higher rate of HeLa cell apoptosis than did nanodisks (19.7 nm) [171]. Needle-shaped PLGA-PEG NPs (30 × 540 nm) induced a higher expression of caspase 3 in HepG2 cells than did spherical NPs (90 nm) [115]. In addition, the hydrophobicity of GNPs (20–25 nm) regulated the rate of A549 cell apoptosis. Hydrophobic GNPs induced higher apoptosis rates than did hydrophilic GNPs [172]. 5.1. Nanoradiosensitizers Based on Apoptosis VPA-loaded mesoporous silica NPs (<200 nm) signiﬁcantly upregulated caspase-3, p53, and cleavage of poly (ADP-ribose) polymerase (PARP) and downregulated Bcl-2, resulting in the apoptosis of C6 and U87 cells under radiation [157]. In another study, the combination of HER2-targeted silica NPs (40 nm) and irradiation induced a higher rate of apoptosis of HER2-overexpressing breast cancer cells than did irradiation alone [158]. Black phosphorus QDs can render tumor cells sensitive to radiotherapy through the overproduction of ROS and elevated apoptosis rates [159]. It was also found that black phosphorus nanosheets (300 nm × 25 nm) enhanced the radiotherapy eﬀects in A375 cells as indicated by higher apoptosis rates and DNA damage levels than were induced by irradiation alone [160]. 17 of 30 Nanomaterials 2020, 10, 504 A series of polymer NPs can be used as nanocarriers to deliver genes, drugs or antibodies that sensitize cancer cells to radiotherapy. Dendrimer NPs (20 nm) loaded with recombinant DNA plasmids enhanced the gene expression of tumor necrosis factor α (TNFα) and herpes simplex virus type 1 thymidine kinase (HSV1-TK) and the apoptosis rates of human choroidal melanomaOCM-1 cells, exhibiting radiosensitization eﬀects under irradiation [161]. The combination of docetaxel-loaded PEG-Pep-PCL NPs (85 nm) and radiation led to higher ROS levels and apoptosis rates in gastric cancer cells than did the combination of docetaxel and radiation or irradiation alone [162]. The MET aberrant activation plays key roles in radiotherapy resistance. The angiogenic inhibitor kringle 1 domain of hepatocyte growth factor (HGFK1) was reported to strongly bind to MET. Cationic copolymer NPs encapsulating a plasmid encoding the HGFK1 gene promoted the radiation-induced apoptosis of U87 and U251 human glioblastoma cells and inhibited tumor growth in vivo [163]. Heat shock protein 70 (Hsp70) mediates the protection of tumor cells against apoptosis. Hsp70-speciﬁc antibody (cmHsp70.1) and survivin-targeting miRNA plasmid-loaded human serum albumin (HSA) NPs (180 nm–220 nm) signiﬁcantly reduced survivin expression and enhanced caspase 3/7 activity in U87 MG and LN229 glioblastoma cells under irradiation [164]. 5.2. The Impact of NP Physicochemical Properties on Apoptosis The size of an NP can determine the apoptosis rates of cancer cells. For example, the apoptosis rate of human colon carcinoma LoVo cells induced by AgNPs (10, 20, 40, 60 and 100 nm) was negatively correlated with NP size [62]. AgNPs (4.7 nm) induced higher expression of caspase 3 and caspase 7 than did AgNPs (42 nm) in HepG2 and HL-60 cells [165]. AgNPs (13 nm) caused a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bax and upregulated the phosphorylation of NF-κB in A549 cells, indicating that AgNPs (13 nm) induced apoptosis via the NF-κB pathway. However, large AgNPs (45 and 92 nm) did not activate the apoptosis signaling pathway [166]. The size of silica NPs (19, 43, 68 nm) is negatively correlated with the apoptosis and necrosis rates of HepG2 cells [61]. However, another study showed that silica NPs (50 nm) induced higher apoptosis rates of HepG2 cells than did silica NPs (20 nm) [60]. ZnO NPs (49.4 nm) induced higher apoptosis rates of human neuroblastoma SHSY5Y cells than did ZnO NPs (90.8 nm) [167]. The translocation of Bax from the cytoplasm to mitochondria is a crucial step in apoptosis. Smaller nano-C60 were more likely to induce the translocation of Bax than were larger NPs in MCF-7 cells [168]. The size of an NP can determine the apoptosis rates of cancer cells. For example, the apoptosis rate of human colon carcinoma LoVo cells induced by AgNPs (10, 20, 40, 60 and 100 nm) was negatively correlated with NP size [62]. AgNPs (4.7 nm) induced higher expression of caspase 3 and caspase 7 than did AgNPs (42 nm) in HepG2 and HL-60 cells [165]. AgNPs (13 nm) caused a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bax and upregulated the phosphorylation of NF-κB in A549 cells, indicating that AgNPs (13 nm) induced apoptosis via the NF-κB pathway. However, large AgNPs (45 and 92 nm) did not activate the apoptosis signaling pathway [166]. The size of silica NPs (19, 43, 68 nm) is negatively correlated with the apoptosis and necrosis rates of HepG2 cells [61]. However, another study showed that silica NPs (50 nm) induced higher apoptosis rates of HepG2 cells than did silica NPs (20 nm) [60]. ZnO NPs (49.4 nm) induced higher apoptosis rates of human neuroblastoma SHSY5Y cells than did ZnO NPs (90.8 nm) [167]. 6.1. Nanoradiosensitizers Based on Autophagy 6.1. Nanoradiosensitizers Based on Autophagy Multiple kinds of NPs can sensitize cancer cells to radiation based on the autophagy mechanism. The combined treatment of AgNPs and irradiation on hypoxic glioma U251 cells led to autophagy. The inhibition of autophagy by 3-MA alleviated cytotoxicity, indicating that autophagy plays key roles in radiosensitization eﬀects [177]. Fe3O4@Ag NPs (11 nm) also exhibited radiosensitization eﬀects in U251 cells through the inhibition of protective autophagy and the eventually increase in calcium-dependent apoptosis [178]. Gadolinium oxide NPs (2–5 nm) sensitized NSCLC cells (A549, NH1299, and NH1650) to radiation and induced cytostatic autophagy [125]. Copper cysteamine NPs sensitized SW620 colorectal cells to X-ray irradiation by diminishing the mitochondrial membrane potential and inducing autophagy [179]. CuO NPs (5.4 nm) induced destructive autophagy, revealing a radiosensitization eﬀect in MCF-7 cells and U14 tumor-bearing nude mice [49]. FePt/GO nanosheets inhibited the proliferation of NSCLC H1975 cells and sensitized H1975 cells to radiation. The radiosensitization mechanisms involved ROS production and autophagy [180]. 6. Autophagy Autophagy is a process related to the degradation of dysfunctional or unnecessary components in cells. Autophagy is categorized into three types: microautophagy, macroautophagy, and chaperone-mediated autophagy [173,174]. Growing evidence has demonstrated that autophagy plays key roles in the pathogenesis of cancer [175]. Moreover, cytostatic autophagy leads to cancer cell death [176]. In this section, we summarize NPs’ radiosensitization eﬀect based on the autophagy mechanism (Table 5). The regulation of autophagy in cancer cells by NPs is also summarized, which may guide the design of nanoradiosensitizers. 18 of 30 Nanomaterials 2020, 10, 504 Table 5. Summary of nanoradiosensitizers based on autophagy mechanism. Table 5. Summary of nanoradiosensitizers based on autophagy mechanism. Composition Size (nm) Surface Chemistry Cell Line/Model Source Energy DEF/SER/Eﬀect Ref. Silver 27 N.A. U251 0–8 Gy 1.78 [177] Fe3O4@Ag 11 N.A. U251 0–8 Gy 1.80 [178] Gadolinium oxide 2–5 N.A. A549, NH1299, NH1650 0–4 Gy 1.10 (A549), 1.11 (NH1299), 1.20 (NH1650) [125] Copper cysteamine N.A. N.A. SW620 1–4 Gy Increased cell death [179] CuO 5.4 N.A. MCF-7 cells, U14 tumor-bearing nude mice 0–8 Gy Increased cell death [49] FePt/GO 3 N.A. H1975 0–8 Gy Inhibited tumor growth [180] Abbreviations: DEF, dose enhancement factor; SER, sensitization enhancement ratio; N.A., not available; Ref., references. 6.2. The Impact of NP Physicochemical Properties on Autophagy Copyright The Royal Society of Chemistry, 2015. 6.2. The Impact of NP Physicochemical Properties on Autophagy The size of NPs can regulate autophagy levels in cancer cells. For example, AgNPs of 10 nm induced higher expression of LC3-II in HepG2 cells, followed by 50 nm and 100 nm AgNPs [181]. The size of palladium nanoparticles (PdNPs, 6 nm, 12 nm, 20 nm) also regulated autophagy in HeLa cells, with 20 nm-sized PdNPs inducing the highest accumulation of autophagosomes through mammalian target of rapamycin (mTOR) signaling pathway inhibition and autophagic ﬂux blockade [17]. Polymeric NPs of 141 nm induced higher autophagy levels in MCF-7 cells than did polymeric NPs (44 nm) [182]. The shape of GNPs can also regulate autophagy levels in cancer cells. Gold nanospheres (20 nm in diameter) induced more autophagosome accumulation in HeLa cells than did GNRs (40 nm in length) through the inhibition of autophagic ﬂux [183]. The surface charge can regulate the autophagy level. The positively charged cetyl trimethyl ammonium bromide (CTAB)-GNRs promoted the transformation of LC3-I to LC3-II in HCT116 cells, while the negatively charged polystyrene sulfonate gold nanorods ((PSS)-GNRs) did not signiﬁcantly enhance the autophagy level [184]. Similarly, CTAB-GNRs (55 × 14 nm) induced AKT-mTOR-dependent autophagy by inducing LC3-II conversion and p62 degradation in A549 cells. However, PSS-GNRs did not induce autophagy (Figure 5) [185]. NH2-GQDs (3.5–5 nm) induced autophagy in A549 cells, as proven by LC3-II conversion and autophagosome accumulation; however, negatively charged COOH-GQDs did not enhance autophagy levels [186]. COOH-SWCNTs induced the autophagic cell death of A549 cells, however, PEG decoration inhibited autophagy [187]. 19 of 30 g y ced the Nanomaterials 2020, 10, 504 charged COOH-GQD Figure 5. The mechanism and signaling pathways in autophagy induced by cetyl trimethyl ammonium bromide gold nanorods (CTAB-GNRs) and polystyrene sulfonate gold nanorods ( PSS- GNRs). Reproduced with permission [185]. Copyright The Royal Society of Chemistry, 2015. Figure 5. The mechanism and signaling pathways in autophagy induced by cetyl trimethyl ammonium bromide gold nanorods (CTAB-GNRs) and polystyrene sulfonate gold nanorods ( PSS-GNRs). Reproduced with permission [185]. Copyright The Royal Society of Chemistry, 2015. Figure 5. The mechanism and signaling pathways in autophagy induced by cetyl trimethyl ammonium bromide gold nanorods (CTAB-GNRs) and polystyrene sulfonate gold nanorods ( PSS- GNRs). Reproduced with permission [185]. Copyright The Royal Society of Chemistry, 2015. Figure 5. The mechanism and signaling pathways in autophagy induced by cetyl trimethyl ammonium bromide gold nanorods (CTAB-GNRs) and polystyrene sulfonate gold nanorods ( PSS-GNRs). Reproduced with permission [185]. 7. Conclusions and Perspectives 7. Conclusions and Perspectives The combination of nanotechnology with radiation therapy significantly increases the precision of therapy and reduces side effects. NPs of different compositions sensitize cancer cells to radiotherapy through multiple mechanisms, including oxidative stress, DNA damage, cell-cycle arrest, apoptosis and autophagy (Figure 6). For successful radiotherapy, some important strategies are considered. First, NPs with high atomic number (Z) are used to enhance radiation therapy efficacy via their photoelectric and Compton effects. Second, targeting cancer cells with specific targeting molecules prolongs the circulation time of the NPs to increase their accumulation in cancer cells. The combination of nanotechnology with radiation therapy signiﬁcantly increases the precision of therapy and reduces side eﬀects. NPs of diﬀerent compositions sensitize cancer cells to radiotherapy through multiple mechanisms, including oxidative stress, DNA damage, cell-cycle arrest, apoptosis and autophagy (Figure 6). For successful radiotherapy, some important strategies are considered. First, NPs with high atomic number (Z) are used to enhance radiation therapy eﬃcacy via their photoelectric and Compton eﬀects. Second, targeting cancer cells with speciﬁc targeting molecules prolongs the circulation time of the NPs to increase their accumulation in cancer cells. Third, the combination of two diﬀerent types of radiosensitizers or the combination of radiosensitizers and anticancer drugs or siRNA in a single nanostructure can result in signiﬁcantly synergistic tumoricidal eﬀects. Finally, the anti-tumor immune response induced during nanotechnology-based radiosensitization has shown great eﬀect in reducing the side-eﬀects of radiation [188] and enhancing the abscopal eﬀects of radiationtherapy [189], which present novel strategies to design radiosensitizers. Despite rapid advancement over the past years, more attention could be paid to the following issues. The radiosensitization eﬀects of NPs are strongly related to their preparation procedures, particle sizes, geometries, surface chemistries and biosafety. At present, most of the nanoradiosensitizers are developed with high-Z metal elements, which are usually undegradable and cause biosafety concerns. Therefore, the impact of the physicochemical properties on the biocompatibility, biodistribution, biodegradability and clearance of the nanoradiosensitizers needs to be systematically evaluated before their clinical applications. Except for the molecule mechanisms of nanoradiosensitization mentioned above, other mechanisms, such as the induction of the anti-tumor immune response, provide novel 20 of 30 ficantly during Nanomaterials 2020, 10, 504 radiosensitizers and a synergistic tumoricid strategies for the design of nanoradiosensitizers. In addition, multifunctional nanoradiosensitizers combining with other anticancer therapies can oﬀer new opportunities for synergistic therapy. 7. Conclusions and Perspectives 7. Conclusions and Perspectives Therefore, the impact of the physicochemical properties on the biocompatibility biodistribution biodegradability and clearance of the nanoradiosensitizers needs Acknowledgments: This research was funded by the National Natural Science Foundation of China (21677090) and the Key R&D Programmes of Zibo (2019ZC010106). biocompatibility, biodistribution, biodegradability and cle to be systematically evaluated before their clinical applicat Conﬂicts of Interest: The authors declare no conﬂict of interest. biocompatibility, biodistribution, biodegradability and cle to be systematically evaluated before their clinical applicati Conﬂicts of Interest: The authors declare no conﬂict of interest. of nanoradio tumor immu References p p g g multifunctional nanoradiosensitizers combining with other anticancer therapies can offer new opportunities for synergistic therapy. 1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 2015, 136, E359–E386. [CrossRef] p p g g multifunctional nanoradiosensitizers combining with other anticancer therapies can offer new opportunities for synergistic therapy. 1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 2015, 136, E359–E386. [CrossRef] Author Contributions: H.S. and S.Z. designed this work of review. H.S. and X.W. performed the literature se . Aly, H.A.A. Cancer therapy and vaccination. J. Immunol. Methods 2012, 382, 1–23. [CrossRef] [PubMed Author Contributions: H.S. and S.Z. designed this work of review. H.S. and X.W. performed the literature search of the databases. H.S., X.W. and S.Z. wrote the manuscript. H.S. and S.Z. revised the manuscript. All author 2. Aly, H.A.A. Cancer therapy and vaccination. J. Immunol. Methods 2012, 382, 1 23. [CrossRef] [PubMed] 3. Brenner, H.; Kloor, M.; Pox, C.P. Colorectal cancer. Lancet 2014, 383, 1490–1502. [CrossRef] of the databases. H.S., X.W. and S.Z. wrote the manuscript. H.S. and S.Z. revised the manuscript. A 3. Brenner, H.; Kloor, M.; Pox, C.P. Colorectal cancer. Lancet 2014, 383, 1490–1502. [CrossRef] of the databases. H.S., X.W. and S.Z. wrote the manuscript. H.S. and S.Z. revised the manuscript. All auth 3. Brenner, H.; Kloor, M.; Pox, C.P. Colorectal cancer. Lancet 2014, 383, 1490–1502. [CrossRef] approved the paper for publication. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. 4. Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, 573–584. [CrossRef] [PubMed] approved the paper for publication. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. 4. Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, 573–584. [CrossRef] [PubMed] Funding: This research received no external funding. Acknowledgements: This research was funded by the National Natural Science Foundation of China (21677090) and the Key R&D Programmes of Zibo (2019ZC010106). 5. 7. Conclusions and Perspectives 7. Conclusions and Perspectives nanotechnology-based radiosensitization has shown great effect in reducing the side-effects of radiation [188] and enhancing the abscopal effects of radiationtherapy [189], which present novel strategies to design radiosensitizers. Fi u e 6 Biolo i al e ha i i ol ed i NP ’ adio e iti atio Figure 6. Biological mechanisms involved in NPs’ radiosensitization. Fi 6 Bi l i l h i i l d i NP ’ di i i i Figure 6. Biological mechanisms involved in NPs’ radiosensitization. Despite rapid advancement over the past years, more attention could be paid to the following issues. The radiosensitization effects of NPs are strongly related to their preparation procedures, particle sizes, geometries, surface chemistries and biosafety. At present, most of the Author Contributions: H.S. and S.Z. designed this work of review. H.S. and X.W. performed the literature search of the databases. H.S., X.W. and S.Z. wrote the manuscript. H.S. and S.Z. revised the manuscript. All authors approved the paper for publication. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding issues. The radiosensitization effects of NPs are strongly related to their preparation procedures, particle sizes, geometries, surface chemistries and biosafety. At present, most of the nanoradiosensitizers are developed with high Z metal elements which are usually undegradable and of the databases. H.S., X.W. and S.Z. wrote the manuscript. H.S. and S.Z. revised the manuscript. All authors approved the paper for publication. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. particle sizes, geometries, surface chemistries nanoradiosensitizers are developed with high-Z metal Funding: This research received no external funding. particle sizes, geometries, surface chemistries and biosafety. At present, most nanoradiosensitizers are developed with high-Z metal elements which are usually undegrada Funding: This research received no external funding. nanoradiosensitizers are developed with high-Z metal elements, which are usually undegradable and cause biosafety concerns. 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https://openalex.org/W1976141396	https://www.scielo.br/j/rcefac/a/YYTbvShHtPkSY3rnLFFzfsQ/?lang=pt&format=pdf	Portuguese	null	Estado mental e impacto do zumbido em idosos	Revista CEFAC	2,014	cc-by	6,848	798 798 R CEFAC 2014 M i J 16(3) 798 809 (1) Universidade Federal de Sergipe – UFS, Aracaju, SE, Brasil. (2) Universidade Federal de Sergipe – UFS, Aracaju, SE, Brasil. (3) Universidade do Estado da Bahia, Salvador, BA, Brasil. (4) Universidade Federal de Sergipe – UFS, Aracaju, SE, Brasil. Conflito de interesses: inexistente RESUMO Objetivo: analisar o desempenho de uma população idosa no Mini Exame do Estado Mental; veri- ficar os resultados da aplicação do Tinnitus Handicap Inventory (THI); averiguar a interferência das variáveis gênero, escolaridade e zumbido no desempenho geral no MEEM e THI; verificar possíveis relações do estado mental com o autorrelato das características psicoacústicas e escala emocional do THI. Métodos: estudo clínico descritivo, exploratório, quantitativo e qualitativo em que se sub- meteram ao MEEM e THI, 108 voluntários, de gênero masculino e feminino, com idade entre 60 a 80 anos encaminhados da Coordenadoria de Atenção Básica de Itabaiana – SE. Para a análise dos dados foram utilizadas a distribuição percentual simples e a correlação de Spearman com p<0,05. Resultados: a média de idade foi 65,63 anos. Os resultados no MEEM agruparam-se em quatro níveis de escolaridade: sem escolaridade (37,0%); ≥1 a ≤8 anos (55,6%); ≥9 a ≤11 anos (4,6%) e ≥12 anos (2,8%); a média no MEEM foi 21,7. Observou-se que 49,1% pontuaram abaixo da nota de corte, enquanto 50,9% apresentaram nota igual ou superior ao parâmetro adotado. No THI, observou- -se que 59,3% apresentavam queixa de zumbido. Verificou-se que o zumbido interfere na qualidade de vida de 89,10% da população estudada. Conclusão: uma parcela expressiva dos participantes apresentou alteração no MEEM. Não existiu associação significante entre gênero, escolaridade e pontuação do MEEM e THI e a maioria dos participantes desta pesquisa referiu prejuízo na qualidade de vida com associação ao zumbido. Inexistiu relação entre queixa das características psicoacústi- cas do zumbido e resultados exibidos pelo MEEM, todavia, os achados apontaram que a maioria da população testada autorrelataram presença de zumbido. DESCRITORES: Idoso; Zumbido; Transtornos Cognitivos; Diagnóstico; Qualidade de Vida RITORES: Idoso; Zumbido; Transtornos Cognitivos; Diagnóstico; Qualidade de Vida INTRODUÇÃO O zumbido é considerado uma percepção de som sem que haja sua presença no meio ambiente11. Consiste de uma sensação definida como ilusória que pode ser caracterizada como barulho semelhante ao ruído da chuva, do mar, de água corrente, de sinos, insetos, apitos, chiado, campainha, pulsação e outros. Esta sensação pode ser contínua ou intermitente, apresentar diferentes características tonais, ser intensa ou suave, além de ser percebida nos ouvidos ou na cabeça12. Esse teste é utilizado no mundo inteiro para avaliar o estado cognitivo. É um teste simples, especialmente indicado para avaliação de grandes grupos populacionais, com fins de análise estatística de incidência e prevalência de demência, de aplicação rápida (de 5 a 10 minutos), com pontuação que varia de 0 a 30 e suas questões avaliam: orientação temporal (5 pontos), orientação espacial (5 pontos), memória imediata (3 pontos), atenção e cálculo (5 pontos), memória de evocação (3 pontos), linguagem (8 pontos) e praxia visual e construtiva (1 ponto). Entretanto, deve-se ter cuidado ao avaliar a memória pelo MEEM, pois o esse exame é altamente influenciado pela idade e escolaridade, o que permite resultados “falso- -positivos” e “falso-negativos”4. O impacto do zumbido na qualidade de vida pode ser avaliado pelo Tinnitus Handicap Inventory (THI) que foi desenvolvido por Newman, Jacobson, Spitzer(1996)13. Este questionário é composto por 25 questões, divididas em três escalas, de fácil aplicação e interpretação, possui confiabilidade para a prática clínica e, ainda, aborda interferências do zumbido sobre a qualidade de vida do paciente: reações funcionais, emocionais e catastróficas ao zumbido. A funcional (F) mede o incômodo provocado pelo zumbido em funções mentais, sociais, ocupacionais e físicas. A escala emocional (E) mede as respostas afetivas como ansiedade, raiva e depressão. A catastrófica (C) quantifica o desespero e a incapacidade referida pelo acometido para conviver ou livrar-se do sintoma. São três as opções de resposta para cada uma das questões, pontuadas da seguinte maneira: para as respostas sim (4 pontos), às vezes (2 pontos) e não (nenhum ponto).13 Os estudos de Almeida (1998) e Brucki et al. (2003)5,6 mostraram a interferência da escolaridade e idade no desempenho desse teste no estado de São Paulo. Almeida (1998)5 traduziu e validou este questionário para o português brasileiro. Comumente, as alterações como perda auditiva e zumbido são frequentes na terceira idade. INTRODUÇÃO contribuintes para a redução da mortalidade foi à melhora nos padrões da qualidade de vida, ocasio- nados por uma melhora no saneamento básico, investimentos em previdência social e infraestrutura urbana, além de um salto significante na saúde pública, e com avanços, por exemplo, da indústria químico-farmacêutica. A população brasileira, segundo o Instituto Brasileiro de Geografia e Estatística1, sofre transfor- mações em seu padrão demográfico. Essas modifi- cações que surgiram nos anos 40 ocorreram de forma morosa, passando a ser mais radicais a partir da década de 60. Isso é evidenciado no declínio das taxas de mortalidade seguido por uma queda dos níveis de fecundidade. Um dos principais fatores O envelhecimento acarreta alterações em vários níveis e sistemas que podem comprometer a qualidade de vida. Dentre os problemas de saúde comuns na terceira idade, encontram-se os trans- tornos mentais, que acometem cerca de um terço da população idosa. Há poucos estudos epidemio- lógicos de morbidade psiquiátrica geral no idoso, apontando prevalências de 26,4% a 33,6% em comunidades brasileiras urbanas2. Rev CEFAC 2014 Mai-Jun; 16(3):798-809 (1) Universidade Federal de Sergipe – UFS, Aracaju, SE, Brasil. (2) Universidade Federal de Sergipe – UFS, Aracaju, SE, Brasil. (3) Universidade do Estado da Bahia, Salvador, BA, Brasil. (4) Universidade Federal de Sergipe – UFS, Aracaju, SE, Brasil. Conflito de interesses: inexistente O estudo do estado mental pode ser avaliado pelo Mini Exame do Estado Mental (MEEM) que é um teste de avaliação cognitiva com o objetivo de Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 799 Estado Mental e Zumbido em Idosos apontam que esse distúrbio acomete de 10 a 15% da população e atinge de 20 a 40% dos idosos. fornecer dados sobre diversos parâmetros cogni- tivos de qualquer população geriátrica. Este teste foi definido como mini por que se preocupa apenas com os aspectos cognitivos da função mental e por isso não serve para identificar a demência. O MEEM avalia: orientação temporal, orientação espacial, registro de três palavras, atenção e cálculo, recor- dação das três palavras, linguagem e capacidade construtiva visual3. fornecer dados sobre diversos parâmetros cogni- tivos de qualquer população geriátrica. Este teste foi definido como mini por que se preocupa apenas com os aspectos cognitivos da função mental e por isso não serve para identificar a demência. O MEEM avalia: orientação temporal, orientação espacial, registro de três palavras, atenção e cálculo, recor- dação das três palavras, linguagem e capacidade construtiva visual3. 1.1.3 - Atenção e Cálculo 1.1.3 - Atenção e Cálculo 5 ( ) Peça ao paciente que conte de trás para frente, começando do nº 100, de 7 em7. Pare depois da 5ª resposta. Alternativamente peça para soletrar “mundo” ao contrário. INTRODUÇÃO O zumbido vem se tornando uma queixa otológica frequente, acometendo cerca de 25 milhões de brasileiros7, e apresenta prevalência elevada, estando presente em 63,3% dos indivíduos acima de 45 anos. Falhas no raciocínio, na memória e na concentração são queixas frequentes, sendo que estas podem prejudicar as atividades de lazer, o repouso, a comunicação, o ambiente social e doméstico, repercutindo na esfera psíquica, provo- cando irritação, ansiedade, depressão e insônia8. Aproximadamente 17% da população é afetada pelo zumbido, e de 15% a 25% referem interferência em sua qualidade de vida9. Axelsson, Ringdahl (1989)10 Este instrumento, traduzido e validado para o português brasileiro em 2005 por Ferreira e colaboradores14, pode ser usado de forma ampla no contexto clínico para avaliação dos pacientes com zumbido, para a quantificação do incômodo relacionado a este sintoma e para avaliação de respostas a tratamentos propostos. Baguley et al. (2000)15 e Berry et al.(2002)16 utilizaram o THI para quantificar a melhora clínica do zumbido e salien- taram a sua utilidade no monitoramento e avaliação do tratamento proposto. De acordo com a proposta de Mccombe et al. (1999)17, o zumbido pode ser: Grau Intensidade Percentual 1 Desprezível 0-16% 2 Leve 18-36% 3 Moderado 38-56% 4 Severo 58-76% 5 Catastrófico 78-100% Figura 1 Classificação do grau da queixa de zumbido segundo Mccombe et al (1999)17 Grau Intensidade Percentual 1 Desprezível 0-16% 2 Leve 18-36% 3 Moderado 38-56% 4 Severo 58-76% 5 Catastrófico 78-100% Figura 1 – Classificação do grau da queixa de zumbido segundo Mccombe et al.(1999)17. Figura 1 – Classificação do grau da queixa de zumbido segundo Mccombe et al.(1999)17. gura 1 – Classificação do grau da queixa de zumbido segundo Mccombe et al.(1999)17 800 Gois RO, Gois BO, Pereira MCCS, Taguchi CK Instruções para administração: Instruções para administração: Foram excluídos os indivíduos com idade inferior a 60 anos, que utilizavam drogas psicotrópicas, com histórico de acidente vascular encefálico, doenças neurológicas degenerativas, depressão, delírio, história de traumatismo cranioencefálico e diagnóstico prévio de demência. MÉTODOS 9 ( ) Mostre um lápis e um relógio, peça-lhe que os nomeie (2 pontos). - Peça que repita o seguinte: “N i ã - Peça que repita o seguinte: “Nem sim, nem não, nem porque” (1 ponto). O projeto desta pesquisa foi apreciado pelo comitê de Ética e Pesquisa do Núcleo de Medicina da Universidade Federal de Sergipe e foi aprovado com o parecer de número CAAE: 0016.0.107.000-10. Todos os pacientes assinaram termo de ciência e consentimento livre e esclarecido, garantindo o anonimato e a desistência a qualquer momento da pesquisa. - Dê as 3 seguintes ordens: “Pegue esta folha de papel com a mão direita, passe a folha para a mão esquerda, coloque a folha no chão” (3 pontos). - “Leia e faça o que está escrito”: “Feche os olhos” (1 ponto). “Feche os olhos” (1 ponto). “Escreva uma frase” (1 ponto). Trata-se de um estudo clínico descritivo, explo- ratório, quantitativo e qualitativo em que foram submetidos ao Mini Exame do Estado Mental (MEEM) e ao Tinnitus Handicap Inventory (THI), 108 voluntários, de gênero masculino e feminino, com faixa etária variando de 60 a 80 anos, avaliados na Associação Atlética de Itabaiana em parceria com a Coordenadoria de Atenção Básica de Itabaiana – SE. “Copie este desenho” (1 ponto). p Total ( ) 1.1.6 - Avalie o nível de consciência: Alerta ( ) sonolento ( ) prostrado ( ) coma ( ) 1.1.6 - Avalie o nível de consciência: Alerta ( ) sonolento ( ) prostrado ( ) coma ( ) 1.1.4 - Memória 3 ( ) Peça que ele repita as três palavras. Dê um ponto para cada resposta correta. 1.1.1 - Máximo Orientação 1.1.1 - Máximo Orientação 5 ( ) Em que ano, mês, estação do ano estamos? 5 ( ) Em que ano, mês, estação do ano estamos? 5 ( ) Onde estamos: estado, país, cidade, hospital? 5 ( ) Onde estamos: estado, país, cidade, hospital? Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 1 - Mini Exame do Estado Mental – MEEM Destaca-se o fato do crescente aumento das pesquisas voltadas para o estudo da qualidade de vida do idoso, porém não se encontram pesquisas que abordam a relação do estado mental e zumbido, levando-se em consideração que os idosos apresentam demandas de cuidados em saúde diferentes daquelas do restante da população, o que também é verdadeiro sob o enfoque fonoau- diológico. Uma população mais envelhecida exige maiores investimentos de recursos em saúde e seguridade social. O Brasil defronta-se com o desafio de elaborar políticas públicas mais eficientes para oferecer melhores condições de vida e saúde à sua crescente população idosa18. 1.1.2 – Registros 3 ( ) Nomeie 3 objetos: diga palavra por palavra, devagar; peça ao paciente que repita as três palavras. Dê um ponto para cada resposta correta. Então repita todas novamente, para que ele aprenda. Frente ao exposto, os objetivos desta pesquisa foram analisar o desempenho de uma população idosa no Mini-Exame do Estado Mental; verificar os resultados da aplicação do Tinnitus Handicap Inventory (THI); averiguar a interferência das variáveis gênero, escolaridade e zumbido no desempenho geral no MEEM e THI; verificar as possíveis relações do estado mental com o autor- relato das características psicoacústicas e escala emocional do THI. 1.1.3 - Atenção e Cálculo 1.2.1 - Orientação 1) Pergunte pela data de hoje. Em seguida, pergunte especificamente pelos dados omitidos, ex.: “Pode me dizer também em que estação do ano nós estamos?” Um ponto para cada resposta correta. Os indivíduos foram submetidos à aplicação do Mini Exame do Estado Mental (MEEM) desen- volvido por Folstein (1975)19. 2) Pergunte por partes: “Pode me dizer o nome deste hospital?” (cidade, país etc.). Um ponto para cada resposta correta. Observação: nesta etapa solicita-se aos voluntários que indicassem Neste estudo utilizou-se a proposta para a língua brasileira de Almeida (1998)5, conforme apresentado abaixo: Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 801 Estado Mental e Zumbido em Idosos o nome do local de avaliação, ou seja, Instituto de Longevidade de Aracaju ou Associação Atlética de Itabaiana. 1.2.3 - Atenção e Cálculo Peça o paciente que conte começando do número 100, de 7 em 7, ao contrário. Peça que pare depois da 5ª subtração (93, 86, 79, 72, 65). Determine o escore pelo total de acertos. Se o paciente não conseguir ou não quiser fazer esta conta, peça-lhe que soletre a palavra “mundo” de trás para frente. O escore será o número de letras que ele disser na ordem correta, ex.: odnum = (5), odunm = (3). Observação: Frente às dificuldades apresen- tadas pela população, nesta etapa apresentou-se ao paciente um desenho de uma casa composta por formas geométricas e solicitou-se que ele copiasse. A aplicação do MEEM foi realizada por dois avaliadores treinados que não possuíam nenhum vínculo de parentesco com os pacientes avaliados, bem como não mantiveram nenhum contato verbal ou visual entre eles. Observação: Frente às dificuldades apresen- tadas pela população, nesta etapa apresentou-se ao paciente um desenho de uma casa composta por formas geométricas e solicitou-se que ele copiasse. A aplicação do MEEM foi realizada por dois avaliadores treinados que não possuíam nenhum vínculo de parentesco com os pacientes avaliados, bem como não mantiveram nenhum contato verbal ou visual entre eles. 1.2.5 - Linguagem Nomeando: Mostre ao paciente um relógio de pulso e pergunte o que é isso. Repita com um lápis. Escore 0 a 2. 1.2.2 - Registro Nesta etapa da avaliação mostra-se ao paciente o telefone celular do avaliador no lugar do relógio de pulso. Pergunte ao paciente se você pode aplicar-lhe um teste para avaliar sua memória. Diga o nome de 3 objetos que não se relacionem entre si, fale lenta e claramente, dê um espaço de um segundo entre cada palavra. Depois de dizer as 3 palavras, peça que ele as repita. Esta primeira repetição determina o escore (0 a 3), continue repetindo as palavras, por no máximo 6 vezes, até que ele repita todas as 3. Se ele não se lembrar das palavras, esta fase do teste deverá ser interrompida, sem insistência. Repetição: Diga uma frase e peça ao paciente que a repita. Repita apenas uma vez. Escore 0 a 1. 3 Ordens: Forneça ao paciente uma folha de papel em branco e peça que ele escreva uma frase para você. Não dite a frase, é preciso que ele escreva espontaneamente. Verifique se a frase contém sujeito, verbo e se faz sentindo. A gramática e a pontuação não é preciso avaliar. Numa folha de papel em branco, desenhe um pentágono dentro do outro, com a diferença de 2 cm e peça que ele copie exatamente. Se os 10 ângulos estiverem presentes e 2 intersecções o escore será de 1 ponto. Tremor e rotação: ignore. 1.2.4 - Memória Você acha difícil tirar a sua atenção do zumbido e se concentrar em outra coisa? 19. Você sente que não tem controle sobre o seu zumbido? 20. Devido ao seu zumbido, você se sente frequentemente cansado? 21. Devido ao seu zumbido, você se sente frequentemente deprimido? 22. O seu zumbido faz com que você sinta ansioso? 23. Você sente que não pode mais suportar o seu zumbido? 24. O seu zumbido piora quando você está estressado? 25. O seu zumbido faz com que você se sinta inseguro? Figura 3 - Questionário do Tinnitus Handicap Inventory (THI)13, traduzido por Ferreira et al.(2005)14 Questão Sim Às vezes Não 1. Devido ao seu zumbido é difícil se concentrar? 2. O volume (intensidade) do seu zumbido faz com que seja difícil escutar as pessoas? 3. O seu zumbido deixa você nervoso? 4. O seu zumbido deixa você confuso? 5. Devido ao seu zumbido, você se sente desesperado? 6. Você se queixa muito do seu zumbido? 7. Devido ao seu zumbido, você tem dificuldade para pegar no sono à noite? 8. Você sente como se não pudesse se livrar do seu zumbido? 9. O seu zumbido interfere na sua capacidade de aproveitar atividades sociais (tais como sair para jantar, ir ao cinema)? 10. Devido ao seu zumbido, você se sente frustrado? 11. Devido ao seu zumbido, você pensa que tem uma doença grave? 12. O seu zumbido torna difícil aproveitar a vida? 13. O seu zumbido interfere nas suas tarefas no serviço e em casa? 14. Devido ao seu zumbido, você se sente frequentemente irritado? 15. Devido ao seu zumbido, você acha difícil ler? 16. O seu zumbido deixa você chateado? 17. Você sente que o seu zumbido atrapalha o seu relacionamento com a sua família e amigos? 18. Você acha difícil tirar a sua atenção do zumbido e se concentrar em outra coisa? 19. Você sente que não tem controle sobre o seu zumbido? 20. Devido ao seu zumbido, você se sente frequentemente cansado? 21. Devido ao seu zumbido, você se sente frequentemente deprimido? 22. O seu zumbido faz com que você sinta ansioso? 23. Você sente que não pode mais suportar o seu zumbido? 24. O seu zumbido piora quando você está estressado? 25. O seu zumbido faz com que você se sinta inseguro? Figura 3 - Questionário do Tinnitus Handicap Inventory (THI)13, traduzido por Ferreira et al.(2005)14 1.2.4 - Memória Pergunte ao paciente se ele pode relembrar as 3 palavras que você lhe pediu que guardasse na memória. Escore 0 a 3. Para a análise dos dados obtidos, utilizou as notas de corte, oriundas da adaptação do estudo de Herrera Jr., Caramelli, Nitrini (1998)20 conforme a Figura 2 abaixo: Escolaridade Sem escolaridade ≥1 e ≤ 8 anos ≥ 9 e ≤ 11 anos ≥ 12 anos Pontuação 19 24 24 28 Figura 2 - Distribuição dos pontos de corte segundo o nível de escolaridade O THI foi aplicado e as respostas foram pontuadas de zero, quando o zumbido não interfere na vida do paciente, até 100 (pontos ou %), quando o grau de incômodo é grave. A somatória dos pontos resultantes das questões foi categorizada em cinco grupos ou graus de gravidade. Para a análise estatística foi utilizado o software SSPS 20.0 e foram utilizadas as medidas resumos para descrição da amostra. Para verificar associação e correlação foram utilizados testes paramétricos com a correlação de Spearman. O nível de significância adotado foi de 5% (p<0,05) que foi destacado com asterisco quando presente. Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 802 802 Gois RO, Gois BO, Pereira MCCS, Taguchi CK Questão Sim Às vezes Não 1. Devido ao seu zumbido é difícil se concentrar? 2. O volume (intensidade) do seu zumbido faz com que seja difícil escutar as pessoas? 3. O seu zumbido deixa você nervoso? 4. O seu zumbido deixa você confuso? 5. Devido ao seu zumbido, você se sente desesperado? 6. Você se queixa muito do seu zumbido? 7. Devido ao seu zumbido, você tem dificuldade para pegar no sono à noite? 8. Você sente como se não pudesse se livrar do seu zumbido? 9. O seu zumbido interfere na sua capacidade de aproveitar atividades sociais (tais como sair para jantar, ir ao cinema)? 10. Devido ao seu zumbido, você se sente frustrado? 11. Devido ao seu zumbido, você pensa que tem uma doença grave? 12. O seu zumbido torna difícil aproveitar a vida? 13. O seu zumbido interfere nas suas tarefas no serviço e em casa? 14. Devido ao seu zumbido, você se sente frequentemente irritado? 15. Devido ao seu zumbido, você acha difícil ler? 16. O seu zumbido deixa você chateado? 17. Você sente que o seu zumbido atrapalha o seu relacionamento com a sua família e amigos? 18. RESULTADOS no MEEM e nota-se que metade das mulheres apresentou alteração no MEEM. Na Tabela 1 observa-se prevalência de indivíduos de gênero feminino se comparado aos de gênero masculino e verifica-se que a maioria da amostra (92,6%) é composta por indivíduos sem e de baixa escolaridade. Na Figura 4 nota-se que nos indivíduos com ≥ 1 e ≤ 8 anos de escolaridade existiu um predomínio de pessoas com alteração no MEEM. Todavia, naqueles sem e com ≥ 9 anos de escolaridade houve uma predominância de indivíduos sem alteração no MEEM. Na Tabela 2 observa-se que 50,9% da amostra pontuaram dentro dos padrões de normalidade Rev. CEFAC. RESULTADOS 2014 Mai-Jun; 16(3):798-809 803 Estado Mental e Zumbido em Idosos 8 Tabela 1 - Classifi cação percentual dos voluntários de gênero masculino e feminino e distribuição da população segundo escolaridade Homens Mulheres Escolaridade Quantidade Percentual Quantidade 4 104 sem escolaridade 40 37,0% ≥ 1 e ≤ 8 anos 60 55,6% Percentual 3,7% 96,30% ≥ 9 e ≤ 11 anos 5 4,6% ≥ 12 anos 3 2,8% Total 3,7% 96,30% 108 100,0% ssifi cação percentual dos voluntários de gênero masculino e feminino e distribuição d gundo escolaridade Tabela 1 - Classifi cação percentual dos voluntários de gênero masculino e feminino e distribuição da população segundo escolaridade houve um equilíbrio entre aqueles que apresen- tavam zumbido e possuíam ou não alteração no MEEM, como também existiu um equilíbrio t l ã t bid Os resultados apontaram que dentre os sujeitos que apresentaram relato de zumbido, 53,1% apresentaram alteração no MEEM; e entre aqueles ã f i i d bid 46 9% Tabela 2 - Distribuição percentual da relação: Gênero x Mini Exame do Estado Mental Gênero MEEM Total Com alteração Sem alteração Feminino Quantidade 52 52 104 Porcentagem 50% 50% 100% Masculino Quantidade 1 3 4 Porcentagem 25% 75% 100% Total Quantidade 53 55 108 Porcentagem 49,1% 50,9% 100% Figura 4 - Distribuição percentual da relação: Escolaridade x Mini Exame do Estado Mental Tabela 2 - Distribuição percentual da relação: Gênero x Mini Exame do Estado Mental Gênero MEEM Total Com alteração Sem alteração Feminino Quantidade 52 52 104 Porcentagem 50% 50% 100% Masculino Quantidade 1 3 4 Porcentagem 25% 75% 100% Total Quantidade 53 55 108 Porcentagem 49,1% 50,9% 100% bela 2 - Distribuição percentual da relação: Gênero x Mini Exame do Estado Mental Figura 4 - Distribuição percentual da relação: Escolaridade x Mini Exame do Estado Mental Figura 4 - Distribuição percentual da relação: Escolaridade x Mini Exame do Estado Mental Figura 4 - Distribuição percentual da relação: Escolaridade x Mini Exame do Estado Mental Os resultados apontaram que dentre os sujeitos que apresentaram relato de zumbido, 53,1% apresentaram alteração no MEEM; e entre aqueles que não referiram queixa de zumbido, 46,9% apresentaram MEEM alterado, denotando-se que houve um equilíbrio entre aqueles que apresen- tavam zumbido e possuíam ou não alteração no MEEM, como também existiu um equilíbrio entre aqueles que não apresentavam zumbido e possuíam ou não alteração no MEEM. Rev. CEFAC. Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 RESULTADOS Tabela 4 - Distribuição percentual da classifi cação quanto ao grau do zumbido e sua interferência na qualidade de vida Classifi cação do grau do zumbido Quantidade Percentual Interferência do zum- bido na qualidade de vida Desprezivel 7 10,9% Não Leve 20 31,3% Sim Moderado 14 21,9% Sim Severo 13 20,3% Sim Catastrófi co 10 15,6% Sim Total 64 100,0% 89,10% Figura 6 - Distribuição percentual da relação: Escolaridade X Classifi cação no Tinnitus Handicap Inventory DISCUSSÃO Nesta pesquisa, a maioria da amostra foi de ê f i i (96 3%) d i di íd d b i Tabela 4 - Distribuição percentual da classifi cação quanto ao grau do zumbido e sua interferência na qualidade de vida Tabela 4 - Distribuição percentual da classifi cação quanto ao grau do zumbido e sua interferência na qualidade de vida Classifi cação do grau do zumbido Quantidade Percentual Interferência do zum- bido na qualidade de vida Desprezivel 7 10,9% Não Leve 20 31,3% Sim Moderado 14 21,9% Sim Severo 13 20,3% Sim Catastrófi co 10 15,6% Sim Total 64 100,0% 89,10% uição percentual da classifi cação quanto ao grau do zumbido e sua interferência na a Tabela 4 - Distribuição percentual da classifi cação quanto ao grau do zumbido e sua qualidade de vida Figura 6 - Distribuição percentual da relação: Escolaridade X Classifi cação no Tinnitus Handicap Inventory Figura 6 - Distribuição percentual da relação: Escolaridade X Classifi cação no Tinnitus Handicap Inventory RESULTADOS 2014 Mai-Jun; 16(3):798-809 Gois RO, Gois BO, Pereira MCCS, Taguchi CK Na Tabela 3 constata-se que 59,30% dos indivíduos submetidos ao THI apresentaram queixa de zumbido, averigua-se que, daqueles que detinham essa queixa, mais da metade da amostra (60,90%) foi composta por indivíduos em que o mesmo era percebido em ambas as orelhas e verifi ca-se que 71,90% dos indivíduos que tinham zumbido, descreviam-no como agudo. Na Figura 5 denota-se que houve uma predo- minância da sensação desprezível/leve do zumbido naqueles com MEEM não alterado. Enquanto que naqueles com MEEM alterado houve uma pequena predominância dos indivíduos com sensação severo/catastrófi ca do zumbido. Figura 5 - Distribuição percentual da relação: Mini Exame do Estado Mental X Classifi cação no Tinnitus Handicap Inventory Figura 5 - Distribuição percentual da relação: Mini Exame do Estado Mental X Classifi cação no Tinnitus Handicap Inventory Tabela 3 - Distribuição percentual dos resultados do Tinnitus Handicap Inventory, classifi cação quanto à orelha predominante do zumbido e sensação sonora ou Pitch Presença de Zumbido Quantidade Percentual Sim 64 59,3% Orelha Pitch Direita Esquerda Ambas Agudo Grave 17,2% 21,9% 60,9% 71,9% 28,10% Não 44 40,7% Total 108 100,0% Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 805 Estado Mental e Zumbido em Idosos Na Tabela 4 averigua-se que quase metade da amostra detentora dessa alteração, (42,20%) foi composta por indivíduos em que o zumbido era percebido de maneira desprezível ou leve e que 89,10% dos indivíduos que se queixam de zumbido, este acaba interferindo na qualidade de vida. Na Figura 6 denota-se que houve um predomínio da sensação desprezível/leve e severo/catastrófi ca do zumbido nos grupos sem escolaridade, ≥1 e ≤ 8 e ≥ 12 anos de escolaridade. Enquanto que no grupo ≥ 9 e ≤ 11 anos de escolaridade somente existiu sensação Desprezível/leve do zumbido. DISCUSSÃO Nesta pesquisa, a maioria da amostra foi de gênero feminino (96,3%) e de indivíduos de baixa ou sem escolaridade (92,6%), o que concordou com estudo similar24. Os resultados relativos ao gênero confi rmaram a tendência encontrada na literatura nos estudos25 relacionados com os idosos segundo os quais, no Brasil, o número absoluto de mulheres Na aplicação do MEEM obteve-se a média de 21,7 pontos, o que foi concordante com as médias alcançadas por alguns estudos 21-24. Observou-se que a média de idade da amostra geral foi de 65,63 anos, aproximando-se dos resul- tados obtidos por alguns pesquisadores 24. Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 806 Gois RO, Gois BO, Pereira MCCS, Taguchi CK idosas tem sido superior quando confrontado com o de homens acima de 65 anos. entrevistada referiu presença de zumbido corrobo- rando com um estudo 31. A média da pontuação no THI da amostra foi de 26,94, o que discordou de estudo32. A análise estatística demonstrou que os resul- tados da aplicação do MEEM não revelaram associação com escolaridade, indo ao encontro de um estudo anterior26. Contudo, discordou de outros,6,24,27 que mostraram que o MEEM apresenta relação com a escolaridade. Nesta pesquisa, o zumbido acompanhado de pitch agudo e em ambas a orelhas foi o mais frequente e o grau do incômodo prevalente foi o leve, o que corrobora com alguns achados relatados na literatura 33-36, Quanto ao desempenho no MEEM, metade da amostra apresentou desempenho inferior à nota de corte estabelecida. A maior concentração ocorreu na faixa ≥ 1 e ≤ 8 anos de escolaridade (32,41%), concordando com alguns estudos23,28 e discordando de outros24,29. Observou-se, ainda, que 89,10% dos indivíduos que possuíam zumbido referiram algum tipo de interferência em suas qualidades de vida, concor- dando com alguns estudos14, 37e discordando de outros 38,39. A análise estatística revelou que não ocorreu relação entre o autorrelato das características psico- acústicas do zumbido e os resultados apresentados pelo MEEM. Porém, os achados apontaram que a maioria da população avaliada queixou-se de zumbido, o que se aproximou dos resultados alcan- çados por um estudo 38. DISCUSSÃO Uma pesquisa22 cujo objetivo era o estudo do MEEM, correlacionando-o com a idade e escola- ridade de idosos vivendo em comunidade, encon- traram a média de 21,97 pontos, sendo que entre os idosos com ≥ 1 e ≤ 8 anos de escolaridade, a média foi 23,85, concordando com os achados desta pesquisa que, nessa faixa de escolaridade, foi de 22,76 pontos. Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 ABSTRACT Purpose: to analyze the performance of the elderly people on the Mini Mental State Examination (MMSE); to verify the results of the Tinnitus Handicap Inventory (THI); to investigate the interference of the variables gender, schooling and tinnitus on the MMSE and THI scores; to verify possible relations with self-reported mental state of psychoacoustic characteristics and emotional domain of THI. Methods: clinical descriptive, exploratory qualitative and quantitative research. It was submitted to the MMSE and THI, 108 volunteers, male and female, aged ranged 60 to 80 years referred from Coordenadoria de Atenção Básica of Itabaiana – SE. The statistical analysis based on sample distribution and Spearman´s correlation with 0,05%. Results: the mean age was 65.63 years. The MMSE results were grouped into four levels of education: no schooling (37.0%), ≥ 1 to ≤ 8 years (55.6%), ≥ 9 to ≤ 11 years (4.6%) and ≥ 12 years (2.8%), the mean MMSE was 21.7 points. It was observed that 49.1% scored below the pattern, while 50.9% presented equals or exceeds the parameter used. On THI, we found that 59.3% presented tinnitus. It was observed that tinnitus interferes with the quality of life on 89.10% of the population. Conclusion: the majority of the elderly people presented abnormal MMSE. It was verify no significant association between gender, schooling and MMSE score and THI and the majority of the participants in this study reported impaired quality of life associated with tinnitus. No relation between abuse of psychoacoustic characteristics of tinnitus and results displayed by the MMSE, however, the data showed that the majority of the population tested complained of tinnitus. KEYWORDS: Aged; Tinnitus; Cognition Disorders; Diagnosis; Quality of Life CONCLUSÃO Depreende-se deste estudo que nem todos os voluntários de baixa escolaridade pontuaram abaixo do corte estabelecido, o que concordou com os resultados verificados em algumas pesquisas30. Uma parcela expressiva dos participantes apresentou alteração no MEEM. Não existiu associação significante entre gênero, escolaridade e pontuação do MEEM e THI e a maioria dos partici- pantes desta pesquisa referiu prejuízo na qualidade de vida com associação ao zumbido. Inexistiu relação entre queixa das características psicoacús- ticas do zumbido e resultados exibidos pelo MEEM, todavia, os achados apontaram que a maioria da população testada autorrelataram presença de zumbido. A distribuição percentual apontou que a maioria dos voluntários avaliados pelo MEEM não apresentou escores indicativo de alteração cognitiva, ressaltando-se que foram adotados padrões diferenciados20 conforme apresentado anteriormente. Em relação ao THI, verificou-se que a média de idade dos voluntários com alteração no THI foi de 65,06 anos, sendo que 59,3% da amostra 807 Estado Mental e Zumbido em Idosos ABSTRACT Purpose: to analyze the performance of the elderly people (MMSE); to verify the results of the Tinnitus Handicap Invento of the variables gender, schooling and tinnitus on the MMS relations with self-reported mental state of psychoacoustic of THI. Methods: clinical descriptive, exploratory qualitati submitted to the MMSE and THI, 108 volunteers, male and referred from Coordenadoria de Atenção Básica of Itabaiana on sample distribution and Spearman´s correlation with 0,05 years. The MMSE results were grouped into four levels of ed 8 years (55.6%), ≥ 9 to ≤ 11 years (4.6%) and ≥ 12 years (2. It was observed that 49.1% scored below the pattern, while the parameter used. On THI, we found that 59.3% presente interferes with the quality of life on 89.10% of the population. people presented abnormal MMSE. It was verify no significant and MMSE score and THI and the majority of the participants of life associated with tinnitus. No relation between abuse of p and results displayed by the MMSE, however, the data show tested complained of tinnitus. 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Bento RF, Sanches TG, Minitti A, Câmara J. Zumbido: características e epidemiologia. Rev Bras Otorrinolaringol. 1997;63(2):229-38. REFERÊNCIAS Correlação entre os achados audiológicos e incômodo com zumbido / Correlation between the audiologic findings and tinnitus disorder. Arq. int.otorrinolaringol. (Impr.) 2011;15(2):172-80. 23. Converso MER, Iartelli I. Caracterização e análise do estado mental e funcional de idosos institucionalizados em instituições públicas de longa permanência. Analysis and characterization of functional capacity and mental state in residents in old folk’s home. J Bras Psiquiatria. 2007;56(4):267-72. 32. Pinto PCL, Sanchez TG, Tomita S. Avaliação da relação entre severidade do zumbido e perda auditiva, sexo e idade do paciente / The impact of gender, age and hearing loss on tinnitus severity. Braz. j. otorhinolaryngol. (Impr.). 2010;76(1):18-24. 33. Morais AA, Gil D. Zumbido em indivíduos sem perda auditiva e sua relação com a disfunção temporomandibular / Tinnitus in individuals without hearing loss and its relation ship with temporomandibular dysfunction. Braz. j. otorhinolaryngol. (Impr.) 2012;78(2):59-65. 24. Machado JC, Ribeiro RCL, Cotta RMM, Leal PFG. Declínio cognitivo de idosos e sua associação com fatores epidemiológicos em Viçosa, Minas Gerais / Cognitive decline ofagedand its association with epidemiological factors in thecityof Viçosa, Minas Gerais. Rev Bras Geriatr Gerontol. 2011;14(1):109-21. 34. Urnau D, Tochetto TM. Características do zumbido e da hiperacusia em indivíduos normo- ouvintes / Characteristics of the tinnitus and hyperacusis in normal hearing individuals. Arq. int. otorrinolaringol. (Impr.) 2011;15(4):468-74. 25. Lebrão ML, Laurenti R. Saúde, bem-estar eenvelhecimento: o estudo Sabe no Município de São Paulo. Rev.Bras. Epidemiol. 2005;8(2):127-41. 26. Nagato AC, Santos MG, Martins TFP, Valença SS, Bezerra FS, Serva MV. Avaliação cognitiva de idosas institucionalizadas através do mini-exame do estado mental com ou sem tratamento fisioterapêutico. Cognitive evaluation of elderly through the mini-mental state exam with or without physical therapy. Fisioterapia Brasil. 2007;8(4):233-8. 35. Pinto PCL, Hoshino AC, Tomita S. Característica dos pacientes com queixa de zumbido atendidos em ambulatório especializado - HUCFF. Cad. Saúde Colet. 2008;16(3):437-48. 36. Sanchez TG, Bento RF, Miniti A, Cârnara J. Zumbido: Características e epidemiologia. Experiência do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Rev Bras Otorrinolaringol. 1997; 63(3):229- 35. 27. Kochhann R, Cerveira MO, Godinho C, Camozzato A, Chaves MLF. Avaliação dos escores 27. Kochhann R, Cerveira MO, Godinho C, Camozzato A, Chaves MLF. Avaliação dos escores Rev. CEFAC. 2014 Mai-Jun; 16(3):798-809 809 Estado Mental e Zumbido em Idosos 37. Casaprima V. 37. Casaprima V. Estudio descriptivo sobre las caracteristicas del acufeno en pacientes adultos que concurren a una clinica privada de ORL de la ciudad de Rosario [Dissertação]. Rosário: Facultad de Ciencias Medicas, Escuela de Fonoaudiologia; 2001. REFERÊNCIAS Estudio descriptivo sobre las caracteristicas del acufeno en pacientes adultos que concurren a una clinica privada de ORL de la ciudad de Rosario [Dissertação]. Rosário: Facultad de Ciencias Medicas, Escuela de Fonoaudiologia; 2001. 39. Almeida TAS, Samelli AG, Mecca FDN, Martino ED.Sensação subjetiva do zumbido pré e pós intervenção nutricional em alterações metabólicas / Tinnitus sensation pre and post nutritional intervention in metabolic disorders. Pró-fono R Atual Cient. 2009;21(4):291-7. 39. Almeida TAS, Samelli AG, Mecca FDN, Martino ED.Sensação subjetiva do zumbido pré e pós intervenção nutricional em alterações metabólicas / Tinnitus sensation pre and post nutritional intervention in metabolic disorders. Pró-fono R Atual Cient. 2009;21(4):291-7. http://dx.doi.org/10.1590/1982-0216201425212 Recebido em: 25/10/2012 Aceito em: 25/04/2013 Endereço para correspondência: Rafael Oliveira Gois Rua Professor Lima Júnior, 801 – Centro Itabaiana – Sergipe - Brasil CEP: 49500-000 E-mail: rafaelgois1989@hotmail.com
https://openalex.org/W3162406794	https://figshare.com/articles/journal_contribution/Estimation_of_shape_constrained_additive_models_with_missing_response_at_random/14593434/1/files/28004760.pdf	English	null	Estimation of shape constrained additive models with missing response at random	Journal of nonparametric statistics	2,021	cc-by	4,179	Supplementary materials for “Estimation of shape constrained additive models with missing response at random” The supplementary materials are organized as follows. Section A presents the conditions re- quired in our asymptotic analysis. Section B includes necessary lemmas and technical proofs. Sec- tion C provides proofs of theorems in the main paper. Appendix A. Assumptions The asymptotic analysis in the main paper requires the following assumptions. (A1) The predictor variables Xi are i.i.d. with support set [0,1]d. Its joint density function, denoted by f (x), is continuous and 0 < c1 ≤f (x) ≤c2 < ∞, for x ∈[0,1]d and positive constants c1 and c2. (A2) The errors {ϵi}n i=1 are i.i.d. with E (ϵi) = 0 and Var (ϵi) = σ2 < ∞. There exists a constant c3 > 0, such that E \|ϵi\|2+ηX ≤c3 a.s. for some η > 0. (A3) The knot sequence 0 = u0 < u1 < ··· < uNn < uNn+1 = 1 is equally spaced on [0,1] with uj = j/(Nn + 1), for j = 0,1,··· , Nn + 1. (A4) The number of interior knots Nn satisﬁes N 3 n log(n0)/n0 →0 and N 2p+5log (n )/n →∞as n →∞ (A5) For 1 ≤l ≤d, the additive function αl is monotone increasing and (p + 1)-times continu- ously differentiable for some integer p ≥1. We assume that there exists a constant c4 > 0, such that α′ l (x) ≥c4, for x ∈[0,1]. (A5) For 1 ≤l ≤d, the additive function αl is concave and (p + 1)-times continuously differ- entiable for some integer p ≥2. Furthermore, we assume that there exists a constant c5 < 0, such that α(2) l (x) ≤c5, for x ∈[0,1]. Assumption (A1) is the same as condition 1 in [1]. Assumption (A2) requires that the error terms are i.i.d. with a common distribution. Assumption (A3) assumes that the interior knots are equally spaced in the interval [0,1], which is required to simplify the linear constraints derived in Lemmas 2 and 3 in the main paper. One can relax this condition to the one considered in [2], [3] and [4], which requires the knot sequence to be pseudo equally spaced. The assumption for the number of interior knots and samples size are give in the assumption (A4). This assumption is satisﬁed by Nn with the optimal order that Nn = O(n 1 2p+3 0 ). Additionally, for the monotone constrained estimator, Assumption (A5) assumes that each additive function is monotone increasing and its ﬁrst order derivative is bounded below from zero. Similarly, for the concave constrained estimator, Assumption (A5∗) requires that each additive function is concave and its second order derivative is bounded above from zero. Appendix B. Lemmas and Proofs Then we have Then we have sup x∈[0,1] \|I2\| = sup x∈[0,1] BT l (x)ABT n0lWY∗ −l = sup x∈[0,1] BT l (x)ABT n0lW(αl + ϵ) = Op Nnn−1 0 n−1/2 × N −1/2 n n1/2 0 = Op N 1/2 n n−1/2 0 n−1/2 , (S.3) sup x∈[0,1] \|I3\| = sup x∈[0,1] BT l (x) BT n0lWBn0l −1 BT n0l ˆW −W Y∗ −l = sup x∈[0,1] BT l (x) BT n0lWBn0l −1 BT n0l ˆW −W (αl + ϵ) = Op Nnn−1 0 × N −1/2 n n1/2 0 n−1/2 = Op N 1/2 n n−1/2 0 n−1/2 , (S.4) sup x∈[0,1] \|I2\| = sup x∈[0,1] BT l (x)ABT n0lWY∗ −l = sup x∈[0,1] BT l (x)ABT n0lW(αl + ϵ) = Op Nnn−1 0 n−1/2 × N −1/2 n n1/2 0 = Op N 1/2 n n−1/2 0 n−1/2 , (S.3) −1 (S.3) p 0 n 0 p n 0 sup x∈[0,1] \|I3\| = sup x∈[0,1] BT l (x) BT n0lWBn0l −1 BT n0l ˆW −W Y∗ −l = sup x∈[0,1] BT l (x) BT n0lWBn0l −1 BT n0l ˆW −W (αl + ϵ) = Op Nnn−1 0 × N −1/2 n n1/2 0 n−1/2 = Op N 1/2 n n−1/2 0 n−1/2 , (S.4) x∈[0,1] x∈[0,1] 0 0 = sup x∈[0,1] BT l (x) BT n0lWBn0l −1 BT n0l ˆW −W (αl + ϵ) = Op Nnn−1 0 × N −1/2 n n1/2 0 n−1/2 = Op N 1/2 n n−1/2 0 n−1/2 , (S.4) (S.4) = sup x∈[0,1] BT l (x)ABT n0l ˆW −W Y∗ −l = sup x∈[0,1] BT l (x)ABT n0l ˆW −W (αl + ϵ) = O N n−1n−1/2 × N −1/2n1/2 0 n−1/2 = O N 1/2n−1/2 0 n−1 (S 5) sup x∈[0,1] I4 = sup x∈[0,1] BT l (x)ABT n0l ˆW −W Y∗ −l = sup x∈[0,1] BT l (x)ABT n0l ˆW −W (αl + ϵ) = Op Nnn−1 0 n−1/2 × N −1/2 n n1/2 0 n−1/2 = Op N 1/2 n n−1/2 0 n−1 . Appendix B. Lemmas and Proofs For each l = 1,..., d, let α∗ l be the one-step backﬁtted unconstrained estimator of αl when all the other additive components are known. In this case, it reduces to the univariate polynomial spline smoothing. Deﬁne Y ∗ i,−l = Yi − P l′̸=l αl′ (Xil′) = αl (Xil) + ϵi, and let Y∗ −l = Y ∗ 1,−l,..., Y ∗ n,−l T , for l = 1,..., d. Then α∗ l (x) = BT l (x)β∗ l where β∗ l = BT n0l ˆWBn0l −1 BT n0l ˆWY∗ −l. Note that α∗ l (x) d l=1 are constructed only for proving the theoretical results. The true sampling weights matrix is denoted by W in the proofs. Lemma A.1 Under regularity conditions (A1)-(A4), for l = 1,..., d, α∗ l −αl 2 = Op Æ Nn/n0 + N −p−1 n . Lemma A.1 Under regularity conditions (A1)-(A4), for l = 1,..., d, α∗ l −αl 2 = Op Æ Nn/n0 + N −p−1 n . Proof. By deﬁnition and Taylor expansion, we have α∗ l (x) = BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆWY∗ −l = BT l (x) BT n0lWBn0l + BT n0l ˆW −W Bn0l −1 BT n0l W + ˆW −W Y∗ −l = BT l (x) h BT n0lWBn0l −1 + A i BT n0l W + ˆW −W Y∗ −l = BT l (x) BT n0lWBn0l −1 BT n0lWY∗ −l + BT l (x)ABT n0lWY∗ −l +BT l (x) BT n0lWBn0l −1 BT n0l ˆW −W Y∗ −l +BT l (x)ABT n0l ˆW −W Y∗ −l = BT l (x) BT n0lWBn0l −1 BT n0lWY∗ −l + BT l (x)ABT n0lWY∗ −l +BT l (x) BT n0lWBn0l −1 BT n0l ˆW −W Y∗ −l +BT l (x)ABT n0l ˆW −W Y∗ −l = I1 + I2 + I3 + I4, in which A = − BT n0lWBn0l −1 BT n0l ˆW −W Bn0l BT n0lWBn0l −1 + ∞ X k=2 (−1)−k BT nlWBnl −1 BT n0l ˆW −W Bn0l k BT n0lWBn0l −1 . Appendix B. Lemmas and Proofs (S.1) (S.1) 2 Theorem 1 in [2] holds when using the weighted least squares estimation, thus one has ∥I1 −αl∥2 = BT l (x) BT n0lWBn0l −1 BT n0lWY∗ −l −αl 2 = Op Æ Nn/n0 + N −p−1 n . (S.2) (S.2) Note that sup x \|Bl (x)\| = Op (1) and the number of nonzero elemente in Bl (x) is bounded. Let λmin and λmax be the smallest and largest eigenvalues of n0 −1BT n0lBn0l, respectively. Then by The- orem 5.4.2 in [5], λmin ≍λmax ≍N −1 n , where ≍means both sides have the same order. Given the weight is bounded away from zero and inﬁnity and sup ˆW −W = Op n−1/2 [6], we have sup BT n0lWBn0l −1 = Op Nnn−1 0 , sup BT n0l ˆW −W Bn0l = Op N −1 n n0n−1/2 , thus sup\|A\| = Op Nnn−1 0 n−1/2 , sup BT n0lWϵ = Op N −1/2 n n1/2 0 , and sup BT n0l ˆW −W ϵ = Op N −1/2 n n1/2 0 n−1/2 . Appendix B. Lemmas and Proofs (S.5) (S.5) From equations (S.1), (S.2), (S.4), (S.4) and (S.5), one has From equations (S.1), (S.2), (S.4), (S.4) and (S.5), one has α∗ l −αl 2 ≤ ∥I1 −αl∥2 + sup\|I2\| + sup\|I3\| + sup I4 = Op Æ Nn/n0 + N −p−1 n , and then Lemma A.1 follows. and then Lemma A.1 follows. and then Lemma A.1 follows. Lemma A.2 Under regularity conditions (A1)-(A4), for l = 1,..., d, ∥˜αl −αl∥2 = Op Æ Nn/n0 + N −p−1 n . Lemma A.2 Under regularity conditions (A1)-(A4), for l = 1,..., d, ∥˜αl −αl∥2 = Op Æ Nn/n0 + N −p−1 n . Lemma A.2 Under regularity conditions (A1)-(A4), for l = 1,..., d, ∥˜αl −αl∥2 = Op Æ Nn/n0 + N −p−1 n . ∥˜αl −αl∥2 = Op Æ Nn/n0 + N −p−1 n . Proof. By deﬁnition, one has Proof. By deﬁnition, one has Proof. By deﬁnition, one has ˜αl (x) = BT l (x) ˜βl = BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆWY−l ˜αl (x) = BT l (x) ˜βl = BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆWY−l 3 = BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW Y − X l′̸=l ˜αl′ = BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW(αl + ϵ) +BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW X l′̸=l (αl′ −˜αl′) = α∗ l + BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW X l′̸=l (αl′ −˜αl′) = α∗ l + I. = BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW(αl + ϵ) +BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW X l′̸=l (αl′ −˜αl′) ̸ = α∗ l + BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW X l′̸ l (αl′ −˜αl′) l′̸=l = α∗ l + I. (S.6) l ̸=l = α∗ l + I. Appendix B. Lemmas and Proofs 4 Applying the inequality sup ϕ∈G(p) 〈ϕ,1〉n0 −〈ϕ,1〉 ∥ϕ∥−1 = Op n−1/2 0 N 1/2 n log1/2 n0 , one has 〈Bl,1〉n0 ≤ \|〈Bl,1〉\| + ∥Bl∥× Op n−1/2 0 N 1/2 n log1/2 n0 〈Bl,1〉n0 ≤ \|〈Bl,1〉\| + ∥Bl∥× Op n−1/2 0 N 1/2 n log1/2 n0 = O N −1 + N −1/2 × n−1/2N 1/2 log1/2 n = O N 〈Bl,1〉n0 ≤ \|〈Bl,1〉\| + ∥Bl∥× Op n−1/2 0 N 1/2 n log1/2 n0 = Op N −1 n + N −1/2 n × n−1/2 0 N 1/2 n log1/2 n0 = Op N −1 n , 0 p n = Op N −1 n + N −1/2 n × n−1/2 0 N 1/2 n log1/2 n0 = Op N −1 n , then we obtains that then we obtains that sup x∈[0,1] \|I1\| = Op Æ Nn/n0 + N −p−1 n , (S.9) sup x∈[0,1] \|I2\| = Op n−1/2Æ Nn/n0 + n−1/2N −p−1 n . (S.10) Finally, Lemma A.2 follows from (S.6), (S.7), (S.8), (S.9), and (S.10). (S.9) (S.10) Finally, Lemma A.2 follows from (S.6), (S.7), (S.8), (S.9), and (S.10). Lemma A.3 For any function α that satisﬁes condition (A5) with p = 2,3 and large enough n, there exists a spline function g ∈G(p) whose coefﬁcients satisfy the concave constraints such that ∥α −g∥∞≤c α(p+1) ∞N −p−1 n , for some constant c > 0. Lemma A.3 For any function α that satisﬁes condition (A5) with p = 2,3 and large enough n, there exists a spline function g ∈G(p) whose coefﬁcients satisfy the concave constraints such that ∥α −g∥∞≤c α(p+1) ∞N −p−1 n , for some constant c > 0. Proof. According to Theorem 1.51 in [7], for any α ∈C p+1 [0,1], there exists a function g ∈G(p), such that ∥α −g∥∞≤c α(p+1) ∞N −p−1 n and α(2) −g(2) ∞≤c α(p+1) ∞N −p+1 n , for some constant c > 0 and p > 1. By condition (A5), α(2) (x) ≤c1 < 0 for some constant c1 and any x ∈[0,1]. Then for large sample size, one has g(2) ≤ α(2) −g(2) ∞+ α(2) ≤c1/2 < 0, therefore g is a concave function. therefore g is a concave function. Given α ∈C p+1 [0,1], we have α′ ∈C p [0,1]. Appendix B. Lemmas and Proofs (S.6) (S.6) Lemma A.1 gives that Lemma A.1 gives that Lemma A.1 gives that α∗ l −αl 2 = Op Æ Nn/n0 + N −p−1 n (S.7) (S.7) By Taylor expansion, we have By Taylor expansion, we have By Taylor expansion, we have I = BT l (x) BT n0l ˆWBn0l −1 BT n0l ˆW X l′̸=l (αl′ −˜αl′) = BT l (x) h BT n0l ˆWBn0l −1 + A i BT n0l W + ˆW −W X l′̸=l (αl′ −˜αl′) = BT l (x) h BT n0l ˆWBn0l −1 + A i BT n0lW X l′̸=l (αl′ −˜αl′) +BT l (x) h BT n0l ˆWBn0l −1 + A i BT n0l ˆW −W X l′̸=l (αl′ −˜αl′) = I1 + I2, (S.8) in which A is deﬁned in (S.1). Since sup x \|Bl (x)\| = Op (1), sup BT n0lWBn0l −1 + A = Op Nnn−1 0 , and sup ˆW −W = Op n−1/2 , one has sup x∈[0,1] \|I1\| = sup x∈[0,1] BT l (x) h BT n0l ˆWBn0l −1 + A i BT n0lW X l′̸=l (αl′ −˜αl′) = Op Nnn−1 0 × n0 × n−1 0 BT n0lW X l′̸=l (αl′ −˜αl′) = Op (Nn) × 〈Bl,1〉n0 × Op Æ Nn/n0 + N −p−1 n , sup x∈[0,1] \|I2\| = sup x∈[0,1] BT l (x) h BT n0l ˆWBn0l −1 + A i BT n0l ˆW −W X l′̸=l (αl′ −˜αl′) = Op Nnn−1 0 × n0 × n−1 0 BT n0l ˆW −W X l′̸=l (αl′ −˜αl′) = Op (Nn) × 〈Bl,1〉n0 × Op n−1/2 × Op Æ Nn/n0 + N −p−1 n . Appendix B. Lemmas and Proofs By Lemma 3 of [4], for p ≤3 and large enough n, there exists a ˆg′ ∈G(p−1) whose coefﬁcients satisfy the monotone constraints, such that ∥α′ −ˆg′∥∞≤c2 α(p+1) ∞N −p n , for some constant c2 > 0. With ˆg (x) = R ˆg′ (x) d x ∈G(p) and follow the similar arguments in the proof of Lemma 3 in [4], we can easily show that ∥g −ˆg∥∞≤c2 α(p+1) ∞N −p−1 n . Furthermore, in the proof of Lemma 3 in [4], we showed that the spline coefﬁcients of ˆg(2) are non-positive which suggests that the coefﬁcients of ˆg satisfy the concave constraints. Therefore, Lemma A.3 follows. Lemma A.4 Under regularity conditions (A1)-(A5), for l = 1,..., d and p ≤3, there exists a spline function gl = JnP j=1 γl jBl j whose coefﬁcients satisfy the monotone constraints such that Lemma A.4 Under regularity conditions (A1)-(A5), for l = 1,..., d and p ≤3, there exists a spline function gl = JnP j=1 γl jBl j whose coefﬁcients satisfy the monotone constraints such that sup j β∗ l j −γl j = Op   v u tN 3 n log n0 n0  . 5 Therefore, the coefﬁcients of α∗ l satisfy the monotone constraints with probability approaching to 1 as n0 →∞. Proof. By Lemma 3 of [4], there exists a spline function gl whose coefﬁcients satisfy the monotone constraints such that ∥αl −gl∥∞= Op N −p−1 n . By deﬁnition, we have Proof. By Lemma 3 of [4], there exists a spline function gl whose coefﬁcients satisfy the monotone constraints such that ∥αl −gl∥∞= Op N −p−1 n . By deﬁnition, we have β∗ l = BT n0l ˆWBn0l −1 BT n0l ˆW(αl + ϵ) = BT n0l ˆWBn0l −1 BT n0l ˆW(gl + αl −gl + ϵ) = BT n0l ˆWBn0l −1 BT n0l ˆWgl + BT n0l ˆWBn0l −1 BT n0l ˆW(αl −gl) + BT n0l ˆWBn0l −1 BT n0l ˆWϵ = I + II + III. (S.11) (S.11) = I + II + III. (S.11) = I + II + III. = I + II + III. Given gl = Bn0lγl, then Given gl = Bn0lγl, then Given gl = Bn0lγl, then I = BT n0l ˆWBn0l −1 BT n0l ˆWgl = γl. Appendix B. Lemmas and Proofs Lemma A.5 Under regularity conditions (A1)-(A4),(A5), for l = 1,..., d and p = 2,3, there exists a spline function gl = JnP j=1 γl jBj whose coefﬁcients satisfy the concave constraints such that Lemma A.5 Under regularity conditions (A1)-(A4),(A5), for l = 1,..., d and p = 2,3, there exists a spline function gl = JnP j=1 γl jBj whose coefﬁcients satisfy the concave constraints such that Lemma A.5 Under regularity conditions (A1)-(A4),(A5*), for l = 1,..., d and p = 2,3, there exists a spline function gl = JnP j=1 γl jBj whose coefﬁcients satisfy the concave constraints such that sup j γl j −β∗ l j = Op   v u tN 3 n log n0 n0  . Therefore, the coefﬁcients of m∗ l satisﬁes the concave constraints with probability approaching to 1 as n0 →∞. Therefore, the coefﬁcients of m∗ l satisﬁes the concave constraints with probability approaching to 1 as n0 →∞. Proof. For p = 2,3, by Lemma A.3, there exists a spline function gl whose coefﬁcients satisfy the concave constraints such that ∥αl −gl∥∞= Op N −p−1 n . By deﬁnition, one has Proof. For p = 2,3, by Lemma A.3, there exists a spline function gl whose coefﬁcients satisfy the concave constraints such that ∥αl −gl∥∞= Op N −p−1 n . By deﬁnition, one has β∗ l = BT n0l ˆWBn0l −1 BT n0l ˆW(αl + ϵ) = BT n0l ˆWBn0l −1 BT n0l ˆW(gl + αl −gl + ϵ) = BT n0l ˆWBn0l −1 BT n0l ˆWgl + BT n0l ˆWBn0l −1 BT n0l ˆW(αl −gl) + BT n0l ˆWBn0l −1 BT n0l ˆWϵ = I + II + III. (S.15) (S.15) = I + II + III. = I + II + III. (S.15) Write gl = Bnlγl, then I = BT n0l ˆWBn0l −1 BT n0l ˆWgl = γl. (S.16) (S.16) Follow the similar arguments in the proof of Lemma A.4, we can easily show that Follow the similar arguments in the proof of Lemma A.4, we can easily show that sup\|II\| = Op N −p−1 n , (S.17) sup\|III\| = Op   v u tN 3 n log n0 n0  . (S.18) (S.17) (S.18) Lemma A.5 follows from equations (S.15), (S.16), (S.17), (S.18), and assumption (A4). Appendix B. Lemmas and Proofs (S.12) (S.12) By Taylor expansion, By Taylor expansion, II = BT n0l ˆWBn0l −1 BT n0l ˆW(αl −gl) = h BT n0lWBn0l −1 + A i BT n0l W + ˆW −W (αl −gl) = h BT n0lWBn0l −1 + A i BT n0lW(αl −gl) + h BT n0lWBn0l −1 + A i BT n0l ˆW −W (αl −gl) = II1 + II2, III = BT n0l ˆWBn0l −1 BT n0l ˆWϵ = h BT n0lWBn0l −1 + A i BT n0l W + ˆW −W ϵ = h BT n0lWBn0l −1 + A i BT n0lWϵ + h BT n0lWBn0l −1 + A i BT n0l ˆW −W ϵ = III1 + III2, in which A is deﬁned in (S.1). Since sup BT n0lWBn0l −1 + A = Op Nnn−1 0 , ∥αl −gl∥∞= Op N −p−1 n , and sup ˆW −W = Op n−1/2 , similar arguments at these in (S.9) and (S.10) gives that sup\|II1\| = Op N −p−1 n and sup\|II2\| = Op n−1/2N −p−1 n , thus sup\|II\| = Op N −p−1 n (S.13) (S.13) Similar arguments in the proof of Lemma 4 in [4] gives that sup n−1 0 BT n0lWϵ = Op Ç Nn log n0 n0 and sup n−1 0 BT n0l ˆW −W ϵ = Op Ç Nn log n0 nn0 , hence one has 6 sup\|III1\| = Op r N3 n log n0 n0 and sup\|III2\| = Op r N3 n log n0 nn0 , then we obtain sup\|III\| = Op   v u tN 3 n log n0 n0   (S.14) sup\|III1\| = Op r N3 n log n0 n0 and sup\|III2\| = Op r N3 n log n0 nn0 , then we obtain sup\|III\| = O   v u tN 3 n log n0   (S sup\|III\| = Op   v u tN 3 n log n0 n0   (S.14) (S.14) From equations (S.11), (S.12), (S.13), (S.14), and assumption (A4), we have sup β∗ l −γl = sup\|II + III\| = Op r N3 n log n0 n0 and then Lemma A.4 follows. Proof of Theorem 1 By deﬁnition and Taylor expansion, we have By deﬁnition and Taylor expansion, we have ˜βl = BT n0l ˆWBn0l −1 BT n0l ˆWY−l = BT n0l ˆWBn0l −1 BT n0l ˆW Y − X l′̸=l ˜αl′ = BT n0l ˆWBn0l −1 BT n0l ˆW(αl + ϵ) + BT n0l ˆWBn0l −1 BT n0l ˆW X l′̸=l (αl′ −˜αl′) = β∗ l + h BT n0l ˆWBn0l −1 + A i BT n0l W + ˆW −W X l′̸=l (αl′ −˜αl′) = β∗ l + h BT n0l ˆWBn0l −1 + A i BT n0lW X l′̸=l (αl′ −˜αl′) + h BT n0l ˆWBn0l −1 + A i BT n0l ˆW −W X l′̸=l (αl′ −˜αl′) = β∗ l + I1 + I2. (S.19) l ̸ l = β∗ l + I1 + I2. (S.19) By Lemma A.4, for any ﬁxed l = 1,...d, the coefﬁcient β∗ l satisﬁes the monotone constraints with probability approaching to 1 as n →∞and sup β∗ l −γl = Op   v u tN 3 n log n0 n0   (S.20) (S.20) Furthermore, similar arguments at these in (S.9) and (S.10) gives that sup\|I1\| = Op p Nn/n0 + N −p−1 n and sup\|I2\| = Op n−1/2p Nn/n0 + n−1/2N −p−1 n , thus sup\|I1 + I2\| = Op Æ Nn/n0 + N −p−1 n , (S.21) Furthermore, similar arguments at these in (S.9) and (S.10) gives that sup\|I1\| = Op p Nn/n0 + N −p−1 n and sup\|I2\| = Op n−1/2p Nn/n0 + n−1/2N −p−1 n , thus sup\|I1 + I2\| = Op Æ Nn/n0 + N −p−1 n , (S.2 (S.21) Finally, Theorem 1 follows From equations (S.19), (S.20), (S.21), and assumption (A4). Appendix B. Lemmas and Proofs Lemma A.5 follows from equations (S.15), (S.16), (S.17), (S.18), and assumption (A4). 7 Proof of Theorem 2 Follow the similar arguments in the proof of Theorem 1 and with Lemma A.5, Theorem 2 can be easily proved. Follow the similar arguments in the proof of Theorem 1 and with Lemma A.5, Theorem 2 can be easily proved. Proof of Theorem 3 When each αl is monotone increasing, Theorem 1 shows that for p ≤3, the coefﬁcients of unconstrained estimator ˜αl satisfy the monotone constraints with probability approaching 1 as n0 → 8 ∞. Similarly, when each αl is concave, for p ≤3 Theorem 2 indicates that the coefﬁcients of the ˜αl satisfy the concave constraints when sample size is large enough. These imply that for p ≤3, the unconstrained estimator ˜αl and the shape constrained estimator ˆαl are identical with probability approaching 1 as n0 →∞. Therefore, ˆαl enjoys the same asymptotic properties as ˜αl. Then by Lemma A.2, we have ∥ˆαl −αl∥= Op p Nn/n0 + N −p−1 n . References [1] Charles J Stone et al. Additive regression and other nonparametric models. The annals of Statistics, 13(2):689–705, 1985. [2] Jianhua Z Huang et al. Projection estimation in multiple regression with application to functional anova models. The annals of statistics, 26(1):242–272, 1998. [3] Lan Xue and Lijian Yang. Additive coefﬁcient modeling via polynomial spline. Statistica Sinica, 16(4):1423, 2006. [4] Lu Wang and Lan Xue. Constrained polynomial spline estimation of monotone additive models. Journal of Statistical Planning and Inference, 167:27–40, 2015. [5] Ronald A DeVore and George G Lorentz. Constructive approximation, volume 303. Springer Science & Business Media, 1993. [6] Erich Leo Lehmann. Elements of large-sample theory. Springer Science & Business Media, 2004. [7] Larry Schumaker. Spline functions: basic theory. Cambridge University Press, 2007. 9 9
https://openalex.org/W2101474141	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/1471-2458-13-1023	English	null	A public private partnership to fight against malaria along the Chad-Cameroon pipeline corridor: I. Baseline data on socio-anthropological aspects, knowledge, attitudes and practices of the population concerning malaria	BMC public health	2,013	cc-by	8,029	Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 * Correspondence: roger.moyou@yahoo.fr 1Department of Microbiology, Hematology, Parasitology & Infectious Diseases, Faculty of Medicine & Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon 2Institute of Medical Research and Medicinal Plants Studies (IMPM), Yaoundé, Cameroon Full list of author information is available at the end of the article A public private partnership to fight against malaria along the Chad-Cameroon pipeline corridor: I. Baseline data on socio-anthropological aspects, knowledge, attitudes and practices of the population concerning malaria A public private partnership to fight against malaria along the Chad-Cameroon pipeline corridor: I. Baseline data on socio-anthropological aspects, knowledge, attitudes and practices of the population concerning malaria Roger Moyou-Somo1,2, Paul Essomba1, Eva Songue1,3, Natacha Nsiewe Tchoubou4, Anita Ntambo5, Huguette Ngo Hiol1, Jacques Pokam Kemajou6, Marie-José Essi5 and Pascal Millet3 RESEARCH ARTICLE Open Access Correspondence: roger.moyou@yahoo.fr 1Department of Microbiology, Hematology, Parasitology & Infectious Diseases, Faculty of Medicine & Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon 2Institute of Medical Research and Medicinal Plants Studies (IMPM), Yaoundé, Cameroon Full list of author information is available at the end of the article © 2013 Moyou-Somo 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. (Continued from previous page) (Continued from previous page) Results: Interviews were conducted in 2597 households (Bipindi 399, Bélabo 835, in Meidougou 820 and Dompta 543). Whatever the study site, 50% of the heads of household were workers of the agro-pastoral sector. Most of the heads of household were men (average 77.4% for men and 22.6% for females). The walls of households were mostly made-up of earth blocks and access to media was low. There were significant differences between mean ages and educational level of the heads of household. Significant differences were also observed between the characteristics of houses and the sites located in the southern regions (Bipindi and Bélabo) and those located in the northern regions (Meidougou and Dompta). The later household heads were younger and less educated than those in the other regions. In most of the study sites, paracetamol was cited as the first intention drug for malaria treatment, followed by chloroquine, a banned drug. More than half of the households studied had a correct knowledge of malaria and its mode of transmission: 120/155 (77.1%) in Bipindi, 244/323 (74.5%) in Bélabo, 171/235 (72.8%) in Meidougou and 118/218 (54.1%) in Dompta. Fever and headache were the malaria signs/symptoms most often cited by the households. An important percentage of pregnant women did not take any malaria prophylaxis during their last pregnancy (up to 43.4% in Bélabo). Conclusion: In all the study sites, there were conditions that indicated the all year round transmission of malaria (characteristics of houses and limited access to media making sensitization campaigns difficult). In general, most households had a good knowledge of malaria and its mode of transmission. However, malaria treatment drugs were most often inappropriate. In this study, recommendations were made in order to guide the implementation of control measures. Keywords: Malaria, Chad-Cameroon pipeline, Knowledge, Attitudes and practices concerning malaria involved in the project. (Continued from previous page) These were the Ministry of Health/ National Malaria Control Programme beneficiary of the project, Cameroon Oil Transportation Company (COTCO) provider of logistics (transportation, lodging, etc.), “Exxon Mobil” for financial support, the University of Yaoundé 1 and the University of Bordeaux 2 responsible for the oper- ational research component, “SANOFI AVENTIS” drug manufacturing company provider of anti-malarial drugs for case management, “Service Medical International” (SMI) provider of malaria kit for community use, and the “Association Camerounaise pour le Marketing Social” (ACMS) responsible for the implementation of control measures (impregnated mosquito nets and malaria kits distribution and training of community health workers (CHW). A research component was included in the Chad-Cameroon pipeline project in order to guide and evaluate the control measures applied along the pipeline corridor. This consisted of epidemiological, entomo- logical and socio-anthropological components. This paper presents the baseline socio-anthropological data as well as the knowledge, attitudes and practices of the local population studied concerning malaria, its transmission, management and prevention. Background g Despite decades of control efforts, malaria remains a major cause of morbidity and mortality in tropical and sub- tropical regions of the world [1]. There are an estimated 300–500 million clinical diseases reported yearly with 90% of them from sub-Saharan Africa, which has an estimated 1–3 million deaths annually [1]. Malaria ranks first as the public health problem in Cameroon, representing 50% of illness in less than five years of age. It also accounts for 40- 45% of medical consultations and 40% of annual home income spent on health [2]. The burden of malaria perpetu- ates poverty and reduces chances of Cameroon government to attain the millennium goals. The Cameroon National Malaria Control Programme of the Ministry of Health is based on the fight against the vector by the use of impreg- nated mosquito nets, the appropriate management of mal- aria cases using artemisinin based combination therapy (ACTs) and communication for changing of behaviour. Earlier studies conducted in 2006 showed that only 13% of children below five years were sleeping under impregnated bed nets and that 37% of pregnant women took correct intermittent preventive treatment (IPT) during their last pregnancy. In the same study, it was also observed that 26.2% of simple malaria cases were properly managed using artemisinin based combination therapies [3]. The Cameroon Oil Transportation Company (COTCO) that ex- ploits the Chad-Cameroon pipeline along the Cameroon territory, Initiated in 2010, a public private partnership pro- ject to control malaria along the pipeline corridor. In order to attain the objective of this study, some partners were Abstract Background: Malaria is ranked as the major public health problem in Cameroon, representing 50% of illness in less than five year old children, 40-45% of medical consultation and 40% of the annual home income spent on health. The Cameroon Oil Transportation Company (COTCO) that exploits the Chad-Cameroon pipeline in Cameroon territory, initiated in 2010, a public private partnership project to control malaria along the pipeline corridor. A research component was included in the project so as to guide and evaluate the control measures applied in this pipeline corridor. This study presents the baseline socio-anthropological data as well as the knowledge, attitudes and practices of the local population concerning malaria, its transmission, management and prevention. Methods: A descriptive cross-sectional survey was undertaken in four sentinel sites (one site per ecological zone) along the Chad-Cameroon pipeline corridor. Three structured questionnaires were used for the survey. Two of them were addressed to the heads of households (one for census and the other to collect information concerning the characteristics of houses and living conditions in households as well as their knowledge, attitudes and practices concerning malaria). The last questionnaire was used to collect information on malaria management and prevention. It was addressed to women who had delivered a living child within the past three years. Interviewers were recruited from each village and trained for two consecutive days on how to fill the different questionnaires. All data were analysed at 5% significant level using Epi-Info, SPSS and Cs PRO 4.0 STATA. Values of p ≤0.05 were considered statistically significant. (Continued on next page) © 2013 Moyou-Somo 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. Page 2 of 10 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Study site N°2 l b h l h Bélabo health area is found in the East Region which is in the humid dense forest ecological zone of Cameroon (13° 18′E, 4°56′N). It is a town lying on the Yaoundé-Ngaoun- déré railway line. Bélabo health area is covered by many rivers including Sesse, Lom, Pangar, Doh yon and the Sanaga The population is estimated at 4,330 inhabitants. Hunting, fishing and farming are the main activities of this Study design This was a cross-sectional, prospective and analytical study. The population is estimated at 3,190 inhabitants. They are merchants, farmers and manage cattle ranches. The climate is tropical which is characterized by two seasons. Annual rainfall varies between 1700 and 3000 mm. Three villages (Dankali, Meidougou and Bounou) were selected in this study site. Study site N°1 Bi i di h l h Bipindi health area has a population estimated at 2,570 in- habitants. Hunting, fishing and farming are the main ac- tivities of this area. It is located in the South Region of Cameroon near the western coast of Africa in the Atlantic littoral evergreen forest (10°28′E, 3°07′N). Three villages (Bipindi rural, Bidjouka and Bikalla) were selected in this study site. Bipindi health area is in the humid forest ecological zone with a mono-modal rainfall climate. The annual rainfall ranges from 2,000 to 10,000 mm, being very high where Cameroon’s volcanic massif comes close to the coast. In general, the highest rainfall occurs be- tween the months of July and September ranging from 400 to 500 mm and declines from about 400 mm, in the wet season, to about 100 mm. Sentinel site N°4 D h l h Dompta health area, found in the North Region of Cameroon, is situated 100 kms away from Touboro, head- quarter of the health district (15°09′E, 71°18′N). The population is estimated at 1,000 inhabitants. They are farmers and manage cattle ranches. It is in the sudana- sahelian ecological zone which is made up of the Mandara Mountains, the far north plains and the Benue valley. The annual rainfall ranges from 800 to 900 mm, from the month July to October while the remaining eight months are dry. There is a dense hydrographic network made up of dry river beds (locally called Mayos) and permanent riv- ers. This zone is at the risk of desertification. Three vil- lages (Mboko, Bougoui and Bemboyo) were selected in this study site. Study population This was made-up of heads of household or their repre- sentatives and women who had given birth to a child during the past three years. Study duration Data were collected during a week in each of the four study sites. This was from the month of September 2010 for Meidougou and Dompta sites, December 2010 for Bipindi site and March 2011 for Bélabo site. An annual average temperature of 23-25°C has been recorded. Sentinel site N°3 Meidougou health area is located in the Adamawa Region of Cameroon (14°13′E, 6°25′N) and classified in the high guinea savannah ecological zone. This zone is dominated by trees and bush savanna found in the high- land plateau of Adamawa Region. Study sites The Chad-Cameroon pipeline crosses 234 villages from Ebomé (Atlantic Ocean) to Mbaï-Mboum (Chad- Cameroon border) and covers a distance of 890 kilometers going through a variety of physical environ- ments (tropical evergreen forest, forest-savannah, and savannah). These physical environments have been di- vided into four ecological zones in this study. Four sentinel sites were chosen according to ecological zone (Figure 1). General objective To identify socio-anthropologic factors that affects the persistence of malaria and determines the knowledge, at- titudes and practices of the population, concerning mal- aria and its transmission. Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Page 3 of 10 Specific objectives Specific objectives area. Six villages (Ndoumba Kanga, Bélabo, Essandjané, Ebaka I, Yébi and Biombé) were selected in this study site. The climate is equatorial with a bimodal rainfall. It has two dry seasons (July to August and mid-November to mid-March) coupled with two rainy seasons (August to mid-November and mid-March to June). An annual aver- age temperature of 30°C has been recorded. – To identify human and environmental factors that favour malaria endemicity, – To evaluate knowledge, attitudes and practices of heads of households as concerns malaria, – To identify mother’s behaviour regarding the management of febrile episodes in infants/children, – To formulate recommendations that will be used as guidelines in the implementation of control measures. Inclusion criteria The inhabitants included in the study were those who had been residents in the village concerned since at least six months and those who had given their informed consent to participate into the study. Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Page 4 of 10 BIPINDI BIPINDI MEIDOUGOU DOMPTA Figure 1 Study sites along the Chad-Cameroon pipeline (Source: COTCO archives). LEGEND. Study sites. BIPINDI Figure 1 Study sites along the Chad-Cameroon pipeline (Source: COTCO archives). LEGEND. Study sites. Filling of questionnaires g q The interviewers, holders of at least a First School Leaving Certificate (Primary education) were recruited from each village with the help of community health workers. In addition, the interviewers were expected to speak and write the French language as well as speak the local language. The selected interviewers were trained for two consecutive days on how to fill the questionnaires and on good behaviour during interviews. Each inter- viewer received a code and was asked to write it on all completed questionnaires and on the main door of each corresponding household where he/she had carried out the interview. This was to prevent duplication by another interviewer. Three types of questionnaires were used in this study: Ethical considerations from east to west, as well as, from the north to south in the following manner. For a population ≤1000 inhabi- tants, one out of two households were included while for a population ranging from 1,001 to 2,000 inhabitants, one out of three households were included. For a popu- lation of more than 2,000 inhabitants, one out of four households was selected to participate in this study. An ethical clearance was obtained from the Cameroon National Ethical Committee (Authorization N° 040/ CNE/SE/2010 of 18/12/2010). A letter of information was given to each selected household to read. The inter- viewer read the letter of information in the French lan- guage or translated it in to the local language. Households willing to participate in the study signed on a consent form. For those unable to write their finger print on the consent form served the purpose. However, households were free to withdraw from the study at any moment without any explanation. All information ob- tained from the households was treated as confidential. Questionnaire N°2 Questionnaire N°2 This questionnaire was also addressed to heads of households. It was used to collect information on the living conditions in households, their knowledge, atti- tudes and practices about malaria. c.) Distribution of ethnic groups per study site In Bipindi, the two main ethnic groups were the Ngoumba (60%) and the Bassa (26%) while in Bélabo, the Bobilis ethnic group (88.4%) was the most represented. In the two northern study sites, the ethnic groups were the same. However, their distribution varied from one study site to the other. Data analysis ld d Field data were checked for validity and entered into the computer using Microsoft Excel 2007 and Access soft- ware. Data were analyzed at 5% significant level using Epi-Info, SPSS Statistical packages, and Cs PRO 4.0 STATA. Values of p ≤0.05 were considered statistically significant. Procedures Before each field collection of data, the interview sched- ule was sent to the different village chiefs and commu- nity health workers concerned. This was to permit them to start sensitizing the population before the arrival of interviewers. On arrival in each village, the administra- tive and traditional rulers were met in order to explain the aim and purpose of the visit and to obtain their dif- ferent authorizations. Questionnaire N°1 Questionnaire N°1 This was addressed to heads of households and served the purpose of a census, (list of people living in the household, their names, ages, sex, educational level and their relationship with the head of the household. b.)Sex distribution of household heads In most of the study sites, the majority of heads of households were men (52.2% in Bipindi, 84.4% in Bélabo, 74% in Meidougou and 89.7% in Dompta). The results obtained showed that men (77.3%) were more represented in the households than women (22.7%). Exclusion criteria In each village of the study sites, households were ran- domly chosen to participate in the interviews. This was initiated from chief of village’s house and then moved The inhabitants excluded in this study were those with uncompleted questionnaires and those who had withdrawn their informed consents. Page 5 of 10 Page 5 of 10 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Socio-demographic characteristics Data obtained from the study showed that interviews were conducted in 2,597 households while the census in- dicated 14,216 inhabitants. Bipindi study site had 399 households with 2565 inhabitants, Bélabo study site had 835 households with 4,429 inhabitants, Meidougou study site had 820 households with 4,027 inhabitants while Dompta study site had 543 households with 3,195 inhabitants. a.) Mean age of heads of households The mean ages (Table 1) were higher in the study sites located in southern region (Bipindi 52 ± 16.5 yrs. and Bélabo 41 ± 18.9 yrs.) than those in the northern regions (in Meidougou 24 ± 2.75 yrs. and in Dompta 34 ± 13.4 yrs.). Questionnaire N°3 On the contrary, the percentage of free union of couples decreased from Bipindi to Dompta (Bipindi, 26.9%, Bélabo 16.7%, Meidougou 2.4% and Dompta 0.8%). Characteristics of the habitats and living conditions in the households Questionnaire N°3 The last questionnaire was used to collect information on malaria management and prevention. It was ad- dressed to women who had delivered a living child within the past three years. Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Page 6 of 10 Table 1 Distribution of household heads according to their ages Age (years) Sites TOTAL Bipindi N (%) Bélabo N (%) Meidougou N (%) Dompta N (%) N (%) <20 1 (0.2) 46 (5.5) 59 (8) 20 (4) 126 (5.1) [20-30[ 24 (6.3) 187 (22.4) 197 (26.5) 110 (22.1) 518 (21.1) [30-40[ 63 (16.6) 217 (26) 146 (19.5) 131 (26.3) 557 (22.6) [40-50[ 88 (23.2) 137 (16.4) 119 (16) 75 (15.1) 419 (17) [50 & above [ 203 (53.5) 247 (29.6) 171 (23) 71 (14.3) 692 (28.1) Do not know (0) 1 (0.1) 53 (7) 91 (18.3) 145(5.9) Mean age 52 ± 16.5 41 ± 18.9 24 ± 2.75 34 ± 13.4 37.74 ± 12.88 Total 379 (100) 835 (100) 745 (100) 498 (100) 2457 (100) Table 1 Distribution of household heads according to their ages southern (Bipindi, 40.7% and Bélabo 22.4%) to the northern regions (Meidougou 14.7% and Dompta 18.9%). g.) Profession of heads of households In most of the study sites, at least 50% of heads of households were workers of the agro-pastoral sector (Bipindi 53.6%, Bélabo 60.1%, Meidougou 70.5% and Dompta 65.8%). to the Baya tribe whereas in Dompta, the Mboum tribe was dominant (42.2% of heads households belonged to this tribe). d.)Matrimonial status In all the study sites, the majority of heads of households were married (Bipindi 48%, Bélabo 48.9%, Meidougou 65.7% and Dompta 77.2%) with an average of 59.6% (p = 0.000). The percentage of married heads of households increased from Bipindi study site in the south region to Dompta study site in the north region. On the contrary, the percentage of free union of couples decreased from Bipindi to Dompta (Bipindi, 26.9%, Bélabo 16.7%, Meidougou 2.4% and Dompta 0.8%). d.)Matrimonial status In all the study sites, the majority of heads of households were married (Bipindi 48%, Bélabo 48.9%, Meidougou 65.7% and Dompta 77.2%) with an average of 59.6% (p = 0.000). The percentage of married heads of households increased from Bipindi study site in the south region to Dompta study site in the north region. Characteristics of the habitats and living conditions in the households a.) Information concerning the habitats a.) Information concerning the habitats Four criteria were considered in their evaluations which are the roof, the ceiling, the walls and the pres- ence of mosquito nets on windows. Study sites located in the southern regions had the majority of their houses roofed with corrugated sheets (Bipindi 95.5% and Bélabo 76.1%) (Table 3). In the Savannah and Sahel regions (Meidougou 58% and Dompta 90%) the roofs were made up of straw. The presence of ceilings in the houses was rare in Bipindi (13.6%) and Bélabo (10.7%). On the con- trary, ceilings were present in Meidougou (66%) and Dompta (84%) study sites. As regards the walls, they were mostly made-up of earth in whatever the study site. The presence of nets on the windows was rare in the houses of study sites located in the forest ecological zones (Bipindi 2.2% and Bélabo 11%). In the northern study sites, more than 30% of the houses had mosquito nets on their windows. e.) Religion e.) Religion It was observed that Christianity was predominant in three study sites (Bipindi 98.2%, Bélabo 90.2%, and Dompta 64.3%). On the contrary, Islam was predominant in Meidougou (49.5%). f.) Educational level In this study, the percentage of heads of households (Table 2) who had no formal education increased from the study sites located in the humid forest ecological zone (Bipindi 13.3% and Bélabo 20.6%) to those found in the savannah ecological zone (Meidougou 51.1%) and to the study site located in the Sahel region (Dompta 54.2%). On the other hand, the percentage of heads of households who had attended secondary school decreased from the b.) Presence of electricity and exposure to media Table 2 Educational level of households heads Educational level Bipindi Bélabo Meidougou Dompta TOTAL N (%) N (%) N (%) N (%) N (%) No level 51 (13.3%) 172 (20.6%) 380 (51.1%) 270 (54.2%) 873 (35.6%) Primary 174 (46%) 470 (56.3%) 257 (34.5%) 134 (26.9%) 1035 (42.2%) Secondary and above 154 (40.7%) 187 (22.4%) 108 (14.7%) 94 (18.9%) 543 (22.1%) TOTAL 379 (100%) 829 (100%) 745 (100%) 498 (100%) 2451 (100%) Table 2 Educational level of households heads Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Page 7 of 10 Page 7 of 10 Table 3 Characteristics of houses per site Sites Bipindi Bélabo Meidougou Dompta TOTAL N = 155 N = 319 N = 275 N = 150 N = 899 Roofs - Zinc (metal sheets) 148 (95.48%) 248 (76.1%) 77 (28%) 6 (4%) 479 (53.28 %) - Straw 4 (2.58%) 22 (6.7%) 160 (58%) 136 (90%) 322 (35.81 %) - Raffia 3 (1.94%) 49 (15%) 38 (14%) 9 (6%) 99 (11.01 %) Ceiling present 21 (13.55 %) 35 (10.70%) 180 (66%) 131 (84%) 367 (40.82%) Walls - Mud 99 (63.87%) 227 (69.60%) 275 (100%) 150 (100%) 751 (83.53 %) - Semi cement 33 (21.29%) 35 (10.7%) 0 0 68 (7.56 %) Windows with nets (%) 3 (2.21 %) 36 (11%) 101 (35.8%) 56 (36.1%) 196 (21.80%) Table 3 Characteristics of houses per site places (mostly from shops), in all the three other study sites, drugs were purchased from private pharmacies and health institutions. Traditional healers and community health workers were rarely cited as drug providers. In all the study sites, the presence of electricity was rare. The highest percentage of households using electri- city was observed in Meidougou (39%). c.) Malaria prevention This study showed that knowledge on the use of impregnated mosquito nets was very high in all the study sites (Bélabo 75.5%, Bipindi 90.3%, Meidougou 90.4% and Dompta 97.4%). As one goes from the southern study sites to the northern ones, the percentage of households owning at least an impregnated mosquito bed net increases (from Bipindi 37.9% to Dompta 75.2%). In addition, the households studied (Bipindi 62.7%, Bélabo 80.9%, Meidougou 55.7% and Dompta 72.4%) reported that the main advantage of using an impregnated mosquito bed net was protection against mosquito Knowledge, attitudes and practices concerning malaria a.) General knowledge of malaria In all the study sites, more than half of the households had a correct knowledge of malaria and its mode of transmission [Bipindi 120/155 (77.08%), Bélabo 244/323 (74.5%), Meidougou 171/235 (72.8%) and Dompta 118/218 (54.1%)]. Fever and headache were the signs/symptoms most often cited by the households whatever the study site and convulsions and diarrhea were the less cited (Table 4). a.) General knowledge of malaria Table 4 Knowledge of signs and symptoms of malaria Sites Nb (%) Signs/ symptoms Bipindi Bélabo Meidougou Dompta TOTAL N 155 323 235 218 931 Fever 43 (27.7%) 263 (80.7%) 48 (20.3%) 53 (24.1%) 407 (43.7%) Headache 41 (26.4%) 235 (72.1%) 52 (21.9%) 48 (22%) 376 (40.3%) Anorexia 17 (11.1%) 237 (72.7%) 13 (5.4%) 13 (6%) 280 (30%) Vomiting 16 (10.1%) 205 (62.9%) 36 (15.5%) 21 (9.5%) 278 (29.8%) Convulsions 1 (0.7%) 73 (22.4%) 4 (1.5%) 5 (2%) 83 (8.9%) Diarrhea 2 (1.5%) 73 (22.4%) 6 (2.5%) 5 (2.5%) 85 (9.1%) Joint pains 16 (10.3%) 113 (34.7%) 24 (10.2%) 32 (14.7%) 185 (19.8%) Sweating/ Shivering - 137 (42.41%) 35 (15%) 29 (13.3%) 201 (21.5%) Table 4 Knowledge of signs and symptoms of malaria b.)Management of fever and malaria Mothers were asked if one of their children had fever/malaria during the last two weeks before the interview. More than 1/3 of the mothers answered positive while a majority sort for appropriate care within 48 hrs from the onset of fever. In most of the study sites, paracetamol was cited as the first intention drug for malaria treatment followed by chloroquine which is a banned drug in Cameroon. Only a few mothers from Bipindi (8.8%), Dompta (5.8%) and Meidougou (1.4%) knew about artemisinin based combination therapies (ACTs), the drug combinations recommended by the Ministry of Health (Figure 2). The places from where malaria drugs were purchased varied from one study site to another. Except for Dompta where 65% of households purchased drugs from inappropriate b.)Management of fever and malaria Mothers were asked if one of their children had fever/malaria during the last two weeks before the interview. More than 1/3 of the mothers answered positive while a majority sort for appropriate care within 48 hrs from the onset of fever. In most of the study sites, paracetamol was cited as the first intention drug for malaria treatment followed by chloroquine which is a banned drug in Cameroon. e.) Religion In general, more than 40% of households had a radio in all the study sites except in the Bélabo study site where only 28.3% had this communication tool. During this study, television sets were found in of homes in Bipindi (18.06%), in Bélabo (23.30%), Meidougou (6.50%) and less in Dompta (0.6%) households. Except in Bélabo where 51.2% house- holds had a mobile phone, this communication tool was used by less than 50% of households in all the other study sites. However, the signal was most often weak. Knowledge, attitudes and practices concerning malaria Only a few mothers from Bipindi (8.8%), Dompta (5.8%) and Meidougou (1.4%) knew about artemisinin based combination therapies (ACTs), the drug combinations recommended by the Ministry of Health (Figure 2). The places from where malaria drugs were purchased varied from one study site to another. Except for Dompta where 65% of households purchased drugs from inappropriate Page 8 of 10 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 obtained from the National Demographic and Health Survey which indicated that 76% of heads of Cameroonian households were men [9,10]. Similar results have also been reported in previous studies [4,8]. In Bipindi and Bélabo study sites, less than 50% of the heads of house- holds were married while in the northern regions (Meidougou and Dompta study sites) more than 60% of the heads of households were married. The discrepancy between the southern and northern regions could be ex- plained by the fact that generally in the south, the Bantu and semi-Bantu cultures are predominant while in the northern regions it is the Sudanese and the Hamite cul- ture [11]. In previous studies, the proportion of heads of households who were married varied from one study site to another with respect to the local culture [4,5,12]. In all the survey sites, most of the heads of households carried out an agro-pastoral activity. This may be due to the fact that the study was conducted in rural zones and the households were sedentary. Other studies carried out in rural zones have also showed that agro-pastoral activities were dominant in this type of setting [4,5,13]. In the Bipindi study site, Ngoumba and Bassa were the majority ethnic groups while in the Bélabo study site; the Bobilis were the dominant tribe. However, in Meiganga and Dompta study sites, it was the Baya, the Fulbé and the Mboum ethnic groups. These observations correspond to the distribution of ethnic groups in Cameroon [10]. These local languages could be used in planning sensitization messages. The level of educational is an important factor which contributes to the improvement of living conditions and it affects procreative behaviour, health and hygienic habits of a population [14]. It was observed that the per- centage of illiterate heads of households increases from the south to the north region. The North Regions had the highest level of illiteracy (Meidougou 51.0% and Dompta 54.2%). Limitations of the study During this study, due to transportation problems during the rainy season and the nature of the roads at this period of the year, certain field trips had to be rescheduled. In addition, the questionnaires did not contain a section to determine the correctness of the local language transla- tion given by the interviewers. This shortcoming could have introduced a bias in the data collected. Knowledge, attitudes and practices concerning malaria This result corroborates those reported in the National Demographic and Health Survey [9] and indi- cates that the Northern Region of Cameroon has the least level of education despite the government efforts. 44.5% 44.5% 8.8% 42.90% 3.30% 0% 38% 62.0% 17.2% 1.4% 6.20% 50.0% 11.5% 5.8% 13.5% 0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0% Paracetamol Chloroquine ACT Don't know Bipindi Bélabo Meidougou Dompta Figure 2 Drugs used for malaria treatment. Figure 2 Drugs used for malaria treatment. bites. Hence, the protection against malaria and other diseases was reported by households (Bélabo, 65.1%, Meidougou 42.6%, Bipindi 37.2% and Dompta 27.1%). The three disadvantages of impregnated mosquito bed nets frequently reported by the households were heat, feeling of being suffocated and high cost. Whatever the study site, an important percentage of pregnant women did not take any prevention against malaria during their last pregnancy (Bélabo 43.4%). However, it was observed that some of women continue to take chloroquine (Bipindi 50%). The percentage of women who had taken, at least, one dose of recommended Sulfadoxin-pyrimethamin Oral (give in full) is low, ranging from 19.5 to 50%. bites. Hence, the protection against malaria and other diseases was reported by households (Bélabo, 65.1%, Meidougou 42.6%, Bipindi 37.2% and Dompta 27.1%). The three disadvantages of impregnated mosquito bed nets frequently reported by the households were heat, feeling of being suffocated and high cost. Whatever the study site, an important percentage of pregnant women did not take any prevention against malaria during their last pregnancy (Bélabo 43.4%). However, it was observed that some of women continue to take chloroquine (Bipindi 50%). The percentage of women who had taken, at least, one dose of recommended Sulfadoxin-pyrimethamin Oral (give in full) is low, ranging from 19.5 to 50%. Socio-demographic characteristics households had electricity, in the other study sites, the percentage of households (16%) that had electricity was higher than those reported for rural zones in Cameroon [9]. Data concerning exposure to the media are important for the broadcasting of information and educative pro- grams. The presence of a TV set in households varied from 0.6% in Dompta to 23.3% in Bélabo. However, 28 to 56% of households had radios though the signals were weak. Mobile phones were also present in 29 to 51% of the households. In order to reach the maximum of people in the community, sensitization programs on the fight against malaria or any other disease could combine many communication tools such as radio spots, mobile phone messages, focus group discussion using French and local languages. Knowledge, attitudes and practices concerning malaria a.) General knowledge of malaria In this study, most of the households had a good knowledge of malaria and its transmission and they (653/931; 70.1%) correctly associated malaria to mosquito bites. These results corroborate those reported for a rural zone in Swaziland [4]. However, they are low when compared to those reported (99 and 75.9%) for Douala, the biggest city in Cameroon [17,18]. In Yaoundé, the capital city of Cameroon, the percentage was also higher (88.8%) than those found in the present study [18]. These differences are probably due to the fact that Douala and Yaoundé are big cities whereas this study was carried out in rural areas. In an urban community of south western Nigeria, 93.2% of households recognized mosquito bites as the cause of malaria [19]. In the present study, knowledge of malaria was independent of the level of education. This observation differs from that obtained in Ndu community of the North West region of Cameroon, where it was reported that the level of education was a significant indicator of malaria knowledge [20]. The most cited signs and symptoms of malaria were fever and headache regardless of the study site. This is consistent with those of a study conducted in Swaziland [4]. Chloroquine, a banned drug, was used by almost 50% of pregnant women for malaria prevention. In addition, 30 to 60% of women did not receive any malaria prevention. It was reported that 47% of pregnant women received drugs for malaria prevention during their last pregnancy, but the distribution rates varied from one zone to another [9]. In the Center and South regions, up to 70% of pregnant women took malaria prevention against 32% in the Adamawa and North regions. In the present study, the same pattern of distribution of women who received malaria prevention during their last pregnancy was observed. Socio-demographic characteristics In Dompta, one of Cameroon Oil Transportation Company’s that has pumping stations, impregnated mosquito nets are distributed to workers and to the entire community. However, there exist some myths about impregnated mosquito nets that limit its use (heat under the nets, feeling of being suffocated, etc.). These ideas should be removed from sensitization campaigns. Since 2004, the Cameroon government has adopted sulfadoxin- pyrimethamin (SP) for the intermittent preventive treatment of malaria in pregnant women. Chloroquine, a banned drug, was used by almost 50% of pregnant women for malaria prevention. In addition, 30 to 60% of women did not receive any malaria prevention. It was reported that 47% of pregnant women received drugs for malaria prevention during their last pregnancy, but the distribution rates varied from one zone to another [9]. In the Center and South regions, up to 70% of pregnant women took malaria prevention against 32% in the Adamawa and North regions. In the present study, the same pattern of distribution of women who received malaria prevention during their last pregnancy was observed. 2002, many households declared that they still used it both for malaria treatment and prevention. However, artesunate-amodiaquine, the ACT recommended by the MoH, is used by very few people. It was also observed that the behavior of in households depended on the closeness of their homes to health facilities. Households living near a health facility had the tendency to buy antimalarial drugs from this structure whereas those living far away had the tendency to go to the nearest shops and street vendors. The percentage of households that own at least one impregnated mosquito net increased gradually from the south to the north. In Meidougou study site, due to the presence of many refugee camps, several NGOs have included in their activities the distribution of impregnated mosquito net which is often also distributed to the local population. In Dompta, one of Cameroon Oil Transportation Company’s that has pumping stations, impregnated mosquito nets are distributed to workers and to the entire community. However, there exist some myths about impregnated mosquito nets that limit its use (heat under the nets, feeling of being suffocated, etc.). These ideas should be removed from sensitization campaigns. Since 2004, the Cameroon government has adopted sulfadoxin- pyrimethamin (SP) for the intermittent preventive treatment of malaria in pregnant women. Socio-demographic characteristics Most of the roofs of households in Bipindi and Bélabo study sites were made up of corrugated sheets while those of Meidougou and Dompta study sites were of straw. Ceil- ings were most often absent in Bipindi and Bélabo study site houses while it was present in Meidougou (66%) and Dompta (84%) houses. The majority of walls were of earth blocks which do not permit the retention of insecticides usually used [15,16]. In all the study sites, it was observed that the characteristics of the habitats favored human- mosquito contact. The highest percentage of electricity supply was noted in Meidougou (39%) and Bipindi (29%) households. Except for Dompta, where only 15% of In this study, 2597 heads of households were interviewed in the four selected study sites. Earlier studies used sample sizes that varied from one study to the other [4-8]. The mean ages of the households varied from 24 years in Dompta to 52 years in Bipindi. In the study sites located in the northern region of Cameroon, the heads of house- holds were younger than in the sites located in the south- ern region. This variability in the mean ages could be due to differences in the local culture of the heads of house- holds [9]. It was observed that most of the household heads were men. This observation corroborates the results Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Page 9 of 10 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 2002, many households declared that they still used it both for malaria treatment and prevention. However, artesunate-amodiaquine, the ACT recommended by the MoH, is used by very few people. It was also observed that the behavior of in households depended on the closeness of their homes to health facilities. Households living near a health facility had the tendency to buy antimalarial drugs from this structure whereas those living far away had the tendency to go to the nearest shops and street vendors. The percentage of households that own at least one impregnated mosquito net increased gradually from the south to the north. In Meidougou study site, due to the presence of many refugee camps, several NGOs have included in their activities the distribution of impregnated mosquito net which is often also distributed to the local population. Author details 1 1Department of Microbiology, Hematology, Parasitology & Infectious Diseases, Faculty of Medicine & Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon. 2Institute of Medical Research and Medicinal Plants Studies (IMPM), Yaoundé, Cameroon. 3University of Bordeaux 2, Victor Ségalen, 146 rue Leo Saignat, 33076, Bordeaux, France. 4East Regional Hospital, Bertoua, Cameroon. 5Department of Public Health, Faculty of Medicine & Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon. 6National Institute of Cartography, Yaoundé, Cameroon. 21. World Health organization: The Roll Back Malaria strategy for improving access to treatment through home management of malaria; 2005:54. www.who.int, Consulted on September 19, 2013. Authors’ contributions RMS, PE, PM and ES planned the study design. RMS, ES, JPK, NNT, HNH and AN performed field activities. ES and RMS drafted the manuscript. All authors read and approved the final manuscript. 17. Chambon R, Lemardeley P, Louis FJ, Foumane V, Louis JP: Knowledge, attitudes, and practices of population faced with culicidea nuisance: results of six Surveys taken in Cameroun in 1994. Bulletin de la Société de Pathologie exotique 1997, 90(5):364–369. Competing interests 16. Etang J, Nwane P, Mbida JA, Piameu M, Manga B, Souop D, Awono-Ambene P: Variations of insecticide residual bio efficacy on different types of walls: result from a community based trial in South Cameroon. Malar J 2011, 10:333. p g The authors declare that they have no competing interests. Conclusions Seconde enquête sur l’emploi et le secteur informel au Cameroun. Rapport principal; 2011:155. www.statistics-cameroon.org, Consulted on September 19, 2013. 9. National demographic and health survey; 2005:319. www.statistics-cameroon. org, Consulted on September 09, 2011. 10. Seconde enquête sur l’emploi et le secteur informel au Cameroun. Rapport principal; 2011:155. www.statistics-cameroon.org, Consulted on September 19, 2013. 10. Seconde enquête sur l’emploi et le secteur informel au Cameroun. Rapport principal; 2011:155. www.statistics-cameroon.org, Consulted on September 19, 2013. – that sensitization messages should insist on the drugs recommended by the MoH for malaria treatment and prevention, 11. Institut National de la Statistique du Cameroun: Enquêtes post campagne sur l’utilisation des moustiquaires imprégnées d’insecticides à longue durée d’action (EPC-Milda); 2013:81. http://www.scbutatistics-cameroon.org/downloads/ MILDA/MILDA-Rapport-complet.pdf consulted on October 28, 2013. – that along the pipeline corridor, indoor spraying should not be used as an anti-vectorial control method, 12. Ahorlu CK, Dunyo SK, Afari EA, Koram KA, Nkrumah FK: Malaria-related beliefs and behaviour in southern Ghana: implications for treatment, prevention and control. Trop Med Int Health 1997, 2:488–499. 13. Nuwaha F: People’s perception of malaria in Mbarara, Uganda. Trop Med Int Health 2002, 7:462–470. – that during sensitization campaigns, the myth about impregnated mosquito nets that limit its use (heat under the nets, feeling of being suffocated, etc.). These ideas should be rolled out 14. Dauletova GS, Karp LL, Absattarova KS: The indicators of reproductive behavior in young families as a criterion of the social and economic level of the society in Kazakhstan. Iran J Public Health 2012, 41(4):19–25. 15. World Health organization: Indoor residual spraying: an operational manual for indoor residual spraying for malaria transmission control and elimination; 2013:116. www.who.int. Consulted on September 19, 2013. Competing interests h h d l h Acknowledgements 18. Ndo C, Menze-Djantio B, Antonio-Nkondjio C: Awareness, attitudes and prevention of malaria in the cities of Douala and Yaoundé (Cameroon). Parasite Vectors 2011, 4:181. This study was granted by Exxon Mobil Foundation. The authors are grateful to COTCO and other project partners, as well as to the community members, the local data collectors (DC), health facilities personnel for their various contributions in the study. Also, we are thankful to Mr. Franklin Sidenou for data analysis and to Dr. Tom Agbor Egbe for reviewing the manuscript. 19. Adedotum AB, Morenikeji OA, Odaibo AB: Knowledge, attitude and practices about malaria in an urban community in South western Nigeria. J Vector Borne Dis 2010, 47(3):155–159. 20. Nsagha Shey D, Njunda Longdoh A, Kamga Fouamno L, Assob Nguelia C, Wiysongue Shey C, Nsagha Mboshi S, et al: Knowledge and practices relating to malaria in Ndu community of Cameroon. Signs and symptoms, causes and prevention. J Public Health Epidemiol 2011, 3(6):294–300. References 1. World Health organization: the African malaria report: WHO regional office for Africa; 2009. www.who.int/malaria/world_report_2009 consulted on October 28, 2013. 2. Ministry of Public Health: le point de la lutte contre le paludisme en; 2008. Rapport de progrès N°2. www.minsante-gov-cm, consulted on Sept.16th 2011. 2. Ministry of Public Health: le point de la lutte contre le paludisme en; 2008. Rapport de progrès N°2. www.minsante-gov-cm, consulted on Sept.16th 2011. 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: doi:10.1186/1471-2458-13-1023 Cite this article as: Moyou-Somo et al.: A public private partnership to fight against malaria along the Chad-Cameroon pipeline corridor: I. Baseline data on socio-anthropological aspects, knowledge, attitudes and practices of the population concerning malaria. BMC Public Health 2013 13:1023. Cite this article as: Moyou-Somo et al.: A public private partnership to fight against malaria along the Chad-Cameroon pipeline corridor: I. Baseline data on socio-anthropological aspects, knowledge, attitudes and practices of the population concerning malaria. BMC Public Health 2013 13:1023. Received: 13 March 2013 Accepted: 8 October 2013 Published: 29 October 2013 Received: 13 March 2013 Accepted: 8 October 2013 Published: 29 October 2013 Conclusions b.)Malaria management and prevention It was observed that the time lapse between the onset of fever in children and that to seek care by mothers was 48 hours. However, this is an acceptable time lapse though the ideal should be 12–24 hours [21]. In all the study sites, the therapeutic means used to treat malaria were not always appropriate. Paracetamol was the first intention drug followed by chloroquine. Though the later was banned in the Cameroonian market, since b.)Malaria management and prevention It was observed that the time lapse between the onset of fever in children and that to seek care by mothers was 48 hours. However, this is an acceptable time lapse though the ideal should be 12–24 hours [21]. In all the study sites, the therapeutic means used to treat malaria were not always appropriate. Paracetamol was the first intention drug followed by chloroquine. Though the later was banned in the Cameroonian market, since In this study, it was observed that there is a discrepancy in almost all the indicators (ethnic groups distribution, ages and the level of education of heads of households, characteristics of the households, etc.), between the study sites located in the southern region (Bipindi and Bélabo) and those located in the northern regions (Meidougou and Dompta). From the results obtained, the following recommenda- tions were made in order to serve as a guide in the con- trol of malaria: Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Moyou-Somo et al. BMC Public Health 2013, 13:1023 http://www.biomedcentral.com/1471-2458/13/1023 Page 10 of 10 – that sensitization programs on the fight against malaria or any other disease could combine many communication tools such as radio spots, mobile phone messages, focus group discussions using French, the official language in the study sites and local languages of the major ethnic groups, – that sensitization programs on the fight against malaria or any other disease could combine many communication tools such as radio spots, mobile phone messages, focus group discussions using French, the official language in the study sites and local languages of the major ethnic groups, 8. Sudan MF, Gayoum FG, El-Amin, El-Rayah, Hayder AG, El-Karim A: Knowledge, practices and perceptions which affects acquiring malaria in man-made malarious area in Khartoum State, Uganda. Malar J 2005, 7:69. 9. National demographic and health survey; 2005:319. www.statistics-cameroon. org, Consulted on September 09, 2011. 10. Submit your next manuscript to BioMed Central and take full advantage of: 3. Malaria indicators cluster survey (MICS): Malaria indicators cluster survey (MICS); 2006. www.childinfo.org. consulted March 21st 2011. 3. Malaria indicators cluster survey (MICS): Malaria indicators cluster survey (MICS); 2006. www.childinfo.org. consulted March 21st 2011. 4. Hlongwana KW, Mabaso ML, Kunene S, Govender D, Maharaj R: Community knowledge, attitudes and practices (KAP) on malaria in Swaziland: a country earmarked for malaria elimination. Malar J 2009, 8(29):1–8. • Convenient online submission • Thorough peer review 5. Paulander J, Olsson H, Lemma H, Getachew A, San Sebastian M: Knowledge attitude and practice about malaria in rural Tigray, Ethiopia. Global Health Action 2008, 2:43–44. 6. Ehrum WO, Abbani EO, Adesanya SO: Malaria prevention, knowledge, attitudes and practices in Southwestern Nigerian community. Afr J Biomed Res 2005, 8:25–29. 7. Nana H: le paludisme dans le bassin du Nyong-Sanaga : connaissances et stratégies de lutte. Yaoundé: Mémoire du diplôme d’ingénieur d’application de la statistique ISSEA; 2008. Submit your manuscript at www.biomedcentral.com/submit

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